Human adenovirus serotype 5 vectors containing E1 and E2B deletions encoding the ebola virus glycoprotein
Methods for generating immune responses to Ebola virus antigens using adenovirus vectors that allow multiple vaccinations with the same adenovirus vector and vaccinations in individuals with preexisting immunity to adenovirus are provided.
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This application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/US2016/012482, filed Jan. 7, 2016, which claims priority to U.S. Provisional Application No. 62/101,968, filed Jan. 9, 2015, which are incorporated herein by reference in their entirety.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 25, 2016, is named 39891-715601_SL.txt and is 166,679 bytes in size.
BACKGROUND OF THE INVENTIONEbola viruses, members of the family Filoviridae, are associated with outbreaks of highly lethal hemorrhagic fever in humans and nonhuman primates. The Bundibugyo ebolavirus, Zaire ebolavirus, and Sudan ebolavirus have all been associated with large outbreaks in Africa, The severity of the current Ebola outbreak in West Africa as highlighted the medical need for long-lasting and compressive Ebola vaccine that covers many strains for at-risk populations that do not have routine access to medical care. While some recombinant adenovirus-based vaccines have conferred good protection against multiple strains of Ebola after a single immunization, their efficacy if often impaired in human by pre-existing immunity to the adenovirus as discussed above.
Of particular interest are Ad5-based immunotherapeutics that have been repeatedly used in humans to induce robust T cell-mediated immune (CMI) responses, all while maintaining an extensive safety profile. In addition, Ad5 vectors can be reliably manufactured in large quantities and are stable for storage and delivery for outpatient administration. Nonetheless, a major obstacle to the use of first generation (E1-deleted) Ad5-based vectors is the high frequency of pre-existing anti-adenovirus type 5 neutralizing antibodies. These antibodies can be present in a potential vaccine receiver due to either prior wild type adenovirus infection and/or induction of adenovirus neutralizing antibodies by repeated injections with Ad5-based vaccines, resulting in inadequate immune stimulation against the target EA.
A major problem with adenovirus vectors has been their inability to sustain long-term transgene expression due largely to the host immune response that eliminates the adenovirus vector and virally transduced cells in immune-competent subjects. Thus, the Use of first generation adenovirus vector vaccines is severely limited by preexisting or induced immunity of vaccines to adenovirus (Ad) (Yang, et al. J Virol 77/799-803 (2003); Casimiro, et al. J Virol 77/6305-6313 (2003)). One group reported that a preponderance of humans have antibody against adenovirus type 5 (Ad5), the most widely used serotype for gene transfer vectors, and that two-thirds of humans studied have lympho-proliferative responses against Ad (Chirmule, et al. Gene Ther 6/1574-1583 (1999)). In another study, an adenovirus vector vaccine carrying an HIV-1 envelope gene was incapable of reimmunizing a primed immune response using non-adjuvanted DNA (Barouch, et al. J. Virol 77/8729-8735 (2003)). Another group reported that non-human primates having pre-existing immunity against Ad5 due to a single immunization with Ad5 were unable to generate transgene-specific antibodies to HIV proteins, as well as altering the overall T-cell responses (McCoy, et al. J. Virol 81/6594-6604 (2007)).
There are numerous mechanisms by which preexisting immunity interferes with adenovirus vector vaccines but one major concern is the presence of neutralizing antibody followed by cell mediated immune elimination of Ad infected antigen harboring cells. Both of these responses can be directed to several Ad proteins. One approach is to increase the vector vaccine dose. Although there is evidence that increasing vaccine doses can increase induction of desired cell mediated immune (CMI) responses in Ad-immune animals (Barouch, et al. J. Virol 77/8729-8735 (2003)), it often results in unacceptable adverse effects in animals and humans. When using first generation Ad5 vector vaccines, one option can be to use the approach of a heterologous prime-boost regimen, using naked (non-vectored) DNA as the priming vaccination, followed by an Ad5 vector immunization. This protocol may result in a subsequent immune response against Ad5 such that one cannot administer a further re-immunization (boost) with the same (or a different) adenovirus vector vaccine that utilizes the same viral backbone. Therefore, with the current First Generation of Ad5 vectors, using this approach can also abrogate any further use of Ad5 vector immunization in the Ad5 immunized vaccinee.
First generation (E1-deleted) adenovirus vector vaccines express Ad late genes, albeit at a decreased level and over a longer time period than wild-type Ad virus (Nevins, et al. Cell 26/213-220 (1981); Gaynor, et al. Cell 33/683-693 (1983); Yang, et al. J Virol 70/7209-7212 (1996)). When using First Generation adenovirus vectors for immunization, vaccine antigens are presented to the immune system simultaneously with highly immunogenic Ad capsid proteins. The major problem with these adenovirus vectors is that the immune responses generated are less likely to be directed to the desired vaccine epitopes (McMichael, et al. Nat Rev Immunol 2/283-291 (2002)) and more likely to be directed to the adenovirus-derived antigens, i.e., antigenic competition. There is controversy about the mechanism by which First Generation adenovirus vectors are potent immunogens. It has been hypothesized that the composition of the Ad capsid or a toxic effect of viral genes creates generalized inflammation resulting in a nonspecific immune stimulatory effect. The E1 proteins of Ad act to inhibit inflammation following infection (Schaack, et al. PNAS 101/3124-3129 (2004)). Removal of the gene segments for these proteins, which is the case for First Generation adenovirus vectors, results in increased levels of inflammation (Schaack, et al. PNAS 101/3124-3129 (2004); Schaack, et al. Viral Immunol 18/79-88 (2005)).
Thus, it is apparent that there remains a need for a more effective Ebola vaccine vector candidate. Ad vaccine vectors that allow for long-term immune response, multiple vaccinations and vaccinations in individuals with preexisting immunity to Ad. The present invention provides this and other advantages.
SUMMARY OF THE INVENTIONThe present invention relates to methods and adenovirus vectors for generating immune responses against target antigens, in particular, those related to Ebola cells. As such, the present invention further provides nucleic acid sequences that encode one or more target antigens of interest, or fragments or variants thereof. As such, the present invention provides polynucleotides that encode target antigens from any source as described further herein, vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
Attempts to overcome anti-Ad immunity have included use of alternative Ad serotypes and/or alternations in the Ad5 viral capsid protein each with limited success and the potential for significantly altering biodistribution of the resultant vaccines. Therefore, a completely novel approach was attempted by further reducing the expression of viral proteins from the E1 deleted Ad5 vectors, proteins known to be targets of pre-existing Ad immunity. Specifically, a novel recombinant Ad5 platform has been described with deletions in the early 1 (E1) gene region and additional deletions in the early 2b (E2b) gene region (Ad5 [E1-, E2b-]). Deletion of the E2b region (that encodes DNA polymerase and the pre-terminal protein) results in decreased viral DNA replication and late phase viral protein expression. This vector platform has been previously reported to successfully induce CMI responses in animal models of cancer and infectious disease and more importantly, this recombinant Ad5 gene delivery platform overcomes the barrier of Ad5 immunity and can be used in the setting of pre-existing and/or vector-induced Ad immunity thus enabling multiple homologous administrations of the vaccine.
The present disclosure provides compositions, methods and kits for generating an immune response against one or multiple Ebola antigens in an individual A composition comprising a replication defective adenovirus vector comprising a nucleic acid sequence encoding an Ebola virus antigen, wherein the Ebola virus antigen encoding sequence has 70%-100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof.
In one aspect, a composition is provided comprising a recombinant nucleic acid vector, wherein the recombinant nucleic acid vector comprises a replication defective adenovirus vector; and wherein upon administration to a human, the composition is capable of inducing an immune response directed towards cells expressing an Ebola virus antigen antigen in said human, wherein the immune response comprises cell mediated immunity.
In some embodiments, the replication defective adenovirus vector comprises a replication defective adenovirus 5 vector. In some embodiments, the replication defective adenovirus vector comprises a deletion in an E2b gene region. In some embodiments, the Ebola virus antigen comprises a modification of 25 or less amino acids.
In one aspect, a composition is provided comprising a recombinant replication defective adenovirus 5 vector having a deletion in an E2b gene region comprising a sequence encoding an Ebola virus antigen, wherein the Ebola virus antigen comprises a modification of 25 or less amino acids. In some embodiments, the Ebola virus antigen comprises a modification 20, 15, 10, 5, or less amino acids. In some embodiments, the Ebola virus antigen comprises a modification in 2, 3, or 4 amino acids. In some embodiments, the Ebola virus antigen comprises a modification in 1 amino acid. In some embodiments, the replication defective adenovirus vector comprises a deletion in an E1 gene region. In some embodiments, the replication defective adenovirus vector comprises a deletion in an E3 gene region. In some embodiments, the replication defective adenovirus vector comprises a deletion in an E4 gene region. In some embodiments, the Ebola virus is selected from the group consisting of EBOV, SUDV, TAFV, BDBV, RESTV, and any combination thereof. In some embodiments, the Ebola virus antigen comprises a sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the Ebola virus antigen is encoded by a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the Ebola virus antigen is encoded by a sequence with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the Ebola virus antigen is encoded by a sequence with at least 97% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the Ebola virus antigen is encoded by a sequence with at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the Ebola virus antigen is encoded by a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and combinations thereof. In some embodiments, the recombinant nucleic acid vector is capable of effecting overexpression of the Ebola virus antigen in transfected cells. In some embodiments, the recombinant nucleic acid vector is capable of inducing a specific immune response against cells expressing the Ebola virus antigen in a human that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 fold over basal. In some embodiments, the human has an inverse Ad5 neutralizing antibody titer of greater than 50, 75, 100, 125, 150, 175, or 200. In some embodiments, the human has an inverse Ad5 neutralizing antibody titer of greater than 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 4767. In some embodiments, the immune response is measured as an Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as a neutralizing Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as Ebola virus antigen specific cell-mediated immunity (CMI). In some embodiments, the immune response is measured as Ebola virus antigen antigen specific IFN-γ secretion. In some embodiments, the immune response is measured as Ebola virus antigen antigen specific IL-2 secretion. In some embodiments, the immune response against the Ebola virus antigen is measured by an ELISspot assay. In some embodiments, the Ebola virus antigen specific CMI is greater than 25, 50, 75, 100, 150, 200, 250, or 300 IFN-γ spot forming cells (SFC) per 106 peripheral blood mononuclear cells (PBMC). In some embodiments, the immune response is measured by T cell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic Ebola virus antigen expressing cells from an Ebola-infected cell line or from an autologous Ebola-infected cell. In some embodiments, the composition further comprises an immunogenic component. In some embodiments, the immunogenic component comprises a cytokine selected from the group consisting of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In some embodiments, the immunogenic component is selected from the group consisting of IL-7, a nucleic acid encoding IL-7, a protein with substantial identity to IL-7, and a nucleic acid encoding a protein with substantial identity to IL-7.
In one aspect, a vial is provided comprising a composition consisting of a therapeutic solution of a volume in the range of 0.8-1.2 mL, the therapeutic solution comprising 2.5-7.5×1011 virus particles; wherein the virus particles comprise a replication defective adenovirus comprising a nucleic acid sequence encoding an Ebola virus antigen.
In some embodiments, the recombinant nucleic acid vector is capable of effecting overexpression of the Ebola virus antigen in transfected cells. In some embodiments, the in transfected cells are E.C7 cells. In some embodiments, the replication defective adenovirus vector comprises a replication defective adenovirus 5 vector. In some embodiments, the replication defective adenovirus comprises a nucleic acid sequence encoding a protein that is capable of inducing a specific immune response against Ebola virus antigen expressing cells in a human. In some embodiments, the immune response is measured as an Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as a neutralizing Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as Ebola virus antigen specific cell-mediated immunity (CMI). In some embodiments, the immune response is measured as Ebola virus antigen specific IFN-γ secretion. In some embodiments, the immune response is measured as Ebola virus antigen specific IL-2 secretion. In some embodiments, the immune response against the Ebola virus antigen is measured by an ELISspot assay In some embodiments, the Ebola virus antigen specific CMI is greater than 25, 50, 75, 100, 150, 200, 250, or 300 IFN-γ spot forming cells (SFC) per 106 peripheral blood mononuclear cells (PBMC). In some embodiments, the immune response is measured by T cell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic Ebola virus antigen expressing cells from an Ebola-infected cell line, or from an autologous Ebola-infected cell. In some embodiments, the therapeutic solution comprises at least 1.0×1011, 1.5×1011, 2.0×1011, 2.5×1011, 3.0×1011, 3.5×1011, 4.0×1011, 4.5×1011, 4.8×1011, 4.9×1011, 4.95×1011, or 4.99×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises at most 7.0×1011, 6.5×1011, 6.0×1011, 5.5×1011, 5.2×1011, 5.1×1011, 5.05×1011, or 5.01×1011, virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 1.0-7.0×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 4.5-5.5×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 4.8-5.2×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 4.9-5.1×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 4.95-5.05×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the therapeutic solution comprises 4.99-5.01×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the vial further comprises an immunogenic component. In some embodiments, the immunogenic component comprises a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In some embodiments, the immunogenic component is selected from the group consisting of IL-7, a nucleic acid encoding IL-7, a protein with substantial identity to IL-7, and a nucleic acid encoding a protein with substantial identity to IL-7.
In one aspect, a method of generating an immune response against an Ebola virus antigen in a human is provided, the method comprising administering to the human a composition described herein.
In one aspect, a method of generating an immune response against an Ebola virus antigen in a human is provided, the method comprising administering to the human the composition a vial described herein.
In some embodiments, the administering step is repeated at least once. In some embodiments, the administering step is repeated after about 3 weeks following a previous administering step. In some embodiments, the administering step is repeated after about 3 months following a previous administering step. In some embodiments, the administering step is repeated twice.
In one aspect, method of generating an immune response against an Ebola virus antigen in a human is provided comprising: a first phase of treatment comprising administering to the human a first composition comprising a first replication defective adenovirus vector encoding an Ebola virus antigen that induces an immune response against cells expressing the Ebola virus antigen antigen in the human; and a subsequent second phase of treatment comprising administering to the human a second composition comprising a second replication defective adenovirus vector encoding an Ebola virus antigen that induces an immune response against cells expressing the Ebola virus antigen in the human.
In one aspect, method of treatment is provided comprising: selecting a first phase of treatment and a second phase of treatment; during the first phase, administering to a human, a total of n times, a first composition comprising a first replication defective adenovirus vector encoding an Ebola virus antigen induces an immune response against cells expressing the Ebola virus antigen antigen in the human; and during the second phase of treatment, administering the human, a total of m times, a second composition comprising a second replication defective adenovirus vector encoding an Ebola virus antigen that induces an immune response against cells expressing the Ebola virus antigen in the human.
In some embodiments, n is greater than 1. In some embodiments, n is 3. In some embodiments, m is greater than 1. In some embodiments, m is 3. In some embodiments, the first phase is at least 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the second phase is at least 2, 3, 4, 5, 6, 7, or 8 months. In some embodiments, the second phase starts 3-16 weeks after first phase ends. In some embodiments, in the first phase two administrations of the replication defective adenovirus are at least 18 days apart. In some embodiments, in the first phase two administrations of the replication defective adenovirus are about 21 days apart. In some embodiments, in the first phase two administrations of the replication defective adenovirus are at most 24 days apart. In some embodiments, in the second phase two administrations of the replication defective adenovirus are at least 10 weeks apart. In some embodiments, in the second phase two administrations of the replication defective adenovirus are about 13 weeks apart. In some embodiments, in the second phase two administrations of the replication defective adenovirus are at most 16 weeks apart.
In one aspect, method of treatment is provided comprising: selecting a first phase and a second phase of treatment; during the first phase, administering to a human a total of 3 times, in about 3 week intervals, a first composition comprising a first replication defective adenovirus vector encoding an Ebola virus antigen that induces an immune response against cells expressing the Ebola virus antigen in the human; and during the second phase, administering to said human a total of 3 times, in about 3 month intervals, a second composition comprising a second replication defective adenovirus vector encoding an an Ebola virus antigen that induces an immune response against cells expressing an Ebola virus antigen in the human; wherein the second phase starts about 3 months after the end of the first phase.
In some embodiments, the Ebola virus antigen encoded by the first replication defective adenovirus vector is the same as the Ebola virus antigen encoded by the second replication defective adenovirus vector. In some embodiments, the Ebola virus antigen encoded by the first replication defective adenovirus vector is different from the Ebola virus antigen encoded by the second replication defective adenovirus vector. In some embodiments, the first replication defective adenovirus vector and the second replication defective adenovirus vector are the same. In some embodiments, the first replication defective adenovirus vector comprises a replication defective adenovirus 5 vector. In some embodiments, the second replication defective adenovirus vector comprises a replication defective adenovirus 5 vector. In some embodiments, the first replication defective adenovirus vector comprises a sequence with 60%-100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and any combination thereof In some embodiments, the second replication defective adenovirus vector comprises a sequence with 60%-100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and any combination thereof. In some embodiments, the Ebola virus antigen encoded by the first or the second replication defective adenovirus vector comprises a modification of 25 amino acids or less. In some embodiments, the Ebola virus antigen encoded by the first or the second replication defective adenovirus vector comprises a modification of 20, 15, 10, or 5 amino acids or less. In some embodiments, the Ebola virus antigen encoded by the first or the second replication defective adenovirus vector comprises a modification of 1 amino acid. In some embodiments, the first replication defective adenovirus vector comprises a deletion in an E2b gene region. In some embodiments, the first replication defective adenovirus vector further comprises a deletion in an E1 gene region. In some embodiments, the first replication defective adenovirus vector further comprises a deletion in an E3 gene region. In some embodiments, the first replication defective adenovirus vector further comprises a deletion in an E4 gene region. In some embodiments, the second replication defective adenovirus vector comprises a deletion in an E2b gene region. In some embodiments, the second replication defective adenovirus vector further comprises a deletion in an E1 gene region. In some embodiments, the second replication defective adenovirus vector further comprises a deletion in an E3 gene region. In some embodiments, the second replication defective adenovirus vector further comprises a deletion in an E4 gene region. In some embodiments, the first composition, the second composition, or both, comprises at least 1.0×1011, 1.5×1011, 2.0×1011, 2.5×1011, 3.0×1011, 3.5×1011, 4.0×1011, 4.5×1011, 4.8×1011, 4.9×1011, 4.95×1011, or 4.99×1011 virus particles comprising the recombinant nucleic acid vector. In some embodiments, the first composition, the second composition, or both, comprises at most 7.0×1011, 6.5×1011, 6.0×1011, 5.5×1011, 5.2×1011, 5.1×1011, 5.05×1011, or 5.01×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 1.0-7.0×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 4.5-5.5×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 4.8-5.2×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 4.9-5.1×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 4.95-5.05×1011 virus particles. In some embodiments, the first composition, the second composition, or both, comprises 4.99-5.01×1011 virus particles. In some embodiments, the immune response to the Ebola virus antigen is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 fold. In some embodiments, the immune response is measured as an Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as a neutralizing Ebola virus antigen specific antibody response. In some embodiments, the immune response is measured as Ebola virus antigen specific cell-mediated immunity (CMI). In some embodiments, the immune response is measured as Ebola virus antigen specific IFN-γ secretion. In some embodiments, the immune response is measured as Ebola virus antigen specific IL-2 secretion. In some embodiments, the immune response against the Ebola virus antigen is measured by ELISspot assay. In some embodiments, the Ebola virus antigen specific CMI is greater than 25, 50, 75, 100, 150, 200, 250, or 300 IFN-γ spot forming cells (SFC) per 106 peripheral blood mononuclear cells (PBMC). In some embodiments, the immune response is measured by T cell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic Ebola virus antigen expressing cells from an Ebola-infected cell line or from an autologous Ebola-infected cell. In some embodiments, a first or a second replication defective adenovirus infects dendritic cells in the human, and wherein the infected dendritic cells present the Ebola virus antigen, thereby inducing the immune response. In some embodiments, the administering steps comprise subcutaneous administration. In some embodiments, the human carries an inverse Ad5 neutralizing antibody titer that is of greater than 50, 75, 100, 125, 150, 160, 175, 200, 225, 250, 275, or 300 prior to the administering step. In some embodiments, the human has an inverse Ad5 neutralizing antibody titer of greater than 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 4767. In some embodiments, the human is not concurrently being treated by any one of steroids, corticosteroids, immunosuppressive agents, and immunotherapy. In some embodiments, the human has not been treated by any one of steroids, corticosteroids, immunosuppressive agents, and immunotherapy prior to the administering step. In some embodiments, the human does not have an autoimmune disease. In some embodiments, the human does not have inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, multiple sclerosis, viral hepatitis, or HIV. In some embodiments, the human has autoimmune related thyroid disease or vitiligo. In some embodiments, the human has cells expressing the Ebola virus antigen. In some embodiments, the human does not have cells expressing the Ebola virus antigen. In some embodiments, the human has at least one, two, or three symptoms of an Ebola virus infection. In some embodiments, the human has received a therapy prior to the administering. In some embodiments, prior to the first phase, the human has received at least one medication selected from the group consisting of: rehydration with oral or intravenous fluids, blood products, immune therapies, drug or therapies for specific symptoms such as fever, fatigue, muscle pain, headache and sore throat, vomiting, diarrhoea, rash, impaired kidney and liver function, and internal and external bleeding. In some embodiments, the human concurrently receives chemotherapy or radiation therapy treatment. In some embodiments, the human concurrently receives a therapy comprising the administration of at least one medication of the group consisting of fluoropyrimidine, irinotecan, oxaliplatin, bevacizunab, Capecitabine, Mitomycin, Regorafenib, cetuxinab, panitumumab, and acetinophen. In some embodiments, the human comprises cells overexpressing the Ebola virus antigen. In some embodiments, the cells overexpressing the Ebola virus antigen overexpress the Ebola virus antigen by at least 2, 3, 4, 5, 10, 15, or 20 times over a baseline expression of an Ebola virus antigen in a non-infected cell In some embodiments, the cells overexpressing the Ebola virus antigen comprise Ebola-infected cells. In some embodiments, the cells overexpressing the Ebola virus antigen comprise immune cells. In some embodiments, the cells overexpressing the Ebola virus antigen comprise blood cells. In some embodiments, the cells overexpressing the Ebola virus antigen comprise epithelium cells. In some embodiments, the Ebola virus antigen is an antigen from EBOV, SUDV, TAFV, BDBV, RESTV, or any combination thereof.
INCORPORATION BY REFERENCEAll publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The following passages describe different aspects of the invention in greater detail. Each aspect of the invention may be combined with any other aspect or aspects of the invention unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature of features indicated as being preferred or advantageous. As used herein, unless otherwise indicated, the article “a” means one or more unless explicitly otherwise provided for. As used herein, unless otherwise indicated, terms such as “contain,” “containing,” “include,” “including,” and the like mean “comprising.” As used herein, unless otherwise indicated, the term “or” can be conjunctive or disjunctive. As used herein, unless otherwise indicated, any embodiment can be combined with any other embodiment.
It has been discovered that Ad5 [E1-, E2b-] vectors are not only are safer than, but appear to be superior to Ad5 [E1-] vectors in regard to induction of antigen specific immune responses, making them much better suitable as a platform to deliver Ebola vaccines that can result in a clinical response. In other cases, immune induction may take months. Ad5 [E1-, E2b-] vectors not only are safer than, but appear to be superior to Ad5 [E1-] vectors in regard to induction of antigen specific immune responses, making them much better suitable as a platform to deliver Ebola vaccines that can result in a clinical response.
Various embodiments of the invention, by taking advantage of the new Ad5 [E1-, E2b-] vector system in delivering a long sought-after need for a develop a therapeutic vaccine against Ebola, overcome barriers found with other Ad5 systems and permit the immunization of people who have previously been exposed to Ad5. In other embodiments of the invention, by taking advantage of the new Ad5 [E1-, E2b-] vector system in delivering a long sought-after need for a develop a therapeutic vaccine against Ebola, overcome barriers found with other Ad5 systems and permit the immunization of people who have previously been exposed to Ad5. In other embodiments of the invention, by taking advantage of the new Ad5 [E1-, E2b-] vector system in delivering a long sought-after need for a develop a therapeutic vaccine against Ebola, overcome barriers found with other Ad5 systems and permit the immunization of people who have previously been exposed to Ad5.
An “adenovirus” (Ad) refers to non-enveloped DNA viruses from the family Adenoviridae. These viruses can be found in, but are not limited to, human, avian, bovine, porcine and canine species. The present invention contemplates the use of any Ad from any of the four genera of the family Adenoviridae (e.g., Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus) as the basis of an E2b deleted virus vector, or vector containing other deletions as described herein. In addition, several serotypes are found in each species. Ad also pertains to genetic derivatives of any of these viral serotypes, including but not limited to, genetic mutations, deletions or transpositions.
A “helper adenovirus” or “helper virus” refers to an Ad that can supply viral functions that a particular host cell cannot (the host may provide Ad gene products such as E1 proteins). This virus is used to supply, in trans, functions (e.g., proteins) that are lacking in a second virus, or helper dependent virus (e.g., a gutted or gutless virus, or a virus deleted for a particular region such as E2b or other region as described herein); the first replication-incompetent virus is said to “help” the second, helper dependent virus thereby permitting the production of the second viral genome in a cell.
An “adenovirus 5 null (Ad5-null)” refers to a non-replicating Ad that does not contain any heterologous nucleic acid sequences for expression.
A “first generation adenovirus” refers to an Ad that has the early region 1 (E1) deleted. In additional cases, the early region 3 (E3) may also be deleted.
“Gutted” or “gutless” refers to an Ad vector that has been deleted of all viral coding regions.
“Transfection” refers to the introduction of foreign nucleic acid into eukaryotic cells. Exemplary means of transfection include calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
“Stable transfection” or “stably transfected” refers to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell. The term “stable transfectant” refers to a cell which has stably integrated foreign DNA into the genomic DNA.
A “reporter gene” indicates a nucleotide sequence that encodes a reporter molecule (e.g., an enzyme). A “reporter molecule” is detectable in any of a variety of detection systems, including, but not limited to, enzyme-based detection assays (e.g., ELISA, histochemical assays), fluorescent, radioactive, and luminescent systems. The E. coli β-galactosidase gene, green fluorescent protein (GFP), the human placental alkaline phosphatase gene, the chloramphenicol acetyltransferase (CAT) gene; and other reporter genes may be employed.
A “heterologous sequence” refers to a nucleotide sequence that is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous nucleic acid may include a naturally occurring nucleotide sequence or some modification relative to the naturally occurring sequence.
A “transgene” refers to any gene coding region, either natural or heterologous nucleic acid sequences or fused homologous or heterologous nucleic acid sequences, introduced into cells or a genome of subject. Transgenes may be carried on any viral vector used to introduce transgenes to the cells of the subject.
A “second generation adenovirus” refers to an Ad that has all or parts of the E1, E2, E3, and, in certain embodiments, E4 DNA gene sequences deleted (removed) from the virus.
A “subject” refers to any animal, including, but not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowls.
An “immunogenic fragment” refers to a fragment of a polypeptide that is specifically recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor resulting in a generation of an immune response specifically against a fragment.
A “target antigen” or “target protein” refers to a molecule, such as a protein, against which an immune response is to be directed.
“E2b deleted” refers to a DNA sequence mutated in such a way so as to prevent expression and/or function of at least one E2b gene product. Thus, in certain embodiments, “E2b deleted” is used in relation to a specific DNA sequence that is deleted (removed) from an Ad genome. E2b deleted or “containing a deletion within an E2b region” refers to a deletion of at least one base pair within an E2b region of an Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, a deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within an E2b region of an Ad genome. An E2b deletion may be a deletion that prevents expression and/or function of at least one E2b gene product and therefore, encompasses deletions within exons of encoding portions of E2b-specific proteins as well as deletions within promoter and leader sequences. In certain embodiments, an E2b deletion is a deletion that prevents expression and/or function of one or both a DNA polymerase and a preterminal protein of an E2b region. In a further embodiment, “E2b deleted” refers to one or more point mutations in a DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations include residues that are replaced with a different residue leading to a change in an amino acid sequence that result in a nonfunctional protein.
“E1-deleted” refers to a DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one E1 gene product. Thus, in certain embodiments, “E1 deleted” is used in relation to a specific DNA sequence that is deleted (removed) from the Ad genome. E1 deleted or “containing a deletion within the E1 region” refers to a deletion of at least one base pair within the E1 region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, the deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E1 region of the Ad genome. An E1 deletion may be a deletion that prevents expression and/or function of at least one E1 gene product and therefore, encompasses deletions within exons of encoding portions of E1-specific proteins as well as deletions within promoter and leader sequences. In certain embodiments, an E1 deletion is a deletion that prevents expression and/or function of one or both of a trans-acting transcriptional regulatory factor of the E1 region. In a further embodiment, “E1 deleted” refers to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.
“Generating an immune response” or “inducing an immune response” refers to a statistically significant change, e.g., increase or decrease, in the number of one or more immune cells (T-cells, B-cells, antigen-presenting cells, dendritic cells, neutrophils, and the like) or in the activity of one or more of these immune cells (CTL activity, HTL activity, cytokine secretion, change in profile of cytokine secretion, etc.).
The terms “nucleic acid” and “polynucleotide” are used essentially interchangeably herein. Polynucleotides of the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (e.g., genomic, cDNA, or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. An isolated polynucleotide, as used herein, means that a polynucleotide is substantially away from other coding sequences. For example, an isolated DNA molecule as used herein does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. This refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment recombinantly in the laboratory.
As will be understood by those skilled in the art, the polynucleotides of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express target antigens as described herein, fragments of antigens, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
When comparing polynucleotide sequences, two sequences are “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA), or by inspection.
One example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program uses as defaults a word length (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.
The “percentage of sequence identity” can be determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence and multiplying the results by 100 to yield the percentage of sequence identity.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a particular antigen of interest, or fragment thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
As would be understood by the skilled artisan upon reading the present disclosure, other regions of the Ad genome can be deleted. Thus to be “deleted” in a particular region of the Ad genome, as used herein, refers to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one gene product encoded by that region. In certain embodiments, to be “deleted” in a particular region refers to a specific DNA sequence that is deleted (removed) from the Ad genome in such a way so as to prevent the expression and/or the function encoded by that region (e.g., E2b functions of DNA polymerase or preterminal protein function). “Deleted” or “containing a deletion” within a particular region refers to a deletion of at least one base pair within that region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted from a particular region. In another embodiment, the deletion is more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within a particular region of the Ad genome. These deletions are such that expression and/or function of the gene product encoded by the region is prevented. Thus deletions encompass deletions within exons encoding portions of proteins as well as deletions within promoter and leader sequences. In a further embodiment, “deleted” in a particular region of the Ad genome refers to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein. Deletions or mutations in the Ad genome can be within one or more of E1a, E1b, E2a, E2b, E3, E4, L1, L2, L3, L4, L5, TP, POL, IV, and VA regions. The deleted adenovirus vectors of the present invention can be generated using recombinant techniques.
As would be recognized by the skilled artisan, the adenovirus vectors for use in the present invention can be successfully grown to high titers using an appropriate packaging cell line that constitutively expresses E2b gene products and products of any of the necessary genes that may have been deleted. In certain embodiments, HEK-293-derived cells that not only constitutively express the E1 and DNA polymerase proteins, but also the Ad-preterminal protein, can be used. In one embodiment, E.C7 cells are used to successfully grow high titer stocks of the adenovirus vectors.
In order to delete critical genes from self-propagating adenovirus vectors, the proteins encoded by the targeted genes can first be coexpressed in HEK-293 cells, or similar, along with the E1 proteins. For example, only those proteins which are non-toxic when coexpressed constitutively (or toxic proteins inducibly-expressed) can be selectively utilized. Coexpression in HEK-293 cells of the E1 and E4 genes is possible. The E1 and protein IX genes, a virion structural protein, can be coexpressed. Further coexpression of the E1, E4, and protein IX genes is also possible. The E1 and 100 k genes can be successfully expressed in transcomplementing cell lines, as can E1 and protease genes.
Cell lines coexpressing E1 and E2b gene products for use in growing high titers of E2b deleted Ad particles are described. The E2b region encodes viral replication proteins, which are essential for Ad genome replication. Useful cell lines constitutively express the approximately 140 kDa Ad-DNA polymerase and/or the approximately 90 kDa preterminal protein. In particular, cell lines that have high-level, constitutive coexpression of the E1, DNA polymerase, and preterminal proteins, without toxicity (e.g., E.C7), are desirable for use in propagating Ad for use in multiple vaccinations. These cell lines permit the propagation of adenovirus vectors deleted for the E1, DNA polymerase, and preterminal proteins.
Further information on viral delivery systems can be found in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.
Heterologous Nucleic Acid
The adenovirus vectors of the present invention typically comprise modified or heterologous nucleic acid sequences that encode one or more target antigens of interest, or variants, fragments or fusions thereof, against which it is desired to generate an immune response. In some embodiments, the adenovirus vectors of the present invention comprise modified or heterologous nucleic acid sequences that encode one or more proteins, variants thereof, fusions thereof, or fragments thereof, that can modulate the immune response. In a further embodiment of the invention, the adenovirus vector of the present invention encodes one or more antibodies against specific antigens, such as anthrax protective antigen, permitting passive immunotherapy. In certain embodiments, the adenovirus vectors of the present invention comprise modified or heterologous nucleic acid sequences encoding one or more proteins having therapeutic effect (e.g., anti-viral, anti-bacterial, anti-parasitic, or anti-Ebola function). Thus the present invention provides the Second Generation E2b deleted adenovirus vectors that comprise a heterologous nucleic acid sequence. In some embodiments, the heterologous modified or nucleic acid sequence is an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, a variant thereof, a fragment thereof, or a combination thereof. In some embodiments, the heterologous modified or nucleic acid sequence is a combination or fusion of an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, a variant thereof, a fragment thereof, or a combination thereof. In some embodiments, the heterologous modified or nucleic acid sequence is a combination or fusion of an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, a variant thereof, a fragment thereof, or a combination thereof.
In particular, the present invention provides an improved adenovirus (Ad)-based vaccine such that multiple vaccinations against one or more antigenic target entity can be achieved. In some embodiments, the improved adenovirus (Ad)-based vaccine comprises a replication defective adenovirus carrying a target antigen, a fragment, a variant or a variant fragment thereof, such as Ad5 [E1-, E2b-]-SEQ. ID. NO.:1, Ad5 [E1-, E2b-]-SEQ. ID. NO.:2, Ad5 [E1-, E2b-]-SEQ. ID. NO.:4, Ad5 [E1-, E2b-]-SEQ. ID. NO.:5, Ad5 [E1-, E2b-]-SEQ. ID. NO.:6. In some embodiments, the improved adenovirus (Ad)-based vaccine comprises a replication defective adenovirus carrying a target antigen, a fragment, a variant or a variant fragment thereof, such as Ad5 [E1-, E2b-]-GP, Ad5 [E1-, E2b-]-NP, Ad5 [E1-, E2b-]-VP40, Ad5 [E1-, E2b-]-VP35, Ad5 [E1-, E2b-]-VP30, and Ad5 [E1-, E2b-]-VP24. Variants and/or fragments of target antigens, for example EBOV, SUDV, TAFV, BDBV, or RESTV antigens, such as GP, NP, VP40, VP35, VP30, or VP24, can be selected based on a variety of factors, including immunogenic potential. Accordingly, a mutant of an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as a mutant of a GP, NP, VP40, VP35, VP30, or VP24 antigen is utilized in various embodiments of the invention for its increased capability to raise an immune response relative to the wild type form. Importantly, vaccination can be performed in the presence of preexisting immunity to the Ad and/or administered to subjects previously immunized multiple times with the adenovirus vector of the present invention or other adenovirus vectors. The adenovirus vectors of the invention can be administered to subjects multiple times to induce an immune response against an antigen of interest, for example an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as GP, NP, VP40, VP35, VP30, or VP24, including but not limited to, the production of antibodies and cell-mediated immune responses against one or more target antigens of EBOV, SUDV, TAFV, BDBV, or RESTV, such as GP, NP, VP40, VP35, VP30, or VP24 and/or one or more Ebola virus strains, as described herein and publically available on GenBank.
The immunogenic polypeptide may be RNA sequence from Ebola Zaire (GenBank: KJ660347.2) or a fragment thereof. In some embodiments, the replication defective adenovirus vector comprises a sequence encoding a nucleotide or polypeptide with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% identity to the immunogenic polypeptide.
In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:1. In some embodiments, the sequence encoding the immunogenic nucleotide or polypeptide comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% identity to SEQ. ID. NO.:1 or a sequence generated from SEQ. ID. NO.:1 by alternative codon replacements optimized for the human genome. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors described herein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type GenBank sequence (Zaire KJ660347).
In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:2. In some embodiments, the sequence encoding the immunogenic nucleotide or polypeptide comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% identity to SEQ. ID. NO.:2 or a sequence generated from SEQ. ID. NO.:2 by alternative codon replacements optimized for the human genome. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors described herein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type GenBank sequence (Sudan KC545392.1).
In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:4. In some embodiments, the sequence encoding the immunogenic nucleotide or polypeptide comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% identity to SEQ. ID. NO.:4 or a sequence generated from SEQ. ID. NO.:4 by alternative codon replacements optimized for the human genome. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors described herein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type NCBI sequence (Tai Forest NC_014372.1).
In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:5. In some embodiments, the sequence encoding the immunogenic nucleotide or polypeptide comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% identity to SEQ. ID. NO.:5 or a sequence generated from SEQ. ID. NO.:5 by alternative codon replacements optimized for the human genome. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors described herein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type NCBI sequence Bundibugyo ebolavirus (NC_014373.1).
In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:6. In some embodiments, the sequence encoding the immunogenic nucleotide or polypeptide comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to SEQ. ID. NO.:6 or a sequence generated from SEQ. ID. NO.:6 by alternative codon replacements optimized for the human genome. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors described herein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type Reston ebolavirus (GenBank: JX477166.1).
In various embodiments, the adenovirus-derived vectors described herein have a deletion in the E2b region, and optionally, in the E1 region, the deletion conferring a variety of advantages to the use of the vectors in immunotherapy as described herein.
Certain regions within the adenovirus genome serve essential functions and may need to be substantially conserved when constructing the replication defective adenovirus vectors of the invention. (See, Lauer et al., J. Gen. Virology, 85, 2615-2625 (2004)); Leza et al., J. Virology, pp. 3003-3013 (1988); and Miralles et al., JBC. Vol. 264, No. 18, pp. 10763-10772 (1983).
First generation, E1-deleted Adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, provide an opportunity to avoid immunological clearance and induce more potent immune responses against the encoded antigen transgene in Ad-immune hosts.
Multiple homologous immunizations with Ad5 [E1-, E2b-]-EA, encoding an Ebola antigen may be used according to the present invention to induce EA-specific cell-mediated immune (CMI) responses with anti-Ebola activity in mice despite the presence of pre-existing or induced Ad5-neutralizing antibody. Cohorts of patients with Ebola can be immunized with escalating doses of Ad5 [E1-, E2b-]-EA. EA-specific CMI responses may be observed despite the presence of pre-existing Ad5 immunity in many or a majority of patients. Importantly, minimal toxicity, and overall patient survival may be similar regardless of pre-existing Ad5 neutralizing antibody titers. In Ebola infected subjects, the novel Ad5 [E1-, E2b-] gene delivery platform can be used to generate significant CMI responses to Ebola antigens in the setting of both naturally acquired and immunization-induced Ad5 specific immunity. An Ebola antigen specific CMI can be, for example, greater than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, or more IFN-γ spot forming cells (SFC) per 106 peripheral blood mononuclear cells (PBMC). Thus, the methods and compositions of the invention relate to a recombinant nucleic acid vector, wherein the recombinant nucleic acid vector comprises a replication defective adenovirus vector, and wherein upon administration to a human, the composition is capable of inducing an immune response directed towards cells expressing an Ebola antigen in said human. The immune response may be induced even in the presence of preexisting immunity against Ad5. In some embodiments, the immune response is raised in a human subject with a preexisting inverse Ad5 neutralizing antibody titer of greater than 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 1000, 12000, 15000 or higher. The immune response may comprise a cell-mediated immunity and/or a humoral immunity as described herein. The immune response may be measured by one or more of intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays, as described herein and to the extent they are available to a person skilled in the art, as well as any other suitable assays known in the art for measuring immune response.
While Ebola immunotherapy achieved by delivering Ebola-associated antigens (EA) provides some survival benefits, limitations to these strategies exist and more immunologically potent vaccines are needed. To address the low immunogenicity a variety of advanced, multi-component vaccination strategies including co-administration of adjuvants and immune stimulating cytokines are provided. The invention relates to recombinant viral vectors that inherently provide innate pro-inflammatory signals, while simultaneously engineered to express the antigen of interest. Of particular interest is adenovirus serotype-5 (Ad5)-based immunotherapeutics that have been repeatedly used in humans to induce robust T cell-mediated immune (CMI) responses, all while maintaining an extensive safety profile. In addition, Ad5 vectors can be reliably manufactured in large quantities and are stable for storage and delivery for outpatient administration. Nonetheless, a major obstacle to the use of first generation (E1-deleted) Ad5-based vectors is the high frequency of pre-existing anti-adenovirus type 5 neutralizing antibodies. These antibodies can be present in a potential vaccinee due to either prior wild type adenovirus infection and/or induction of adenovirus neutralizing antibodies by repeated injections with Ad5-based vaccines, each resulting in inadequate immune stimulation against the target EA.
Provided herein is an Ad5 [E1-, E2b-] platform containing a gene insert for a EBOV, SUDV, TAFV, BDBV, or RESTV antigen with a modification that enhances T cell responses and is used in various embodiments of the invention for therapies raising an immune response against at least one EBOV, SUDV, TAFV, BDBV, or RESTV antigen. Multiple immunizations with this Ad5 platform can be used to induce EBOV, SUDV, TAFV, BDBV, or RESTV antigen specific CMI responses with anti-Ebola activity despite the presence of existing Ad5 immunity in mice. In some embodiments the Ad5 [E1-, E2B-] comprises SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, or a combination thereof. In some embodiments the Ad5 [E1-, E2B-] comprises an EBOV, SUDV, TAFV, BDBV, or RESTV antigen encoding sequence from GenBank. In some embodiments the Ad5 [E1-, E2B-] comprises a sub-species of EBOV, SUDV, TAFV, BDBV, or RESTV antigen encoding sequence from a human isolate from GenBank or NCBI. It is contemplated, that a phase I/II clinical trial of EBOV, SUDV, TAFV, BDBV, or RESTV immunotherapies, as provided herein, would demonstrate safety and immunogenicity in humans CMI can be induced without a substantial effect on clinical outcome relative to the existence of pre-existing Ad5-immunity.
EBOV as Target for Immune Response
Ebola virus (EBOV) is a member of the family Filoviridae. Its genome comprises a single-stranded, RNA molecule of approximately 19-kb in size. Ebola virions are filamentous particles that may appear in the shape of a shepherd's crook, of a “U” or of a “6,” and they may be coiled, toroid or branched. In general, Ebola virions are 80 nanometers (nm) in width and may be as long as 14,000 nm. EBOV can be subdivided into at least five distinct species with different levels of pathogenicities. The genomes of the five different Ebolaviruses (BDBV, EBOV, RESTV, SUDV and TAFV) differ in sequence and the number and location of gene overlaps.
Ebola viruses display filamentous particles that give the virus its characteristic name, are enveloped, non-segmented, have negative stranded RNA and varying morphology. The Ebola virus genome contains seven genes, the nucleoprotein (NP), virion protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, and an RNA-dependent RNA polymerase (L). Except for GP, all genes are monocistronic and encode one structural protein. The inner ribonucleoprotein complex of the virus contains the RNA genome that is encapsulated by the NP, which associates with VP35, VP30, and RNA-dependent RNA polymerase to the functional transcriptase-replicase complex. Proteins of the ribonucleoprotein complex have additional functions such as VP35 that is an interferon antagonist. VP40 is a matrix protein and mediates virus particle formation. VP24 is another structural protein associated with the membrane and interferes with interferon signaling. The GP is the only transmembrane surface protein and forms trimeric spikes consisting of GP-1 and GP-2 that are two disulphide linked furin-cleavage fragment. An important feature of the Ebola virus as compared to other Mononegavirales is the production of soluble GP (from the GP gene) secreted out of infected cells.
In some aspects, the disclosure provides for a recombinant vector as provided herein comprising at least one target virus antigen from the BDBV. In some aspects, the disclosure provides for a recombinant vector as provided herein comprising at least one target virus antigen from EBOV. In some aspects, the disclosure provides for a recombinant vector as provided herein comprising at least one target virus antigen from RESTV. In another aspect the disclosure provides for a recombinant vector as provided herein comprising at least one target virus antigen from SUDV. In another aspect the disclosure provides for a recombinant vector as provided herein comprising at least one target virus antigen from TAFV. In another aspect the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens from Ebola viruses described in GenBank and NCBI.
In another aspect the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens from a combination of Ebola strains, for example BDBV, EBOV, RESTV, SUDV and TAFV, and others as described in GenBank and NCBI.
Ebola virions, like virions of other filoviruses, can contain seven proteins (see
The glycoprotein of Ebola virus is unusual in that it is encoded in two open reading frames. Transcriptional editing is needed to express the transmembrane form that is incorporated into the virion. The unedited form produces a nonstructural secreted glycoprotein (sGP) that is synthesized in large amounts early during the course of infection. In some cases, the encoded protein is cut after translation, generating a mature secreted form that sits on the surfaces of viral particles, as well as a sugar-coated smaller part.
In some aspects, the disclosure provides for a recombinant vector as provided herein comprising a target virus antigen from the glycoprotein (GP) of at least one Ebola virion. In some aspects the disclosure provides for a recombinant vector as provided herein comprising a target virus antigen from a nucleoprotein (NP) of at least one Ebola virion. In some aspects, the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens from at least one of the four virion structural proteins (VP40, VP35, VP30, and VP24), from at least one Ebola virion. For example, a recombinant vector can comprise a target virus antigen from VP40 of at least one Ebola virion. For example, a recombinant vector can comprise a target virus antigen from VP35 of at least one Ebola virion. For example, a recombinant vector can comprise a target virus antigen from VP30 of at least one Ebola virion. For example, a recombinant vector can comprise a target virus antigen from VP24 of at least one Ebola virion. In some aspects, the disclosure provides for a recombinant vector as provided herein comprising a target virus antigen from the L protein of at least one Ebola virion.
The Ebola life cycle is thought to begin with a virion contacting a host cell. The structural glycoprotein (known as GP1,2) is responsible for the virus' ability to bind to and infect targeted cells. The virion is thought to attach to specific cell-surface receptors on the host cell such as, for example, C-type lectins, DC-SIGN, or integrins, which is followed by fusion of the viral envelope with host cell's cellular membranes. After the virions taken up by the host cell they then travel to acidic endosomes and lysosomes where the viral envelope glycoprotein GP is cleaved.
The viral RNA polymerase, encoded by the L gene, partially uncoats the nucleocapsid and transcribes the genes into positive-strand mRNAs, which are then translated into structural and nonstructural proteins that comprise the virion. The most abundant protein produced is the nucleoprotein, whose concentration in the host cell determines when L switches from gene transcription to genome replication. Replication of the viral genome results in full-length, positive-strand antigenomes that are, in turn, transcribed into genome copies of negative-strand virus progeny. Newly synthesized structural proteins and genomes self-assemble and accumulate near the inside of the cell membrane. Virions bud off from the host cell, gaining their envelopes from the cellular membrane from which they bud from. The mature viral progeny particles then infect other cells to repeat the cycle.
In some aspects, the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens from the L gene of at least one Ebola virion. In one aspect the disclosure provides for a recombinant vector as provided herein comprising the target virus that inhibits the production of the nucleoprotein of at least one Ebola virion. In one aspect the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens that inhibit the genome replication of at least one Ebola virion. In one aspect the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens that inhibit the budding process of at least one Ebola virion. In one aspect, the disclosure provides for a recombinant vector as provided herein comprising the target virus antigens that inhibit the infection process of at least one Ebola virion.
In various embodiments, Ad5 [E1-, E2B-]-SEQ. ID. NO.:1, Ad5 [E1-, E2B-]-SEQ. ID. NO.:2, Ad5 [E1-, E2B-]-SEQ. ID. NO.:4, Ad5 [E1-, E2B-]-SEQ. ID. NO.:5, and/or Ad5 [E1-, E2B-]-SEQ. ID. NO.:6 increase the capability to transduce dendritic cells, improving antigen specific immune responses in the vaccine by taking advantage of the reduced inflammatory response against Ad5 [E1-, E2b-] vector viral proteins and the resulting evasion of pre-existing Ad immunity.
For example, Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L can increase the capability to transduce dendritic cells, improving antigen specific immune responses in the vaccine by taking advantage of the reduced inflammatory response against Ad5 [E1-, E2b-] vector viral proteins and the resulting evasion of pre-existing Ad immunity.
In various embodiments Ad5 [E1-, E2B-]-SEQ. ID. NO.:1, Ad5 [E1-, E2B-]-SEQ. ID. NO.:2, Ad5 [E1-, E2B-]-SEQ. ID. NO.:4, Ad5 [E1-, E2B-]-SEQ. ID. NO.:5, Ad5 [E1-, E2B-]-SEQ. ID. NO.:6 therapeutic and preventative vaccines can be used to increase overall survival (OS) of a human and have a toxicity profile bounds of technical safety. For example, Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L therapeutic and preventative vaccines can be used to increase overall survival (OS) of a human and have a toxicity profile bounds of technical safety.
Further, in various embodiments, the composition and methods of the invention lead to clinical responses, such as altered disease progression or life expectancy of human infected with Ebola. Further, in various embodiments, the composition and methods of the invention lead to clinical responses, such as altered disease progression or life expectancy of human at low, medium and high risk for infection with Ebola.
In some aspects, the disclosure provides compositions and methods using adenovirus based vectors expressing at least one antigen selected from the group consisting of: an EBOV, SUDV, TAFV, BDBV, and RESTV antigen. For example, the disclosure provides compositions and methods using adenovirus based vectors expressing at least one antigen selected from the group consisting of GP, NP, VP40, VP35, VP30, VP24, and L antigens.
Further, in various embodiments, the composition and methods of the invention lead to clinical responses, such as altered disease progression or life expectancy of human infected with Ebola. Further, in various embodiments, the composition and methods of the invention lead to clinical responses, such as altered disease progression or life expectancy of human at low, medium and high risk for infection with Ebola.
Ad5-Based Ebola Vaccines
Adenoviruses are a family of DNA viruses characterized by an icosahedral, non-enveloped capsid containing a linear double-stranded genome. Of the human Ads, none are associated with any neoplastic disease, and only cause relatively mild, self-limiting illness in immunocompetent individuals. The first genes expressed by the virus are the E1 genes, which act to initiate high-level gene expression from the other Ad5 gene promoters present in the wild type genome. Viral DNA replication and assembly of progeny virions occur within the nucleus of infected cells, and the entire life cycle takes about 36 hr with an output of approximately 104 virions per cell. The wild type Ad5 genome is approximately 36 kb, and encodes genes that are divided into early and late viral functions, depending on whether they are expressed before or after DNA replication. The early/late delineation is nearly absolute, since it has been demonstrated that super-infection of cells previously infected with an Ad5 results in lack of late gene expression from the super-infecting virus until after it has replicated its own genome. Without bound by theory, this is likely due to a replication dependent cis-activation of the Ad5 major late promoter (MLP), preventing late gene expression (primarily the Ad5 capsid proteins) until replicated genomes are present to be encapsulated. The composition and methods of the invention take advantage of feature in the development of advanced generation Ad vectors/vaccines.
Ad5 Vectors
First generation, or E1-deleted adenovirus vectors Ad5 [E1-] are constructed such that a transgene replaces only the E1 region of genes. Typically, about 90% of the wild-type Ad5 genome is retained in the vector. Ad5 [E1-] vectors have a decreased ability to replicate and cannot produce infectious virus after infection of cells not expressing the Ad5 E1 genes. The recombinant Ad5 [E1-] vectors are propagated in human cells (typically 293 cells) allowing for Ad5 [E1-] vector replication and packaging. Ad5 [E1-] vectors have a number of positive attributes; one of the most important is their relative ease for scale up and cGMP production. Currently, well over 220 human clinical trials utilize Ad5 [E1-] vectors, with more than two thousand subjects given the virus sc, im, or iv. Additionally, Ad5 vectors do not integrate; their genomes remain episomal. Generally, for vectors that do not integrate into the host genome, the risk for insertional mutagenesis and/or germ-line transmission is extremely low if at all. Conventional Ad5 [E1-] vectors have a carrying capacity that approaches 7 kb.
Ad5 [E1-] Vectors Used as a Vaccine
Ad5 [E1-] vectors encoding a variety of antigens can efficiently transduce 95% of ex vivo exposed DCs to high titers of the vector. Importantly, increasing levels of foreign gene expression were noted in the DCs with increasing multiplicities of infection (MOI) with the vector. DCs infected with Ad5 [E1-] vectors encoding a variety of antigens (including the tumor antigens MART-1, MAGE-A4, DF3/MUC1, p53, hugp100 melanoma antigen, polyoma virus middle-T antigen) have the propensity to induce antigen specific CTL responses, have an enhanced antigen presentation capacity, and have an improved ability to initiate T-cell proliferation in mixed lymphocyte reactions. Immunization of animals with DCs transduced by Ad5 vectors encoding tumor specific antigens have been demonstrated to result in significant levels of protection for the animals when challenged with tumor cells expressing the respective antigen. Interestingly, intra-tumoral injection of Ads encoding IL-7 was less effective than injection of DCs transduced with IL-7 encoding Ad5 vectors at inducing antitumor immunity, further heightening the interest in ex vivo transduction of DCs by Ad5 vectors. Ex vivo DC transduction strategies have also been used to attempt to induce tolerance in recipient hosts, for example, by Ad5 mediated delivery of the CTLA4Ig into DCs, blocking interactions of the DCs CD80 with the CD28 molecule present on T-cells.
Ad5 vector capsid interactions with DCs in and of themselves may trigger several beneficial responses, which may be enhancing the propensity of DCs to present antigens encoded by Ad5 vectors. For example, immature DCs, though specialized in antigen uptake, are relatively inefficient effectors of T-cell activation. DC maturation coincides with the enhanced ability of DCs to drive T-cell immunity. In some instances, the compositions and methods of the invention take advantage of an Ad5 infection resulting in direct induction of DC maturation. Studies of immature bone marrow derived DCs from mice suggest that Ad vector infection of immature bone marrow derived DCs from mice resulted may upregulate cell surface markers normally associated with DC maturation (MHC I and II, CD40, CD80, CD86, and ICAM-1) as well as down-regulation of CD11c, an integrin known to be down regulated upon myeloid DC maturation. In some instances, Ad vector infection triggers IL-12 production by DCs, a marker of DC maturation. Without being bound by theory, these events may possibly be due to Ad5 triggered activation of NF-κB pathways. Mature DCs can be efficiently transduced by Ad vectors, and did not lose their functional potential to stimulate the proliferation of naive T-cells at lower MOI, as demonstrated by mature CD83+ human DC (derived from peripheral blood monocytes. However, mature DCs may also be less infectable than immature ones. Modification of capsid proteins can be used as a strategy to optimize infection of DC by Ad vectors, as well as enhancing functional maturation, for example using the CD40L receptor as a viral vector receptor, rather than using the normal CAR receptor infection mechanisms.
Most dramatically, when the potential of non-viral vectors to induce anti-HIV immune responses was directly compared to Ad5 based vectoring systems, the Ad5 based systems were found to be far superior. For example, in Ad5 naïve primate models, vaccination with a Ad5 [E1-] expressing the HIV gag was superior in protecting the animals from SHIV infections as compared to similar efforts utilizing naked DNA vaccines expressing HIV-gag. Thus, viral vectors can be superior to naked DNA approaches. Combined strategies (building upon their clinical experiences with naked DNA-gag vectors alone) using naked DNA-gag vaccines as a priming vaccination, followed by boosting with the Ad5 [E1-]-gag vaccine further improved T cell responses in human trials than those previously noted with the DNA-HIV-gag encoding vector alone.
In a recent phase 1 clinical trial evaluating safety and immunogenicity, 20 healthy adults were vaccinated once intramuscularly with a recombinant chimpanzee Ad-based (cAd) vaccine containing the GP component of the Zaire and Sudan strains of Ebola virus (cAd-EBO). GP-specific antibodies were induced in all 20 subjects. The antibody titers were highest in the group (n=10) that received 2×1011 virus particles (VP) as compared to the group (n=10) that received 2×1010 VP. The antibody responses attained were observed to be in the range reported to be associated with vaccine-induced protective immunity in challenge studies involving nonhuman primates (NHP). GP-specific T-cell responses were also more frequent among those who received the 2×1011 VP dose as compared to those that received the 2×1010 VP dose. The vaccine was safely tolerated and no serious adverse effects were observed. Importantly, it was demonstrated that anti-chimpanzee Ad antibodies were also induced in the subjects and these antibodies will prevent further vaccination (boost) that may be required to maintain protective immune responses. In order to circumvent the boosting challenge, these investigators propose to perform an additional clinical trial evaluating the safety and immunogenicity of the cAd-EBO vaccine combined with a booster vaccine composed of a recombinant modified vaccinia virus Ankara (MVA) containing GP of the Ebola virus (MVA-EBO). Even if this approach is successful, the development of neutralizing antibodies to both cAd and MVA must be considered, as that could mitigate further immunizations using these two recombinant vaccines.
Ad5 vectors offer a unique opportunity to allow for high level and efficient transduction of EAs such as EBOV, SUDV, TAFV, BDBV, and RESTV antigens, such as GP, NP, VP40, VP35, VP30, VP24, and L. One of the major problems facing Ad5 based vectors is the high propensity of pre-existing immunity to Ads in the human population, and how this may preclude the use of conventional, Ad5 [E1-] deleted (first generation Ads) in most human populations, for any additional vaccine application.
Ad5 [E1-, E2B-]-Ebola Vaccine: The Use of Ad5 [E1-, E2b-] Vaccines to Overcome the Challenge of Pre-Existing Anti-Ad5 Immunity
Studies in humans and animals have demonstrated that pre-existing immunity against Ad5 can be an inhibitory factor to commercial use of Ad-based vaccines. The preponderance of humans have antibody against Ad5, the most widely used subtype for human vaccines, with two-thirds of humans studied having lympho-proliferative responses against Ad5. This pre-existing immunity can inhibit immunization or re-immunization using typical Ad5 vaccines and may preclude the immunization of a vaccinee against a second antigen, using an Ad5 vector, at a later time. Overcoming the problem of pre-existing anti-vector immunity has been a subject of intense investigation. Investigations using alternative human (non-Ad5 based) Ad5 subtypes or even non-human forms of Ad5 have been examined. Even if these approaches succeed in an initial immunization, subsequent vaccinations may be problematic due to immune responses to the novel Ad5 subtype. To avoid the Ad5 immunization barrier, and improve upon the limited efficacy of first generation Ad5 [E1-] vectors to induce optimal immune responses, various embodiments of the invention relate to a next generation Ad5 vector based vaccine platform. The new Ad5 platform has additional deletions in the E2b region, removing the DNA polymerase and the preterminal protein genes. The Ad5 [E1-, E2b-] platform has an expanded cloning capacity that is sufficient to allow inclusion of many possible genes. Ad5 [E1-, E2b-] vectors have up to about 12 kb gene-carrying capacity as compared to the 7 kb capacity of Ad5 [E1-] vectors, providing space for multiple genes if needed. In some embodiments, an insert of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 kb is introduced into an Ad5 vector, such as the Ad5 [E1-, E2b-] vector. Deletion of the E2b region confers advantageous immune properties on the Ad5 vectors of the invention, often eliciting potent immune responses to target transgene antigens while minimizing the immune responses to Ad viral proteins.
In various embodiments, Ad5 [E1-, E2b-] vectors of the invention induce a potent CMI, as well as antibodies against the vector expressed vaccine antigens even in the presence of Ad immunity. Ad5 [E1-, E2b-] vectors also have reduced adverse reactions as compared to Ad5 [E1-] vectors, in particular the appearance of hepatotoxicity and tissue damage. A key aspect of these Ad5 vectors is that expression of Ad late genes is greatly reduced. For example, production of the capsid fiber proteins could be detected in vivo for Ad5 [E1-] vectors, while fiber expression was ablated from Ad5 [E1-, E2b-] vector vaccines. The innate immune response to wild type Ad is complex. Proteins deleted from the Ad5 [E1-, E2b-] vectors generally play an important role. Specifically, Ad5 [E1-, E2b-] vectors with deletions of preterminal protein or DNA polymerase display reduced inflammation during the first 24 to 72 hours following injection compared to Ad5 [E1-] vectors. In various embodiments, the lack of Ad5 gene expression renders infected cells invisible to anti-Ad activity and permits infected cells to express the transgene for extended periods of time, which develops immunity to the target.
Various embodiments of the invention contemplate increasing the capability for the Ad5 [E1-, E2b-] vectors to transduce dendritic cells, improving antigen specific immune responses in the vaccine by taking advantage of the reduced inflammatory response against Ad5 [E1-, E2b-] vector viral proteins and the resulting evasion of pre-existing Ad immunity.
In some cases, this immune induction may take months. Ad5 [E1-, E2b-] vectors not only are safer than, but appear to be superior to Ad5 [E1-] vectors in regard to induction of antigen specific immune responses, making them much better suitable as a platform to deliver Ebola vaccines that can result in a clinical response.
Various embodiments of the invention, by taking advantage of the new Ad5 [E1-, E2b-] vector system in delivering a long sought-after need for a develop a therapeutic vaccine against Ebola, overcome barriers found with other Ad5 systems and permit the immunization of people who have previously been exposed to Ad5.
In various embodiments the compositions and methods of the invention comprising an Ad5 [E1-, E2b-] vector Ebola vaccine effect of increased overall survival (OS) within the bounds of technical safety.
Adenovirus Vectors
Compared to first generation adenovirus vectors, certain embodiments of the Second Generation E2b deleted adenovirus vectors of the present invention contain additional deletions in the DNA polymerase gene (pol) and deletions of the pre-terminal protein (pTP). E2b deleted vectors have up to a 13 kb gene-carrying capacity as compared to the 5 to 6 kb capacity of First Generation adenovirus vectors, easily providing space for nucleic acid sequences encoding any of a variety of target antigens. The E2b deleted adenovirus vectors also have reduced adverse reactions as compared to First Generation adenovirus vectors. E2b deleted vectors have reduced expression of viral genes, and this characteristic leads to extended transgene expression in vivo.
The innate immune response to wild type Ad can be complex, and it appears that Ad proteins expressed from adenovirus vectors play an important role. Specifically, the deletions of pre-terminal protein and DNA polymerase in the E2b deleted vectors appear to reduce inflammation during the first 24 to 72 hours following injection, whereas First Generation adenovirus vectors stimulate inflammation during this period. In addition, it has been reported that the additional replication block created by E2b deletion also leads to a 10,000 fold reduction in expression of Ad late genes, well beyond that afforded by E1, E3 deletions alone. The decreased levels of Ad proteins produced by E2b deleted adenovirus vectors effectively reduce the potential for competitive, undesired, immune responses to Ad antigens, responses that prevent repeated use of the platform in Ad immunized or exposed individuals. The reduced induction of inflammatory response by second generation E2b deleted vectors results in increased potential for the vectors to express desired vaccine antigens during the infection of antigen presenting cells (i.e. dendritic cells), decreasing the potential for antigenic competition, resulting in greater immunization of the vaccine to the desired antigen relative to identical attempts with First Generation adenovirus vectors. E2b deleted adenovirus vectors provide an improved Ad-based vaccine candidate that is safer, more effective, and more versatile than previously described vaccine candidates using First Generation adenovirus vectors.
Thus, first generation, E1-deleted Adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Without being bound by theory, Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, for example by virtue of diminished late phase viral protein expression, may avoid immunological clearance and induce more potent immune responses against the encoded antigen transgene in Ad-immune hosts.
The present invention contemplates the use of E2b deleted adenovirus vectors, such as those described in U.S. Pat. Nos. 6,063,622; 6,451,596; 6,057,158; 6,083,750; and 8,298,549. The vectors with deletions in the E2b regions in many cases cripple viral protein expression and/or decrease the frequency of generating replication competent Ad (RCA). Propagation of these E2b deleted adenovirus vectors can be done utilizing cell lines that express the deleted E2b gene products. The present invention also provides such packaging cell lines; for example E.C7 (formally called C-7), derived from the HEK-293 cell line.
Further, the E2b gene products, DNA polymerase and preterminal protein, can be constitutively expressed in E.C7, or similar cells along with the E1 gene products. Transfer of gene segments from the Ad genome to the production cell line has immediate benefits: (1) increased carrying capacity; and, (2) a decreased potential of RCA generation, typically requiring two or more independent recombination events to generate RCA. The E1, Ad DNA polymerase and/or preterminal protein expressing cell lines used in the present invention can enable the propagation of adenovirus vectors with a carrying capacity approaching 13 kb, without the need for a contaminating helper virus. In addition, when genes critical to the viral life cycle are deleted (e.g., the E2b genes), a further crippling of Ad to replicate or express other viral gene proteins occurs. This can decrease immune recognition of virally infected cells, and allow for extended durations of foreign transgene expression.
E1, DNA polymerase, and preterminal protein deleted vectors are typically unable to express the respective proteins from the E1 and E2b regions. Further, they may show a lack of expression of most of the viral structural proteins. For example, the major late promoter (MLP) of Ad is responsible for transcription of the late structural proteins L1 through L5. Though the MLP is minimally active prior to Ad genome replication, the highly toxic Ad late genes are primarily transcribed and translated from the MLP only after viral genome replication has occurred. This cis-dependent activation of late gene transcription is a feature of DNA viruses in general, such as in the growth of polyoma and SV-40. The DNA polymerase and preterminal proteins are important for Ad replication (unlike the E4 or protein IX proteins). Their deletion can be extremely detrimental to adenovirus vector late gene expression, and the toxic effects of that expression in cells such as APCs.
In certain embodiments, the adenovirus vectors contemplated for use in the present invention include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally, the E1 region. In some cases, such vectors do not have any other regions of the Ad genome deleted. In another embodiment, the adenovirus vectors contemplated for use in the present invention include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally, deletions in the E1 and E3 regions. In some cases, such vectors have no other regions deleted. In a further embodiment, the adenovirus vectors contemplated for use in the present invention include adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally, deletions in the E1, E3 and, also optionally, partial or complete removal of the E4 regions. In some cases, such vectors have no other deletions. In another embodiment, the adenovirus vectors contemplated for use in the present invention include adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally deletions in the E1 and/or E4 regions. In some cases, such vectors contain no other deletions. In an additional embodiment, the adenovirus vectors contemplated for use in the present invention include adenovirus vectors that have a deletion in the E2a, E2b and/or E4 regions of the Ad genome. In some cases, such vectors have no other deletions. In one embodiment, the adenovirus vectors for use herein comprise vectors having the E1 and/or DNA polymerase functions of the E2b region deleted. In some cases, such vectors have no other deletions. In a further embodiment, the adenovirus vectors for use herein have the E1 and/or the preterminal protein functions of the E2b region deleted. In some cases, such vectors have no other deletions. In another embodiment, the adenovirus vectors for use herein have the E1, DNA polymerase and/or the preterminal protein functions deleted. In some cases, such vectors have no other deletions. In one particular embodiment, the adenovirus vectors contemplated for use herein are deleted for at least a portion of the E2b region and/or the E1 region. In some cases, such vectors are not “gutted” adenovirus vectors. In this regard, the vectors may be deleted for both the DNA polymerase and the preterminal protein functions of the E2b region. In an additional embodiment, the adenovirus vectors for use in the present invention include adenovirus vectors that have a deletion in the E1, E2b and/or 100 K regions of the adenovirus genome. In one embodiment, the adenovirus vectors for use herein comprise vectors having the E1, E2b and/or protease functions deleted. In some cases, such vectors have no other deletions. In a further embodiment, the adenovirus vectors for use herein have the E1 and/or the E2b regions deleted, while the fiber genes have been modified by mutation or other alterations (e.g., to alter Ad tropism). Removal of genes from the E3 or E4 regions may be added to any of the mentioned adenovirus vectors. In certain embodiments, the adenovirus vector may be a “gutted” adenovirus vector.
The present invention also provides compositions and methods for immunotherapy against Ebola using a viral gene delivery platform to immunize against Ebola genes combined with an immune pathway checkpoint modulator. For example, compositions and methods for immunotherapy against Ebola using a viral gene delivery platform can be used immunize against Ebola genes combined with an immune pathway checkpoint modulator, such as an inhibitor of PD1. These compositions and methods can utilize an Ad5 [E1-, E2b-]-Ebola vaccine combined with an immune pathway checkpoint modulator, such as an inhibitor of a checkpoint inhibitor.
The compositions and methods can be used to generate an immune response against a target antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate immune responses against an Ebola antigen, such as an Ebola antigen expressed or presented by a cell. For example, the compositions and methods can be used to generate an immune response against an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as GP, NP, VP40, VP35, VP30, VP24, L, or any combination thereof, expressed or presented by a cell. For example, the compositions and methods can be used to generate an immune response against an EBOV antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against a SUDV antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against a TAFV antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against a BDBV antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against a RESTV antigen expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against GP expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against NP expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against VP40 expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against VP35 expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against VP30 expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against VP24 expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against L expressed and/or presented by a cell.
The compositions and methods can be used to generate an immune response against multiple target antigens expressed and/or presented by a cell. For example, the compositions and methods can be used to generate an immune response against two or more EBOV, SUDV, TAFV, BDBV, or RESTV antigens, such as two or more of GP, NP, VP40, VP35, VP30, VP24, and L antigens. For example, the compositions and methods can be used to generate an immune response against an EBOV antigen and a SUDV antigen. For example, the compositions and methods can be used to generate an immune response against a TAFV antigen and a BDBV antigen. For example, the compositions and methods can be used to generate an immune response against a RESTV antigen and an EBOV antigen. For example, the compositions and methods can be used to generate an immune response against GP and NP. For example, the compositions and methods can be used to generate an immune response against GP and VP40. For example, the compositions and methods can be used to generate an immune response against GP and VP35. For example, the compositions and methods can be used to generate an immune response against GP and VP30. For example, the compositions and methods can be used to generate an immune response against GP and VP24. For example, the compositions and methods can be used to generate an immune response against GP and L. For example, the compositions and methods can be used to generate an immune response against NP and VP40. For example, the compositions and methods can be used to generate an immune response against NP and VP35. For example, the compositions and methods can be used to generate an immune response against NP and VP30. For example, the compositions and methods can be used to generate an immune response against NP and VP24. For example, the compositions and methods can be used to generate an immune response against NP and L. For example, the compositions and methods can be used to generate an immune response against VP40 and VP35. For example, the compositions and methods can be used to generate an immune response against VP40 and VP30. For example, the compositions and methods can be used to generate an immune response against VP40 and VP24. For example, the compositions and methods can be used to generate an immune response against VP40 and L. For example, the compositions and methods can be used to generate an immune response against VP35 and VP30. For example, the compositions and methods can be used to generate an immune response against VP35 and VP24. For example, the compositions and methods can be used to generate an immune response against VP35 and L. For example, the compositions and methods can be used to generate an immune response against VP30 and VP24. For example, the compositions and methods can be used to generate an immune response against VP30 and L. For example, the compositions and methods can be used to generate an immune response against VP24 and L.
A modified form of an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as GP, NP, VP40, VP35, VP30, VP24, or L can be used in a vaccine directed to raising an immune response against an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as GP, NP, VP40, VP35, VP30, VP24, or L; or cells expressing and/or presenting an EBOV, SUDV, TAFV, BDBV, or RESTV antigen, such as GP, NP, VP40, VP35, VP30, VP24, or L. Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the epitope of the polypeptide encoded by the variant polynucleotide or such that the immunogenicity of the heterologous target protein is not substantially diminished relative to a polypeptide encoded by the native polynucleotide sequence. In some cases, said one or more substitutions, additions, deletions and/or insertions may result in an increased immunogenicity of the epitope of the polypeptide encoded by the variant polynucleotide. As described elsewhere herein, the polynucleotide variants can encode a variant of the target antigen, or a fragment (e.g., an epitope) thereof wherein the propensity of the variant polypeptide or fragment (e.g., epitope) thereof to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished relative to the native polypeptide. The polynucleotide variants can encode a variant of the target antigen, or a fragment thereof wherein the propensity of the variant polypeptide or fragment thereof to react with antigen-specific antisera and/or T-cell lines or clones is substantially increased relative to the native polypeptide.
In particular, the present invention provides an improved Ad-based vaccine such that multiple vaccinations against one or more antigenic target entity can be achieved. In some embodiments, the improved Ad-based vaccine comprises a replication defective adenovirus carrying a target antigen, a fragment, a variant or a variant fragment thereof, such as Ad5 [E1-, E2b-]-EBOV. Variants or fragments of target antigens, such as GP, NP, VP40, VP35, VP30, VP24, or L, can be selected based on a variety of factors, including immunogenic potential. A mutant GP, NP, VP40, VP35, VP30, VP24, or L can utilized for its increased capability to raise an immune response relative to the wild-type GP, NP, VP40, VP35, VP30, VP24, or L, respectively. Importantly, vaccination can be performed in the presence of preexisting immunity to the Ad or administered to subjects previously immunized multiple times with the Ad vector of the present invention or other Ad vectors. The Ad vectors can be administered to subjects multiple times to induce an immune response against an antigen of interest, such as GP, NP, VP40, VP35, VP30, VP24, or L, including but not limited to, the production of antibodies and CMI responses against one or more target antigens. In particular embodiments, variants or fragments of target antigens are modified such that they have one or more reduced biological activities. For example, an Ebola protein target antigen may be modified to reduce or eliminate the viral activity of the protein, or a viral protein may be modified to reduce or eliminate one or more activities or the viral protein. An example of a modified Ebola protein is a GP, NP, VP40, VP35, VP30, VP24, or L, having a point mutation that results in a variant protein, such as a variant protein with increased immunogenicity.
In order to express a desired target antigen polypeptide or fragment or variant thereof, or fusion protein comprising any of the above, as described herein, the nucleotide sequences encoding the polypeptide, or functional equivalents, are inserted into an appropriate Ad as described elsewhere herein using recombinant techniques known in the art.
Compositions
Viral Vectors for Ebola Immunotherapies and Vaccines
Recombinant viral vectors can be used to express protein coding genes or antigens (e.g., EAs). The advantages of recombinant viral vector based vaccines and immunotherapy include high efficiency gene transduction, highly specific delivery of genes to target cells, induction of robust immune responses, and increased cellular immunity. The present disclosure provides for recombinant adenovirus vectors comprising deletions or insertions of crucial regions of the viral genome. The viral vectors of provided by the present disclosure can comprise heterologous nucleic acid sequences that encode one or more target antigens of interest, or variants, fragments or fusions thereof, against which it is desired to generate an immune response.
Suitable viral vectors that can be used with the methods and compositions of the present disclosure include but are not limited to retroviruses, lentiviruses, provirus, Vaccinia virus, adenoviruses, adeno-associated viruses, self-complementary adeno-associated virus, Cytomegalovirus, or Sendai virus. In some embodiments, the viral vector can be replication-competent. In some embodiments, the viral vector can be replication-defective. For replication-defective viral vectors, the viruses' genome can have the coding regions necessary for additional rounds of replication and packaging replaced with other genes, or deleted. These viruses are capable of infecting their target cells and delivering their viral payload, but then fail to continue the typical lytic pathway that leads to cell lysis and death. Depending on the viral vector, the typical maximum length of an allowable DNA or cDNA insert in a replication-defective viral vector is can be about 8-10 kilobases (kb).
Retroviruses have been used to express antigens, such as an enveloped, single-stranded RNA virus that contains reverse transcriptase. Retrovirus vectors can be replication-defective. Retrovirus vectors can be of murine or avian origin. Retrovirus vectors can be from Moloney murine leukemia virus (MoMLV). Retrovirus vectors can be used that require genome integration for gene expression. Retrovirus vectors can be used to provide long-term gene expression. For example, retrovirus vectors can have a genome size of approximately 7-11 kb and the vector can harbor 7-8 kb long foreign DNA inserts. Retrovirus vectors can be used to display low immunogenicity and most patients do not show pre-existing immunity to retroviral vectors. Retrovirus vectors can be used to infect dividing cells. Retrovirus vectors can be used to not infect non-dividing cells.
Lentivirus vectors have been used to express antigens. Lentiviruses constitute a subclass of retroviruses. Lentivirus vectors can be used to infect non-dividing cells. Lentivirus vectors can be used to infect dividing cells. Lentivirus vectors can be used to infect both non-dividing and dividing cells. Lentiviruses generally exhibit broader tropism than retroviruses. Several proteins such as tat and rev regulate the replication of lentiviruses. These regulatory proteins are typically absent in retroviruses. HIV is an exemplary lentivirus that can be engineered into a transgene delivery vector. The advantages of lentivirus vectors are similar to those of retroviral vectors. Although lentiviruses can potentially trigger tumorigenesis, the risk is lower than that of retroviral vectors, as the integration sites of lentiviruses are away from the sites harboring cellular promoters. HIV-based vectors can be generated, for example, by deleting the HIV viral envelope and some of the regulatory genes not required during vector production. Instead of parental envelope, several chimeric or modified envelope vectors are generated because it determines the cell and tissue specificity.
Cytomegalovirus (CMV) vectors have been used to express antigens. CMV is a member of the herpesviruses. Species-specific CMVs can be used (e.g., human CMV (HCMV), e.g., human herpesvirus type 5. HCMV contains a 235 kb double-stranded linear DNA genome surrounded by a capsid. The envelope contains glycoproteins gB and gH, which bind to cellular receptors.
Sendai virus (SeV) vectors have been used to express antigens. SeV is an enveloped, single-stranded RNA virus of the family Paramyxovirus. The SeV genome encodes six protein and two envelope glycoproteins, HN and F proteins, that mediate cell entry and determine its tropism. SeV vectors that lack F protein can be used as a replication-defective virus to improve the safety of the vector. SeV vector produced in a packaging cell can be used to expresses the F protein. An F gene-deleted and transgene-inserted genome can be transfected into a packaging cell. SeV contains RNA dependent RNA polymerase and viral genome localizes to the cytoplasm. This ensures that fast gene expression occurs soon after infection and the genotoxic advantage of SeV. SeV vectors can be used to exhibit highly efficient gene transfer. SeV vectors can be used to transduce both dividing and non-dividing cells. SeV vectors can be used to transduce non-dividing cells. SeV vectors can be used to transduce dividing cells. SeV vectors can be used, for example, to efficiently transduce human airway epithelial cells. SeV vectors can be, for example, administered by a mucosal (e.g., oral and nasal) route. Intranasal administration can be used to potentially reduce the influence of a pre-existing immunity to SeV, as compared to intramuscular administration. Compared to other viral vectors, its transgene capacity (3.4 kb) is low. SeV is highly homologous to the human parainfluenza type 1 (hPIV-1) virus; thus, a pre-existing immunity against hPIV-1 can work against the use of SeV.
Adenovirus Vectors
In general, adenoviruses are attractive for clinical because they can have a broad tropism, they can infect a variety of dividing and non-dividing cell types and hey can be used systemically as well as through more selective mucosal surfaces in a mammalian body. In addition, their relative thermostability further facilitates their clinical use. Adenoviruses are a family of DNA viruses characterized by an icosahedral, non-enveloped capsid containing a linear double-stranded genome. Generally, adenoviruses are found as non-enveloped viruses comprising double-stranded DNA genome approximated ˜30-35 kilobases in size. Of the human Ads, none are associated with any neoplastic disease, and only cause relatively mild, self-limiting illness in immunocompetent individuals. The first genes expressed by the virus are the E1 genes, which act to initiate high-level gene expression from the other Ad5 gene promoters present in the wild type genome. Viral DNA replication and assembly of progeny virions occur within the nucleus of infected cells, and the entire life cycle takes about 36 hr with an output of approximately 104 virions per cell. The wild type Ad5 genome is approximately 36 kb, and encodes genes that are divided into early and late viral functions, depending on whether they are expressed before or after DNA replication. The early/late delineation is nearly absolute, since it has been demonstrated that super-infection of cells previously infected with an Ad5 results in lack of late gene expression from the super-infecting virus until after it has replicated its own genome. Without bound by theory, this is likely due to a replication dependent cis-activation of the Ad5 major late promoter (MLP), preventing late gene expression (primarily the Ad5 capsid proteins) until replicated genomes are present to be encapsulated. The composition and methods of the invention take advantage of feature in the development of advanced generation Ad vectors/vaccines. The linear genome of the adenovirus is generally flanked by two origins for DNA replication (ITRs) and has eight units for RNA polymerase II-mediated transcription. The genome carries five early units E1A, E1B, E2, E3, E4, and E5, two units that are expressed with a delay after initiation of viral replication (IX and IVa2), and one late unit (L) that is subdivided into L1-L5. Some adenoviruses can further encode one or two species of RNA called virus-associated (VA) RNA.
Adenoviruses that induce innate and adaptive immune responses in human patient are provided. By deletion or insertion of crucial regions of the viral genome, recombinant vectors are provided that have been engineered to increase their predictability and reduce unwanted side effects. In some aspects, the invention provides for an adenovirus vector comprising the genome deletion or insertion selected from the group consisting of: E1A, E1B, E2, E3, E4, E5, IX, IVa2, L1, L2, L3, L4, and L5, and any combination thereof.
The present disclosure provides for recombinant adenovirus vectors comprising an altered capsid. Generally, the capsid of an adenovirus is primarily comprises 20 triangular facets of an icosahedron each icosahedron contains 12 copies of hexon trimers. In addition there are also other several additional minor capsid proteins, IIIa, VI, VIII, and IX.
The present disclosure provides for recombinant adenovirus vectors comprising one or more altered fiber proteins. In general the fiber proteins, which also form trimers, are inserted at the 12 vertices into the pentameric penton bases. The fiber can comprise of a thin N-terminal tail, a shaft, and a knob domain. The shaft can comprise a variable numbers of β-strand repeats. The knob can comprise one or more loops A, B, C, D, E, F, G, H, I, J. The fiber knob loops can bind to cellular receptors. The present disclosure provides for adenovirus vectors to be used in vaccine systems for the treatment of Ebola
Suitable adenoviruses that can be used with the present methods and compositions of the disclosure include but are not limited to species-specific adenovirus including human subgroups A, B1, B2, C, D, E and F or their crucial genomic regions as provided herein, which subgroups can further classified into immunologically distinct serotypes. Further, suitable adenoviruses that can be used with the present methods and compositions of the disclosure include, but are not limited to, species-specific adenovirus or their crucial genomic regions identified from primates, bovines, fowls, reptiles, or frogs.
Some adenoviruses serotypes preferentially target distinct organs. Serotypes such as AdHu1, AdHu2, and AdHu5 (subgenus C), generally effect the infect upper respiratory, while subgenera A and F effect gastrointestinal organs. The present disclosure provides for recombinant adenovirus vectors to be used in preferentially target distinct organs for the treatment of Ebola. In some applications the recombinant adenovirus vector is altered to reduce tropism to a specific organ in a mammal. In some applications the recombinant adenovirus vector is altered to increase tropism to a specific organ in a mammal.
The tropism of an adenovirus can be determined by their ability to attach to host cell receptors. In some instances the process of host cell attachment can involve the initial binding of the distal knob domain of the fiber to a host cell surface molecule followed by binding of the RGD motif within the penton base with αV integrins. The present disclosure provides for recombinant adenovirus vectors with altered tropism such that they can be genetic engineered to infect specific cell types of a host. The present disclosure provides for recombinant adenovirus vectors with altered tropism for the treatment of cell-specific Ebola infections. The present disclosure provides for recombinant adenovirus vectors with altered fiber knob from one or more adenoviruses of subgroups A, B, C, D, or F, or a combination thereof or the insertion of RGD sequences. In some applications the recombinant adenovirus vectors comprising an altered fiber knob results in a vector with reduced tropism for one or more particular cell types. In some applications the recombinant adenovirus vectors comprising an altered fiber knob results in a vector with enhanced tropism for one or more particular cell types. In some applications the recombinant adenovirus vectors comprising an altered fiber knob results in a vector with reduced product-specific B or T-cell responses. In some applications the recombinant adenovirus vectors comprising an altered fiber knob results in a vector with enhanced product-specific B or T-cell responses.
The present disclosure provides for recombinant adenovirus vectors that are coated with other molecules to circumvent the effects of virus-neutralizing antibodies or improve transduction in to a host cell. The present disclosure provides for recombinant adenovirus vectors that are coated with an adaptor molecule that aids in the attachment of the vector to a host cell receptor. By way of example an adenovirus vector can be coated with adaptor molecule that connects coxsackie Ad receptor (CAR) with CD40L resulting in increased transduction of dendritic cells, thereby enhancing immune responses in a subject. Other adenovirus vectors similarly engineered for enhancing the attachment to other target cell types are also included by the present disclosure.
Ad5 Vectors
Studies in humans and animals have demonstrated that pre-existing immunity against Ad5 can be an inhibitory factor to commercial use of Ad-based vaccines. The preponderance of humans have antibody against Ad5, the most widely used subtype for human vaccines, with two-thirds of humans studied having lympho-proliferative responses against Ad5. This pre-existing immunity can inhibit immunization or re-immunization using typical Ad5 vaccines and may preclude the immunization of a vaccine against a second antigen, using an Ad5 vector, at a later time. Overcoming the problem of pre-existing anti-vector immunity has been a subject of intense investigation. Investigations using alternative human (non-Ad5 based) Ad5 subtypes or even non-human forms of Ad5 have been examined. Even if these approaches succeed in an initial immunization, subsequent vaccinations may be problematic due to immune responses to the novel Ad5 subtype. To avoid the Ad5 immunization barrier, and improve upon the limited efficacy of first generation Ad5 [E1-] vectors to induce optimal immune responses, various embodiments of the invention relate to a next generation Ad5 vector based vaccine platform.
First generation, or E1-deleted adenovirus vectors Ad5 [E1-] are constructed such that a transgene replaces only the E1 region of genes. Typically, about 90% of the wild-type Ad5 genome is retained in the vector. Ad5 [E1-] vectors have a decreased ability to replicate and cannot produce infectious virus after infection of cells that do not express the Ad5 E1 genes. The recombinant Ad5 [E1-] vectors are propagated in human cells (e.g., 293 cells) allowing for Ad5 [E1-] vector replication and packaging. Ad5 [E1-] vectors have a number of positive attributes; one of the most important is their relative ease for scale up and cGMP production. Currently, well over 220 human clinical trials utilize Ad5 [E1-] vectors, with more than two thousand subjects given the virus sc, im, or iv. Additionally, Ad5 vectors do not integrate; their genomes remain episomal. Generally, for vectors that do not integrate into the host genome, the risk for insertional mutagenesis and/or germ-line transmission is extremely low if at all. Conventional Ad5 [E1-] vectors have a carrying capacity that approaches 7 kb.
Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, provide an opportunity to avoid immunological clearance and induce more potent immune responses against the encoded Ebola antigen transgene in Ad-immune hosts. The new Ad5 platform has additional deletions in the E2b region, removing the DNA polymerase and the preterminal protein genes. The Ad5 [E1-, E2b-] platform has an expanded cloning capacity that is sufficient to allow inclusion of many possible genes. Ad5 [E1-, E2b-] vectors have up to about 12 kb gene-carrying capacity as compared to the 7 kb capacity of Ad5 [E1-] vectors, providing space for multiple genes if needed. In some embodiments, an insert of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 kb is introduced into an Ad5 vector, such as the Ad5 [E1-, E2b-] vector. Deletion of the E2b region confers advantageous immune properties on the Ad5 vectors of the invention, often eliciting potent immune responses to target transgene antigens while minimizing the immune responses to Ad viral proteins.
In various embodiments, Ad5 [E1-, E2b-] vectors of the invention induce a potent CMI, as well as antibodies against the vector expressed vaccine antigens even in the presence of Ad immunity. Ad5 [E1-, E2b-] vectors also have reduced adverse reactions as compared to Ad5 [E1-] vectors, in particular the appearance of hepatotoxicity and tissue damage. A key aspect of these Ad5 vectors is that expression of Ad late genes is greatly reduced. For example, production of the capsid fiber proteins could be detected in vivo for Ad5 [E1-] vectors, while fiber expression was ablated from Ad5 [E1-, E2b-] vector vaccines. The innate immune response to wild type Ad is complex. Proteins deleted from the Ad5 [E1-, E2b-] vectors generally play an important role. Specifically, Ad5 [E1-, E2b-] vectors with deletions of preterminal protein or DNA polymerase display reduced inflammation during the first 24 to 72 h following injection compared to Ad5 [E1-] vectors. In various embodiments, the lack of Ad5 gene expression renders infected cells invisible to anti-Ad activity and permits infected cells to express the transgene for extended periods of time, which develops immunity to the target.
Various embodiments of the invention contemplate increasing the capability for the Ad5 [E1-, E2b-] vectors to transduce dendritic cells, improving antigen specific immune responses in the vaccine by taking advantage of the reduced inflammatory response against Ad5 [E1-, E2b-] vector viral proteins and the resulting evasion of pre-existing Ad immunity.
Replication Defective Ad5 Vector
Attempts to overcome anti-Ad immunity have included use of alternative Ad serotypes and/or alternations in the Ad5 viral capsid protein each with limited success and the potential for significantly altering biodistribution of the resultant vaccines. Therefore, a completely novel approach was attempted by further reducing the expression of viral proteins from the E1 deleted Ad5 vectors, proteins known to be targets of pre-existing Ad immunity. Specifically, a novel recombinant Ad5 platform has been described with deletions in the early 1 (E1) gene region and additional deletions in the early 2b (E2b) gene region (Ad5 [E1-, E2b-]). Deletion of the E2b region (that encodes DNA polymerase and the pre-terminal protein) results in decreased viral DNA replication and late phase viral protein expression. This vector platform can be used to induce CMI responses in animal models of Ebola infection and more importantly, this recombinant Ad5 gene delivery platform overcomes the barrier of Ad5 immunity and can be used in the setting of pre-existing and/or vector-induced Ad immunity thus enabling multiple homologous administrations of the vaccine. In particular embodiments, the present invention relates to a replication defective adenovirus vector of serotype 5 comprising a sequence encoding an immunogenic polypeptide. The immunogenic polypeptide may be a mutant, natural variant, or a fragment thereof.
In some embodiments, the replication defective adenovirus vector comprises a modified sequence encoding a polypeptide with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to a wild-type immunogenic polypeptide or a fragment thereof. In some embodiments, the replication defective adenovirus vector comprises a modified sequence encoding a subunit of a wild-type polypeptide. The compositions and methods of the invention, in some embodiments, relate to an adenovirus-derived vector comprising at least 60% sequence identity to SEQ. ID. NO.:1, 2, 4, 5, or 6.
In some embodiments, an adenovirus-derived vector, optionally relating to a replication defective adenovirus, comprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% identity to SEQ. ID. NO.: 1, 2, 4, 5, or 6 or a sequence generated from SEQ. ID. NO.:3 by alternative codon replacements. In various embodiments, the adenovirus-derived vectors described herein have a deletion in the E2b region, and optionally, in the E1 region, the deletion conferring a variety of advantages to the use of the vectors in immunotherapy as described herein.
Certain regions within the adenovirus genome serve essential functions and may need to be substantially conserved when constructing the replication defective adenovirus vectors of the invention. These regions are further described in Lauer et al., J. Gen. Virol., 85, 2615-25 (2004), Leza et al., J. Virol., p. 3003-13 (1988), and Miralles et al., J. Bio Chem., Vol. 264, No. 18, p. 10763-72 (1983), which are incorporated by reference in their entirety. Recombinant nucleic acid vectors comprising a sequence with identity values of at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% to a portion of SEQ. ID. NO.: 1, 2, 4, 5, or 6, such as a portion comprising at least about 100, 250, 500, 1000 or more bases of SEQ. ID. NO.: 1, 2, 4, 5, or 6 are within the bounds of the invention.
The present invention contemplates the use of E2b deleted adenovirus vectors, such as those described in U.S. Pat. Nos. 6,063,622; 6,451,596; 6,057,158; 6,083,750; and 8,298,549. The vectors with deletions in the E2b regions in many cases cripple viral protein expression and/or decrease the frequency of generating replication competent Ad (RCA). Propagation of these E2b deleted adenovirus vectors can be done utilizing cell lines that express the deleted E2b gene products. Such packaging cell lines are provided herein; e.g., E.C7 (formally called C-7), derived from the HEK-293 cell line.
Further, the E2b gene products, DNA polymerase and preterminal protein, can be constitutively expressed in E.C7, or similar cells along with the E1 gene products. Transfer of gene segments from the Ad genome to the production cell line has immediate benefits: (1) increased carrying capacity; and, (2) a decreased potential of RCA generation, typically requiring two or more independent recombination events to generate RCA. The E1, Ad DNA polymerase and/or preterminal protein expressing cell lines used in the present invention can enable the propagation of adenovirus vectors with a carrying capacity approaching 13 kb, without the need for a contaminating helper virus. In addition, when genes critical to the viral life cycle are deleted (e.g., the E2b genes), a further crippling of Ad to replicate or express other viral gene proteins occurs. This can decrease immune recognition of infected cells, and extend durations of foreign transgene expression.
E1, DNA polymerase, and preterminal protein deleted vectors are typically unable to express the respective proteins from the E1 and E2b regions. Further, they may show a lack of expression of most of the viral structural proteins. For example, the major late promoter (MLP) of Ad is responsible for transcription of the late structural proteins L1 through L5. Though the MLP is minimally active prior to Ad genome replication, the highly toxic Ad late genes are primarily transcribed and translated from the mLP only after viral genome replication has occurred. This cis-dependent activation of late gene transcription is a feature of DNA viruses in general, such as in the growth of polyoma and SV-40. The DNA polymerase and preterminal proteins are important for Ad replication (unlike the E4 or protein IX proteins). Their deletion can be extremely detrimental to adenovirus vector late gene expression, and the toxic effects of that expression in cells such as APCs.
The adenovirus vectors can include a deletion in the E2b region of the Ad genome and, optionally, the E1 region. In some cases, such vectors do not have any other regions of the Ad genome deleted. The adenovirus vectors can include a deletion in the E2b region of the Ad genome and deletions in the E1 and E3 regions. In some cases, such vectors have no other regions deleted. The adenovirus vectors can include a deletion in the E2b region of the Ad genome and deletions in the E1, E3 and partial or complete removal of the E4 regions. In some cases, such vectors have no other deletions. The adenovirus vectors can include a deletion in the E2b region of the Ad genome and deletions in the E1 and/or E4 regions. In some cases, such vectors contain no other deletions. The adenovirus vectors can include a deletion in the E2a, E2b and/or E4 regions of the Ad genome. In some cases, such vectors have no other deletions. The adenovirus vectors can have the E1 and/or DNA polymerase functions of the E2b region deleted. In some cases, such vectors have no other deletions. The adenovirus vectors can have the E1 and/or the preterminal protein functions of the E2b region deleted. In some cases, such vectors have no other deletions. The adenovirus vectors can have the E1, DNA polymerase and/or the preterminal protein functions deleted. In some cases, such vectors have no other deletions. The adenovirus vectors can have at least a portion of the E2b region and/or the E1 region. In some cases, such vectors are not gutted adenovirus vectors. In this regard, the vectors may be deleted for both the DNA polymerase and the preterminal protein functions of the E2b region. The adenovirus vectors can have a deletion in the E1, E2b and/or 100 K regions of the adenovirus genome. The adenovirus vectors can comprise vectors having the E1, E2b and/or protease functions deleted. In some cases, such vectors have no other deletions. The adenovirus vectors can have the E1 and/or the E2b regions deleted, while the fiber genes have been modified by mutation or other alterations (for example to alter Ad tropism). Removal of genes from the E3 or E4 regions may be added to any of the adenovirus vectors mentioned. In certain embodiments, the adenovirus vector may be a gutted adenovirus vector.
Other regions of the Ad genome can be deleted. A “deletion” in a particular region of the Ad genome refers to a specific DNA sequence that is mutated or removed in such a way so as to prevent expression and/or function of at least one gene product encoded by that region (e.g., E2b functions of DNA polymerase or preterminal protein function). Deletions encompass deletions within exons encoding portions of proteins as well as deletions within promoter and leader sequences. A deletion within a particular region refers to a deletion of at least one base pair within that region of the Ad genome. More than one base pair can be deleted. For example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs can be deleted from a particular region. The deletion can be more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within a particular region of the Ad genome. These deletions can prevent expression and/or function of the gene product encoded by the region. For example, a particular region of the Ad genome can include one or more point mutations such that one or more encoded proteins is non-functional. Such mutations include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein. Exemplary deletions or mutations in the Ad genome include one or more of E1a, E1b, E2a, E2b, E3, E4, L1, L2, L3, L4, L5, TP, POL, IV, and VA regions. Deleted adenovirus vectors can be made, for example, using recombinant techniques.
Ad vectors for use in the present invention can be successfully grown to high titers using an appropriate packaging cell line that constitutively expresses E2b gene products and products of any of the necessary genes that may have been deleted. HEK-293-derived cells that not only constitutively express the E1 and DNA polymerase proteins, but also the Ad-preterminal protein, can be used. E.C7 cells can be used, for example, to grow high titer stocks of the adenovirus vectors.
To delete critical genes from self-propagating adenovirus vectors, proteins encoded by the targeted genes can first be coexpressed in HEK-293 cells, or similar, along with E1 proteins. For example, those proteins which are non-toxic when coexpressed constitutively (or toxic proteins inducibly-expressed) can be selectively utilized. Coexpression in HEK-293 cells of the E1 and E4 genes is possible (for example utilizing inducible, not constitutive, promoters). The E1 and protein IX genes, a virion structural protein, can be coexpressed. Further coexpression of the E1, E4, and protein IX genes is also possible. E1 and 100 K genes can be expressed in trans-complementing cell lines, as can E1 and protease genes.
Cell lines coexpressing E1 and E2b gene products for use in growing high titers of E2b deleted Ad particles can be used. Useful cell lines constitutively express the approximately 140 kDa Ad-DNA polymerase and/or the approximately 90 kDa preterminal protein. Cell lines that have high-level, constitutive coexpression of the E1, DNA polymerase, and preterminal proteins, without toxicity (e.g., E.C7), are desirable for use in propagating Ad for use in multiple vaccinations. These cell lines permit the propagation of adenovirus vectors deleted for the E1, DNA polymerase, and preterminal proteins.
The recombinant Ad of the present invention can be propagated using, for example, tissue culture plates containing E.C7 cells infected with Ad vector virus stocks at an appropriate MOI (e.g., 5) and incubated at 37° C. for 40-96 h. The infected cells can be harvested, resuspended in 10 mM Tris-Cl (pH 8.0), and sonicated, and the virus can be purified by two rounds of cesium chloride density centrifugation. The virus containing band can be desalted over a column, sucrose or glycerol can be added, and aliquots can be stored at −80° C. Virus can be placed in a solution designed to enhance its stability, such as A195. The titer of the stock can be measured (e.g., by measurement of the optical density at 260 nm of an aliquot of the virus after lysis). Plasmid DNA, either linear or circular, encompassing the entire recombinant E2b deleted adenovirus vector can be transfected into E.C7, or similar cells, and incubated at 37° C. until evidence of viral production is present (e.g., cytopathic effect). Conditioned media from cells can be used to infect more cells to expand the amount of virus produced before purification. Purification can be accomplished, for example, by two rounds of cesium chloride density centrifugation or selective filtration. Virus may be purified by chromatography using commercially available products or custom chromatographic columns.
The compositions of the present invention can comprise enough virus to ensure that cells to be infected are confronted with a certain number of viruses. Thus, in various embodiments, the present invention provides a stock of recombinant Ad, such as an RCA-free stock of recombinant Ad. Viral stocks can vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. Viral stocks can have a titer of at least about 106, 107, or 108 pfu/ml, or higher, such as at least about 109, 1010, 1011, or 1012 pfu/ml. Depending on the nature of the recombinant virus and the packaging cell line, a viral stock of the present invention can have a titer of even about 1013 particles/ml or higher.
Polynucleotides and Variants Encoding Antigen Targets
The present disclosure further provides nucleic acid sequences, also referred to herein as polynucleotides that encode one or more target antigens of interest, or fragments or variants thereof. As such, the present invention provides polynucleotides that encode target antigens from any source as described further herein, vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors. In order to express a desired target antigen polypeptide, nucleotide sequences encoding the polypeptide, or functional equivalents, can be inserted into an appropriate Ad vector (e.g., using recombinant techniques). The appropriate adenovirus vector may contain the necessary elements for the transcription and translation of the inserted coding sequence and any desired linkers. Methods which are well known to those skilled in the art may be used to construct these adenovirus vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a target antigen polypeptide/protein/epitope of the invention or a portion thereof) or may comprise a sequence that encodes a variant, fragment, or derivative of such a sequence. Polynucleotide sequences can encode target antigen proteins. In some embodiments, polynucleotides represent a novel gene sequence optimized for expression in specific cell types that may substantially vary from the native nucleotide sequence or variant but encode a similar protein antigen.
In other related embodiments, polynucleotide variants have substantial identity to native sequences encoding proteins (e.g., target antigens of interest), for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a native polynucleotide sequence encoding the polypeptides (e.g., BLAST analysis using standard parameters). These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Polynucleotides can encode a protein comprising for example at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a protein sequence encoded by a native polynucleotide sequence.
Polynucleotides can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 11, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or more contiguous nucleotides encoding a polypeptide (e.g., target protein antigens), and all intermediate lengths there between. “Intermediate lengths”, in this context, refers to any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like. A polynucleotide sequence may be extended at one or both ends by additional nucleotides not found in the native sequence encoding a polypeptide, such as an epitope or heterologous target protein. This additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides or more, at either end of the disclosed sequence or at both ends of the disclosed sequence.
The polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, expression control sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. Illustrative polynucleotide segments with total lengths of about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
A mutagenesis approach, such as site-specific mutagenesis, can be employed to prepare target antigen sequences. Specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. Site-specific mutagenesis can be used to make mutants through the use of oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. For example, a primer comprising about 14 to about 25 nucleotides or so in length can be employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered. Mutations may be made in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
Mutagenesis of polynucleotide sequences can be used to alter one or more properties of the encoded polypeptide, such as the immunogenicity of an epitope comprised in a polypeptide or the immunogenicity of a target antigen. Assays to test the immunogenicity of a polypeptide include, but are not limited to, T-cell cytotoxicity assays (CTL/chromium release assays), T-cell proliferation assays, intracellular cytokine staining, ELISA, ELISpot, etc. Other ways to obtain sequence variants of peptides and the DNA sequences encoding them can be employed. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
Polynucleotide segments or fragments encoding the polypeptides of the present invention may be readily prepared by, for example, directly synthesizing the fragment by chemical means. Fragments may be obtained by application of nucleic acid reproduction technology, such as PCR, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
A variety of vector/host systems may be utilized to contain and produce polynucleotide sequences. Exemplary systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA vectors; yeast transformed with yeast vectors; insect cell systems infected with virus vectors (e.g., baculovirus); plant cell systems transformed with virus vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
Control elements or regulatory sequences present in an Ad vector may include those non-translated regions of the vector-enhancers, promoters, and 5′ and 3′ untranslated regions. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, sequences encoding a polypeptide of interest may be ligated into an Ad transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells. In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest (e.g., ATG initiation codon and adjacent sequences). Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used. Specific termination sequences, either for transcription or translation, may also be incorporated in order to achieve efficient translation of the sequence encoding the polypeptide of choice.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products (e.g., target antigens), can be used (e.g., using polyclonal or monoclonal antibodies specific for the product). Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed.
The Ad vectors can comprise a product that can be detected or selected for, such as a reporter gene whose product can be detected, such as by fluorescence, enzyme activity on a chromogenic or fluorescent substrate, and the like, or selected for by growth conditions. Exemplary reporter genes include green fluorescent protein (GFP), β-galactosidase, chloramphenicol acetyltransferase (CAT), luciferase, neomycin phosphotransferase, secreted alkaline phosphatase (SEAP), and human growth hormone (HGH). Exemplary selectable markers include drug resistances, such as neomycin (G418), hygromycin, and the like.
The Ad vectors can also comprise a promoter or expression control sequence. The choice of the promoter will depend in part upon the targeted cell type and the degree or type of control desired. Promoters that are suitable within the context of the present invention include, without limitation, constitutive, inducible, tissue specific, cell type specific, temporal specific, or event-specific. Examples of constitutive or nonspecific promoters include the SV40 early promoter, the SV40 late promoter, CMV early gene promoter, bovine papilloma virus promoter, and adenovirus promoter. In addition to viral promoters, cellular promoters are also amenable within the context of this invention. In particular, cellular promoters for the so-called housekeeping genes are useful (e.g., β-actin). Viral promoters are generally stronger promoters than cellular promoters. Inducible promoters may also be used. These promoters include MMTV LTR, inducible by dexamethasone, metallothionein, inducible by heavy metals, and promoters with cAMP response elements, inducible by cAMP, heat shock promoter. By using an inducible promoter, the nucleic acid may be delivered to a cell and will remain quiescent until the addition of the inducer. This allows further control on the timing of production of the protein of interest. Event-type specific promoters (e.g., HIV LTR) can be used, which are active or upregulated only upon the occurrence of an event, such as Ebola infection, for example. The HIV LTR promoter is inactive unless the tat gene product is present, which occurs upon viral infection. Some event-type promoters are also tissue-specific. Preferred event-type specific promoters include promoters activated upon viral infection.
Examples of promoters include promoters for α-fetoprotein, α-actin, myo D, carcinoembryonic antigen, VEGF-receptor; FGF receptor; TEK or tie 2; tie; urokinase receptor; E- and P-selectins; VCAM-1; endoglin; endosialin; αV-β3 integrin; endothelin-1; ICAM-3; E9 antigen; von Willebrand factor; CD44; CD40; vascular-endothelial cadherin; notch 4, high molecular weight melanoma-associated antigen; prostate specific antigen-1, probasin, FGF receptor, VEGF receptor, erb B2; erb B3; erb B4; MUC-1; HSP-27; int-1; int-2, CEA, HBEGF receptor; EGF receptor; tyrosinase, MAGE, IL-2 receptor; prostatic acid phosphatase, probasin, prostate specific membrane antigen, α-crystallin, PDGF receptor, integrin receptor, α-actin, SM1 and SM2 myosin heavy chains, calponin-hl, SM22 α-angiotensin receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, immunoglobulin heavy chain, immunoglobulin light chain, and CD4.
Repressor sequences, negative regulators, or tissue-specific silencers may be inserted to reduce non-specific expression of the polynucleotide. Multiple repressor elements may be inserted in the promoter region. Repression of transcription is independent of the orientation of repressor elements or distance from the promoter. One type of repressor sequence is an insulator sequence. Such sequences inhibit transcription and can silence background transcription. Negative regulatory elements can be located in the promoter regions of a number of different genes. The repressor element can function as a repressor of transcription in the absence of factors, such as steroids, as does the NSE in the promoter region of the ovalbumin gene. These negative regulatory elements can bind specific protein complexes from oviduct, none of which are sensitive to steroids. Three different elements are located in the promoter of the ovalbumin gene. Oligonucleotides corresponding to portions of these elements can repress viral transcription of the TK reporter. One of the silencer elements shares sequence identity with silencers in other genes (TCTCTCCNA (SEQ. ID. NO.:7)).
Further, repressor elements can be located in the promoter region of a variety of genes, including the collagen II gene, for example. Nuclear factors from HeLa cells can bind specifically to DNA fragments containing the silencer region. Repressor elements may play a role regulating transcription in the carbamyl phosphate synthetase gene. This gene is expressed in only two different cell types, hepatocytes and epithelial cells of the intestinal mucosa. Negative regulatory regions are also found in the promoter region of the choline acetyltransferase gene, the albumin promoter, phosphoglycerate kinase (PGK-2) gene promoter, and in the 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase gene, in which the negative regulatory element inhibits transcription in non-hepatic cell lines. Furthermore, the negative regulatory element Tse-1 is located in a number of liver specific genes, including tyrosine aminotransferase (TAT). TAT gene expression is liver specific and inducible by both glucocorticoids and the cAMP signaling pathway. The cAMP response element (CRE) can ask as the target for repression by Tse-1 and hepatocyte-specific elements. Accordingly, it is clear that varieties of such elements are known or are readily identified.
In certain embodiments, Elements that increase the expression of the desired target antigen can be incorporated into the nucleic acid sequence of the Ad vectors described herein. Exemplary elements include internal ribosome binding sites (IRESs). IRESs can increase translation efficiency. As well, other sequences may enhance expression. For some genes, sequences especially at the 5′ end may inhibit transcription and/or translation. These sequences are usually palindromes that can form hairpin structures. In some cases, such sequences in the nucleic acid to be delivered are deleted. Expression levels of the transcript or translated product can be assayed to confirm or ascertain which sequences affect expression. Transcript levels may be assayed by any known method, including Northern blot hybridization, RNase probe protection and the like. Protein levels may be assayed by any known method, including ELISA.
Ebola Antigen-Specific Immunotherapies and Vaccines
The present disclosure provides for single antigen or combination antigen immunization against Ebola antigens, such as GP, NP, VP40, VP35, VP30, VP24, and/or L, utilizing such vectors and other vectors as provided herein. The present disclosure provides for therapeutic vaccines against Ebola antigens. The present disclosure provides for prophylactic vaccines against Ebola antigens. Further, in various embodiments, the composition and methods provide herein can lead to clinical responses, such as altered disease progression or life expectancy.
Ad5 [E1-] vectors encoding a variety of antigens can be used to efficiently transduce 95% of ex vivo exposed DC's to high titers of the vector. Importantly, the inventors have discovered increasing levels of foreign gene expression in the DC with increasing multiplicities of infection (MOI) with the vector. DCs infected with Ad5 [E1-] vectors can encode a variety of Ebola antigens that have the propensity to induce antigen specific CTL responses, have an enhanced antigen presentation capacity, and/or have an improved ability to initiate T-cell proliferation in mixed lymphocyte reactions. Immunization of animals with dendritic cells (DCs) previously transduced by Ad5 vectors encoding tumor specific antigens can be used to induce significant levels of protection for the animals when challenged with tumor cells expressing the respective antigen. Interestingly, intra-tumoral injection of Ads encoding IL-7 is less effective than injection of DCs transduced with IL-7 encoding Ad5 vectors at inducing anti-tumor immunity. Ex vivo transduction of DCs by Ad5 vectors is contemplated by the present disclosure. Ex vivo DC transduction strategies can been used to induce recipient host tolerance. For example, Ad5 mediated delivery of the CTLA4Ig into DCs can block interactions of the DCs CD80 with CD28 molecules present on T-cells.
Ad5 vector capsid interactions with DCs may trigger several beneficial responses, which may be enhancing the propensity of DCs to present antigens encoded by Ad5 vectors. For example, immature DCs, though specialized in antigen uptake, are relatively inefficient effectors of T-cell activation. DC maturation coincides with the enhanced ability of DCs to drive T-cell immunity. In some instances, the compositions and methods of the invention take advantage of an Ad5 infection resulting in direct induction of DC maturation Ad vector infection of immature bone marrow derived DCs from mice may upregulate cell surface markers normally associated with DC maturation (MHC I and II, CD40, CD80, CD86, and ICAM-1) as well as down-regulation of CD11c, an integrin down regulated upon myeloid DC maturation. In some instances, Ad vector infection triggers IL-12 production by DCs, a marker of DC maturation. Without being bound by theory, these events may possibly be due to Ad5 triggered activation of NF-κB pathways. Mature DCs can be efficiently transduced by Ad vectors, and do not lose their functional potential to stimulate the proliferation of naive T-cells at lower MOI, as demonstrated by mature CD83+ human DC (derived from peripheral blood monocytes). However, mature DCs may also be less infectable than immature ones.
Modification of capsid proteins can be used as a strategy to optimize infection of DC by Ad vectors, as well as enhancing functional maturation, for example using the CD40L receptor as a viral vector receptor, rather than using the normal CAR receptor infection mechanisms.
In various embodiments the compositions and methods of the invention comprising an Ad5 [E1-, E2b-]-GP, NP, VP40, VP35, VP30, VP24, L, or any combination thereof, vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) GP vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) NP vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) VP40 vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) VP35 vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) VP30 vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) VP24 vaccine effect of increased overall survival (OS) within the bounds of technical safety. For example, the compositions and methods of the invention can comprise an Ad5 [E1-, E2b-] vector(s) L vaccine effect of increased overall survival (OS) within the bounds of technical safety.
As noted above, the adenovirus vectors of the present invention comprise nucleic acid sequences that encode one or more target proteins or antigens of interest. In this regard, the vectors may contain nucleic acid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different target antigens of interest. The target antigens may be a full length protein or may be a fragment (e.g., an epitope) thereof. The adenovirus vectors may contain nucleic acid sequences encoding multiple fragments or epitopes from one target protein of interest or may contain one or more fragments or epitopes from numerous different target proteins of interest.
A target antigen may comprise any substance against which it is desirable to generate an immune response but generally, the target antigen is a protein. A target antigen may comprise a full length protein, a subunit of a protein, an isoform of a protein, or a fragment thereof that induces an immune response (i.e., an immunogenic fragment). A target antigen or fragment thereof may be modified, e.g., to reduce one or more biological activities of the target antigen or to enhance its immunogenicity.
In certain embodiments, immunogenic fragments bind to an MHC class I or class II molecule. An immunogenic fragment may “bind to” an MHC class I or class II molecule if such binding is detectable using any assay known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of 125I labeled β-2-microglobulin (β-2m) into MHC class I/β2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994). Alternatively, functional peptide competition assays that are known in the art may be employed. Immunogenic fragments of polypeptides may generally be identified. Representative techniques for identifying immunogenic fragments include screening polypeptides for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An immunogenic fragment of a particular target polypeptide is a fragment that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length target polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). In other words, an immunogenic fragment may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may be performed using methods known in the art.
In some embodiments, the viral vectors of the present invention comprise heterologous nucleic acid sequences that encode one or more proteins, variants thereof, fusions thereof, or fragments thereof, that can modulate the immune response. In some embodiments, the viral vector of the present invention encodes one or more antibodies against specific antigens, such as anthrax protective antigen, permitting passive immunotherapy. In some embodiments, the viral vectors of the present invention comprise heterologous nucleic acid sequences encoding one or more proteins having therapeutic effect (e.g., anti-viral, anti-bacterial, anti-parasitic, or anti-Ebola function). In some embodiments the Second Generation E2b deleted adenovirus vectors comprise a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence is GP, NP, VP40, VP35, VP30, VP24, L, a variant, a portion, or any combination thereof.
Target antigens include, but are not limited to, antigens derived from a variety of Ebola viruses. In some embodiments, parts or variants of Ebola proteins are employed as target antigens. In some embodiments, parts or variants of Ebola proteins being employed as target antigens have a modified, for example, increased ability to effect and immune response against the Ebola protein or cells containing the same. A vaccine of the present invention can vaccinate against an antigen. A vaccine can also target an epitope. An antigen can be an Ebola virus antigen. An epitope can be an Ebola virus epitope. Such a Ebola virus epitope may be derived from a wide variety of Ebola viruses, such as antigens from Ebola viruses resulting from mutations and shared Ebola species specific antigens. Ebola antigens (EAs) may be antigens not normally expressed by the host. Ebola-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, other biological molecules or any combinations thereof.
Illustrative Ebola proteins useful in the present invention include, but are not limited to any one or more of GP, NP, VP40, VP35, VP30, VP24, and L. In some embodiments, the viral vector comprises a target antigen sequence encoding a modified polypeptide selected from GP, NP, VP40, VP35, VP30, VP24, and L wherein the polypeptide or a fragment thereof has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to the described sequence.
The inventors have discovered that multiple homologous immunizations with Ad5 [E1-, E2b-]-GP, NP, VP40, VP35, VP30, VP24, and/or L, induced GP, NP, VP40, VP35, VP30, VP24, and/or L-specific cell-mediated immune (CMI) responses with anti-Ebola activity in animals despite the presence of pre-existing or induced Ad5-neutralizing antibody. Cohorts of patients with Ebola can be immunized with escalating doses of Ad5 [E1-, E2b-]-GP, NP, VP40, VP35, VP30, VP24, and/or L. In subjects with Ebola infections, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the EAs GP, NP, VP40, VP35, VP30, VP24, and/or L in the setting of both naturally acquired and immunization-induced Ad5 specific immunity.
GP, NP, VP40, VP35, VP30, VP24, and/or L antigen specific CMI can be, for example, greater than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, or more IFN-γ spot forming cells (SFC) per 106 peripheral blood mononuclear cells (PBMC). In some embodiments, the immune response is raised in a human subject with a preexisting inverse Ad5 neutralizing antibody titer of greater than 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 1000, 12000, 15000 or higher. The immune response may comprise a cell-mediated immunity and/or a humoral immunity as described herein. The immune response may be measured by one or more of intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T-cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays, as described herein and to the extent they are available to a person skilled in the art, as well as any other suitable assays known in the art for measuring immune response.
In some embodiments, the replication defective adenovirus vector comprises a modified sequence encoding a subunit with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to a wild-type subunit of the polypeptide. The immunogenic polypeptide may be a mutant GP, NP, VP40, VP35, VP30, VP24, or L, or a fragment thereof. In some embodiments, the replication defective adenovirus vector comprises a sequence encoding a polypeptide with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to the immunogenic polypeptide. In some embodiments, the sequence encoding the immunogenic polypeptide comprises the sequence of SEQ. ID. NO.:1.
In some embodiments, the sequence encoding the immunogenic polypeptide comprises a sequence with at least 70% 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% identity to SEQ. ID. NO.:1, 2, 4, 5, or 6, or a sequence generated from SEQ. ID. NO.: 1, 2, 4, 5, or 6 by alternative codon replacements. In some embodiments, the immunogenic polypeptide encoded by the adenovirus vectors comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such as single amino acid substitutions or deletions, as compared to a wild-type human GP, NP, VP40, VP35, VP30, VP24, or L sequence.
In certain embodiments Ebola antigens may be identified directly from an individual infected with an Ebola virus. In this regard, screens can be carried out using a variety of known technologies. For example, in one embodiment, a cell or tissue biopsy is taken from a patient, RNA is isolated from the sample cells and screened using a gene chip (for example, from Affymetrix, Santa Clara, Calif.) and a Ebola virus antigen is identified. Once the Ebola virus target antigen is identified, it may then be cloned, expressed and purified using techniques known in the art.
This target antigen can then linked to one or more epitopes or incorporated or linked to cassettes or viral vectors described herein and administered to the patient in order to alter the immune response to the target molecule isolated from a sample from he Ebola infected patient. In this manner, “personalized” immunotherapy and vaccines are contemplated within the context of the invention. In some embodiments, a personalized Ebola antigen related to SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6 or a combination thereof is characterized from a patient and further utilized as the target antigen as a whole, in part or as a variant.
Combination Immunotherapies and Vaccines
The present disclosure provides for a combination immunotherapy and vaccine compositions for the treatment of Ebola infections. In some aspects, combination immunotherapies and vaccines provided herein can comprise a multi-targeted immunotherapeutic approach against antigens associated with Ebola infections. In some aspects, combination immunotherapies and vaccines provided herein can comprise a multi-targeted antigen signature immunotherapeutic approach against antigens associated with Ebola infections. The compositions and methods of the invention, in various embodiments, provide viral based vectors expressing a wild-type or variant of GP, NP, VP40, VP35, VP30, VP24, and/or L for immunization of Ebola, as provided herein. These vectors can raise an immune response against GP, NP, VP40, VP35, VP30, VP24, and/or L.
In some aspects, the vector comprises at least one antigen. In some aspects, the vector comprises at least two antigens. In some aspects, the vector comprises at least three antigens. In some aspects, the vector comprises more than three antigens. In some aspects, the vaccine formulation comprises 1:1 ratio of vector to antigen. In some aspects, the vaccine comprises 1:2 ratio of vector to antigen. In some aspects, the vaccine comprises 1:3 ratio of vector to antigen. In some aspects, the vaccine comprises 1:4 ratio of vector to antigen. In some aspects, the vaccine comprises 1:5 ratio of vector to antigen. In some aspects, the vaccine comprises 1:6 ratio of vector to antigen. In some aspects, the vaccine comprises 1:7 ratio of vector to antigen. In some aspects, the vaccine comprises 1:8 ratio of vector to antigen. In some aspects, the vaccine comprises 1:9 ratio of vector to antigen. In some aspects, the vaccine comprises 1:10 ratio of vector to antigen.
In some aspects, the vaccine is a combination vaccine, wherein the vaccine comprises at least two vectors each containing at least a single antigen. In some aspects the vaccine is a combination vaccine, wherein the vaccine comprises at least three vectors each containing at least a single antigen target. In some aspects the vaccine is a combination vaccine, wherein the vaccine comprises more than three vectors each containing at least a single antigen.
In some aspects, the vaccine is a combination vaccine, wherein the vaccine comprises at least two vectors, wherein a first vector of the at least two vectors comprises at least a single antigen and wherein a second vector of the at least two vectors comprises at least two antigens. In some aspects, the vaccine is a combination vaccine, wherein the vaccine comprises at least three vectors, wherein a first vector of the at least three vectors comprises at least a single antigen and wherein a second vector of the at least three vectors comprises at least two antigens. In some aspects, the vaccine is a combination vaccine, wherein the vaccine comprises three or more vectors, wherein a first vector of the three or more vectors comprises at least a single antigen and wherein a second vector of the three or more vectors comprises at least two antigens. In some aspects the vaccine is a combination vaccine, wherein the vaccine comprises more than three vectors each containing at least two antigens.
When a mixture of different antigens are simultaneously administered or expressed from a same or different vector in an individual, they may compete with one another. As a result the formulations comprising different concentration and ratios of expressed antigens in a combination immunotherapy or vaccine must be evaluated and tailored to the individual or group of individuals to ensure that effective and sustained immune responses occur after administration.
Composition that comprises multiple antigens can be present at various ratios. For example, formulations with more than vector can have various ratios. For example, immunotherapies or vaccines can have two different vectors in a stoichiometry of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3: 1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4: 1, 4:3, 4:5, 4:6, 4:7, 4:8, 5: 1, 5:3, 5:4, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:7, 6:8, 7: 1, 7:3, 7:4, 7:5, 7:6, 7:8, 8: 1, 8:3, 8:4, 8:5, 8:6, or 8:7. For example, immunotherapies or vaccines can have three different vectors in a stoichiometry of: 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 2:1:1, 2:3:1, 2:4:1, 2:5:1, 2:6:1, 2:7:1, 2:8:1, 3:1, 3:3:1, 3:4:1, 3:5:1, 3:6:1, 3:7:1, 3:8:1, 3:1:1, 3:3:1, 3:4:1, 3:5:1, 3:6:1, 3:7:1, 3:8:1, 4:1:1, 4:3:1, 4:4:1, 4:5:1, 4:6:1, 4:7:1, 4:8:1, 5:1:1, 5:3:1, 5:4:1, 5:5:1, 5:6:1, 5:7:1, 5:8:1, 6:1:1, 6:3:1, 6:4:1, 6:5:1, 6:6:1, 6:7:1, 6:8:1, 7:1:1, 7:3:1, 7:4:1, 7:5:1, 7:6:1, 7:7:1, 7:8:1, 8:1:1, 8:3:1, 8:4:1, 8:5:1, 8:6:1, 8:7:1, 8:8:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:5:2, 1:6:2, 1:7:2, 1:8:2, 2:1:2, 2:3:2, 2:4:2, 2:5:2, 2:6:2, 2:7:2, 2:8:2, 3:1:2, 3:3:2, 3:4:2, 3:5:2, 3:6:2, 3:7:2, 3:8:2, 3:1:2, 3:3:2, 3:4:2, 3:5:2, 3:6:2, 3:7:2, 3:8:2, 4:1:2, 4:3:2, 4:4:2, 4:5:2, 4:6:2, 4:7:2, 4:8:2, 5:1:2, 5:3:2, 5:4:2, 5:5:2, 5:6:2, 5:7:2, 5:8:2, 6:1:2, 6:3:2, 6:4:2, 6:5:2, 6:6:2, 6:7:2, 6:8:2, 7:1:2, 7:3:2, 7:4:2, 7:5:2, 7:6:2, 7:7:2, 7:8:2, 8:1:2, 8:3:2, 8:4:2, 8:5:2, 8:6:2, 8:7:2, 8:8:2, 1:1:3, 1:2:3, 1:3:3, 1:4:3, 1:5:3, 1:6:3, 1:7:3, 1:8:3, 2:1:3, 2:3:3, 2:4:3, 2:5:3, 2:6:3, 2:7:3, 2:8:3, 3:1:3, 3:3:3, 3:4:3, 3:5:3, 3:6:3, 3:7:3, 3:8:3, 3:1:3, 3:3:3, 3:4:3, 3:5:3, 3:6:3, 3:7:3, 3:8:3, 4:1:3, 4:3:3, 4:4:3, 4:5:3, 4:6:3, 4:7:3, 4:8:3, 5:1:3, 5:3:3, 5:4:3, 5:5:3, 5:6:3, 5:7:3, 5:8:3, 6:1:3, 6:3:3, 6:4:3, 6:5:3, 6:6:3, 6:7:3, 6:8:3, 7:1:3, 7:3:3, 7:4:3, 7:5:3, 7:6:3, 7:7:3, 7:8:3, 8:1:3, 8:3:3, 8:4:3, 8:5:3, 8:6:3, 8:7:3, 8:8:3, 1:1:4, 1:2:4, 1:3:4, 1:4:4, 1:5:4, 1:6:4, 1:7:4, 1:8:4, 2:1:4, 2:3:4, 2:4:4, 2:5:4, 2:6:4, 2:7:4, 2:8:4, 3:1:4, 3:3:4, 3:4:4, 3:5:4, 3:6:4, 3:7:4, 3:8:4, 3:1:4, 3:3:4, 3:4:4, 3:5:4, 3:6:4, 3:7:4, 3:8:4, 4:1:4, 4:3:4, 4:4:4, 4:5:4, 4:6:4, 4:7:4, 4:8:4, 5:1:4, 5:3:4, 5:4:4, 5:5:4, 5:6:4, 5:7:4, 5:8:4, 6:1:4, 6:3:4, 6:4:4, 6:5:4, 6:6:4, 6:7:4, 6:8:4, 7:1:4, 7:3:4, 7:4:4, 7:5:4, 7:6:4, 7:7:4, 7:8:4, 8:1:4, 8:3:4, 8:4:3, 8:5:4, 8:6:4, 8:7:4, 8:8:4, 1:1:5, 1:2:5, 1:3:5, 1:4:5, 1:5:5, 1:6:5, 1:7:5, 1:8:5, 2:1:5, 2:3:5, 2:4:5, 2:5:5, 2:6:5, 2:7:5, 2:8:5, 3:1:5, 3:3:5, 3:4:5, 3:5:5, 3:6:5, 3:7:5, 3:8:5, 3:1:5, 3:3:5, 3:4:5, 3:5:5, 3:6:5, 3:7:5, 3:8:5, 4:1:5, 4:3:5, 4:4:5, 4:5:5, 4:6:5, 4:7:5, 4:8:5, 5:1:5, 5:3:5, 5:4:5, 5:5:5, 5:6:5, 5:7:5, 5:8:5, 6:1:5, 6:3:5, 6:4:5, 6:5:5, 6:6:5, 6:7:5, 6:8:5, 7:1:5, 7:3:5, 7:4:5, 7:5:5, 7:6:5, 7:7:5, 7:8:5, 8:1:5, 8:3:5, 8:4:5, 8:5:5, 8:6:5, 8:7:5, 8:8:5, 1:1:6, 1:2:6, 1:3:6, 1:4:6, 1:5:6, 1:6:6, 1:7:6, 1:8:6, 2:1:6, 2:3:6, 2:4:6, 2:5:6, 2:6:6, 2:7:6, 2:8:6, 3:1:6, 3:3:6, 3:4:6, 3:5:6, 3:6:6, 3:7:6, 3:8:6, 3:1:6, 3:3:6, 3:4:6, 3:5:6, 3:6:6, 3:7:6, 3:8:6, 4:1:6, 4:3:6, 4:4:6, 4:5:6, 4:6:6, 4:7:6, 4:8:6, 5:1:6, 5:3:6, 5:4:6, 5:5:6, 5:6:6, 5:7:6, 5:8:6, 6:1:6, 6:3:6, 6:4:6, 6:5:6, 6:6:6, 6:7:6, 6:8:6, 7:1:6, 7:3:6, 7:4:6, 7:5:6, 7:6:6, 7:7:6, 7:8:6, 8:1:6, 8:3:6, 8:4:6, 8:5:6, 8:6:5, 8:7:6, 8:8:6, 1:1:7, 1:2:7, 1:3:7, 1:4:7, 1:5:7, 1:6:7, 1:7:7, 1:8:7, 2:1:7, 2:3:7, 2:4:7, 2:5:7, 2:6:7, 2:7:7, 2:8:7, 3:1:7, 3:3:7, 3:4:7, 3:5:7, 3:6:7, 3:7:7, 3:8:7, 3:1:7, 3:3:7, 3:4:7, 3:5:7, 3:6:7, 3:7:7, 3:8:7, 4:1:7, 4:3:7, 4:4:7, 4:5:7, 4:6:7, 4:7:7, 4:8:7, 5:1:7, 5:3:7, 5:4:7, 5:5:7, 5:6:7, 5:7:7, 5:8:7, 6:1:7, 6:3:7, 6:4:7, 6:5:7, 6:6:7, 6:7:7, 6:8:7, 7:1:7, 7:3:7, 7:4:7, 7:5:7, 7:6:7, 7:7:7, 7:8:7, 8:1:7, 8:3:7, 8:4:7, 8:5:7, 8:6:5, 8:7:7, or 8:8:7.
The present disclosure provides for a combination immunotherapies or vaccines comprising: at least two, at least three, or more than three different target antigens comprising a sequence encoding a GP, NP, VP40, VP35, VP30, VP24, and/or L. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein a wild-type or modified target antigen comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:1. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein a wild-type or modified target antigen comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:2. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein a wild-type or modified target antigen comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:4. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein a wild-type or modified target antigen comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:5. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein a wild-type or modified target antigen comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:6.
In some aspects the present disclosure provides combination immunotherapies comprising multi-targeted immunotherapeutic directed to EAs and molecular compositions comprising an immune pathway checkpoint modulator that targets at least one immune-checkpoint protein of the immune inhibitory pathway. The present disclosure provides for a combination immunotherapies or vaccines comprising: at least two, at least three, or more than three different target antigens comprising a sequence encoding a wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, and at least one molecular composition comprising an immune pathway checkpoint modulator. For example, a combination immunotherapy or vaccine can comprise at least two, at least three, or more than three different target antigens comprising a sequence encoding a modified wild-type or modified GP, NP, VP40, VP35, VP30, VP24, and/or L, wherein the modified target antigen comprises a sequence with an at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ. ID. NO.:1, 2, 4, 5, and/or 6, and at least one molecular composition of the immune-checkpoint inhibitory pathway.
In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets CTLA4. In some embodiments, the least one molecular comprises an immune pathway checkpoint modulator that targets PD1. In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets PDL1. In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets LAG3. In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets B7-H3. In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets B7-H4. In some embodiments, the least one molecular composition comprises an immune pathway checkpoint modulator that targets TIM3. In some embodiment the molecular composition comprises an immune pathway checkpoint modulator that is a monoclonal or polyclonal antibody directed to PD1, PDL1, PDL2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7-H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3, CD137, CD137L, OX40, OX40L, CD27, CD70, CD40, CD40L, TIM3 (i.e., HAVcr2), GALS, and A2aR.
Immunological Fusion Partner Antigen Targets
The viral vectors of the present invention may also include nucleic acid sequences that encode proteins that increase the immunogenicity of the target antigen. In this regard, the protein produced following immunization with the adenovirus vector containing such a protein may be a fusion protein comprising the target antigen of interest fused to a protein that increases the immunogenicity of the target antigen of interest.
In one embodiment, such an immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences are described in U.S. Patent Application 60/158,585 and U.S. Pat. No. 7,009,042. Briefly, Ra12 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 kDa encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (see, e.g., U.S. Patent Application 60/158,585; Skeiky et al., Infection and Immun. 67:3998-4007 (1999)). C-terminal fragments of the MTB32A coding sequence express at high levels and remain as soluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One Ra12 fusion polypeptide comprises a 14 kDa C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other Ra12 polynucleotides generally comprise at least about 15, 30, 60, 100, 200, 300, or more nucleotides that encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ra12 polypeptide. Variants can have at least about 70%, 80%, or 90% identity, or more, to a polynucleotide sequence that encodes a native Ra12 polypeptide or a portion thereof.
An immunological fusion partner can be derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B. In some cases, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids). A protein D derivative may be lipidated. Within certain embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes, which may increase the expression level in E. coli and may function as an expression enhancer. The lipid tail may ensure optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenza virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
The immunological fusion partner can be the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus can be employed. Within another embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion can, for example, be found in the C-terminal region starting at residue 178. One particular repeat portion incorporates residues 188-305.
In some embodiments, the antigen target comprises an immunogenic component comprising a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In some embodiments, the antigen target further comprises one or more immunogenic component comprises a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In some embodiments, the antigen target comprises an immunogenic component comprising a nucleic acid encoding of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, a protein with substantial identity to of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, and a nucleic acid encoding a protein with substantial identity to of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.
In some embodiments, the antigen target is fused or linked to an immunogenic component comprising a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In some embodiments, the antigen target is co-expressed in a cell with an immunogenic component comprising a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.
Immune Pathway Checkpoint Modulators
In some embodiments, compositions of the present invention are administered with one or more immune pathway checkpoint modulators. A balance between activation and inhibitory signals regulates the interaction between T lymphocytes and disease cells, wherein T-cell responses are initiated through antigen recognition by the T-cell receptor (TCR). The inhibitory pathways and signals are referred to as immune checkpoints. In normal circumstances, immune checkpoints play a critical role in control and prevention of autoimmunity and also protect from tissue damage in response to pathogenic infection.
The present disclosure provides combination immunotherapies comprising viral vector based vaccines and compositions for modulating immune checkpoint inhibitory pathways for the treatment of Ebola infections. In some embodiments, modulating is increasing expression or activity of a gene or protein. In some embodiments, modulating is decreasing expression or activity of a gene or protein. In some embodiments, modulating affects a family of genes or proteins.
In general, the immune inhibitory pathways are initiated by ligand-receptor interactions. It is now clear that in diseases, the disease can co-opt immune-checkpoint pathways as mechanism for inducing immune resistance in a subject.
The induction of immune resistance or immune inhibitory pathways in a subject by a given disease can be blocked by molecular compositions such as siRNAs, antisense, small molecules, mimic, a recombinant form of ligand, receptor or protein, or antibodies (which can be an Ig fusion protein) that are known to modulate one or more of the Immune Inhibitory Pathways. For example, preliminary clinical findings with blockers of immune-checkpoint proteins, such as Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) have shown promise for enhancing anti-tumor immunity.
Because diseased cells can express multiple inhibitory ligands, and disease-infiltrating lymphocytes express multiple inhibitory receptors, dual or triple blockade of immune checkpoints proteins may enhance anti-disease immunity. Combination immunotherapies as provide herein can comprise one or more molecular compositions comprising an immune pathway checkpoint modulator that targets one or more of the following immune-checkpoint proteins: PD1, PDL1, PDL2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7-H3 (also known as CD276), B7-H4 (also known as B7-S1, B7x and VCTN1), BTLA (also known as CD272), HVEM, KIR, TCR, LAG3 (also known as CD223), CD137, CD137L, OX40, OX40L, CD27, CD70, CD40, CD40L, TIM3 (also known as HAVcr2), GALS, A2aR and Adenosine. In some embodiments, the molecular composition comprises a siRNAs. In some embodiments, the molecular composition comprises a small molecule. In some embodiments, the molecular composition comprises a recombinant form of a ligand. In some embodiments, the molecular composition comprises a recombinant form of a receptor. In some embodiments, the molecular composition comprises an antibody. In some embodiments, the combination therapy comprises more than one molecular composition and/or more than one type of molecular composition. As it will be appreciated by those in the art, future discovered proteins of the immune checkpoint inhibitory pathways are also envisioned to be encompassed by the present disclosure.
In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of CTLA4. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of PD1. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of PDL1. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of LAG3. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of B7-H3. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of B7-H4. In some embodiments, combination immunotherapies comprise molecular compositions for the modulation of TIM3. In some embodiments, modulation is an increase or enhancement of expression. In other embodiments, modulation is the decrease of absence of expression.
Two exemplary immune checkpoint inhibitors include the cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and the programmed cell death protein-1 (PD1). CTLA-4 can be expressed exclusively on T-cells where it regulates early stages of T-cell activation. CTLA-4 interacts with the co-stimulatory T-cell receptor CD28 which can result in signaling that inhibits T-cell activity. Once TCR antigen recognition occurs, CD28 signaling may enhances TCR signaling, in some cases leading to activated T-cells and CTLA-4 inhibits the signaling activity of CD28. The present disclosure provides immunotherapies as provided herein in combination with anti-CTLA-4 monoclonal antibody for the treatment of Ebola. The present disclosure provides immunotherapies as provided herein in combination with CTLA-4 molecular compositions for the treatment of Ebola.
Programmed death cell protein ligand-1 (PDL1) is a member of the B7 family and is distributed in various tissues and cell types. PDL1 can interact with PD1 inhibiting T-cell activation and CTL mediated lysis. Significant expression of PDL1 has been demonstrated on various human tumors and PDL1 expression is one of the key mechanisms in which tumors evade host anti-tumor immune responses. Programmed death-ligand 1 (PDL1) and programmed cell death protein-1 (PD1) interact as immune checkpoints. This interaction can be a major tolerance mechanism which results in the blunting of anti-tumor immune responses and subsequent tumor progression. PD1 is present on activated T cells and PDL1, the primary ligand of PD1, is often expressed on tumor cells and antigen-presenting cells (APC) as well as other cells, including B cells. Significant expression of PDL1 has been demonstrated on various human tumors including HPV-associated head and neck cancers. PDL1 interacts with PD1 on T cells inhibiting T cell activation and cytotoxic T lymphocyte (CTL) mediated lysis. The present disclosure provides immunotherapies as provided herein in combination with anti-PD1 or anti-PDL1 monoclonal antibody for the treatment of Ebola. The present disclosure provides immunotherapies as provided herein in combination with PD1 or anti-PDL1 molecular compositions for the treatment of Ebola. The present disclosure provides immunotherapies as provided herein in combination with anti-CTLA-4 and anti-PD1 monoclonal antibodies for the treatment of Ebola. The present disclosure provides immunotherapies as provided herein in combination with anti-CTLA-4 and PDL1 monoclonal antibodies for the treatment of Ebola. The present disclosure provides immunotherapies as provided herein in combination with anti-CTLA-4, anti-PD1, PDL1, monoclonal antibodies, or a combination thereof, for the treatment of Ebola.
Immune checkpoint molecules can be expressed by T cells. Immune checkpoint molecules can effectively serve as “brakes” to down-modulate or inhibit an immune response. Immune checkpoint molecules include, but are not limited to Programmed Death 1 (PD1, also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as IVSTM3, accession number: NM_173799), LAIR1 (also known as CD305, GenBank accession number: CR542051.1), SIGLECIO (GenBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, ILIORA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 which directly inhibit immune cells. For example, PD1 can be combined with an adenoviral vaccine of the present invention to treat a patient in need thereof.
Table 1, without being exhaustive, shows exemplary immune checkpoint genes that can be inactivated to improve the efficiency of the adenoviral vaccine of the present invention. Immune checkpoints gene can be selected from such genes listed in Table 1 and others involved in co-inhibitory receptor function, cell death, cytokine signaling, arginine tryptophan starvation, TCR signaling, Induced T-reg repression, transcription factors controlling exhaustion or anergy, and hypoxia mediated tolerance.
The combination of an adenoviral-based vaccine and an immune pathway checkpoint modulator may result in reduction in Ebola infection, progression, or symptoms in treated patients, as compared to either agent alone. In another embodiment of this invention the combination of an adenoviral-based vaccine and an immune pathway checkpoint modulator may result improved overall survival of treated patients, as compared to either agent alone. In some cases, the combination of an adenoviral vaccine and an immune pathway checkpoint modulator may increase the frequency or intensity of Ebola-specific T cell responses in treated patients as compared to either agent alone.
The present invention also discloses the use of immune checkpoint inhibition to improve performance of an adenoviral vector-based vaccine. Said immune checkpoint inhibition may be administered at the time of the vaccine. Said immune checkpoint inhibition may also be administered after a vaccine. Immune checkpoint inhibition may occur simultaneously to an adenoviral vaccine administration. Immune checkpoint inhibition may occur 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 minutes after vaccination. Immune checkpoint inhibition may also occur 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post vaccination. In some cases, immune inhibition may occur 1, 2, 3, 4, 5, 6, or 7 days after vaccination. Immune checkpoint inhibition may occur at any time before or after vaccination.
In another aspect, the invention pertains to a vaccine comprising an antigen and an immune pathway checkpoint modulator. The invention can pertain to a method for treating a subject having a condition that would benefit from downregulation of an immune checkpoint protein, PD1 for example, and its natural binding partner(s) on cells of the subject.
An immune pathway checkpoint modulator may be combined with an adenoviral vaccine comprising any antigen. For example, an antigen can be GP, NP, VP40, VP35, VP30, VP24, and/or L. An immune pathway checkpoint modulator may produce a synergistic effect when combined with a vaccine. An immune pathway checkpoint modulator may also produce an additive effect when combined with a vaccine.
Formulations
The present invention provides pharmaceutical compositions comprising a vaccination regime that can be administered either alone or together with a pharmaceutically acceptable carrier or excipient, by any routes, and such administration can be carried out in both single and multiple dosages. More particularly, the pharmaceutical composition can be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hand candies, powders, sprays, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, such oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes. The compositions described throughout can be formulated into a pharmaceutical medicament and be used to treat a human or mammal, in need thereof, diagnosed with a disease, e.g., Ebola.
For administration, the adenovirus vector stock can be combined with an appropriate buffer, physiologically acceptable carrier, excipient or the like. In certain embodiments, an appropriate number of virus vector particles (VP) are administered in an appropriate buffer, such as, sterile PBS or saline. In certain embodiment's viral vector compositions disclosed herein are provided in specific formulations for administration subcutaneously, parenterally, intravenously, intramuscularly, or even intraperitoneally. In certain embodiments, formulations in a solution of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, squalene-based emulsion, Squalene-based oil-in-water emulsions, water-in-oil emulsions, oil-in-water emulsions, nonaqueous emulsions, water-in-paraffin oil emulsion, and mixtures thereof and in oils. In other embodiments, viral vectors may are provided in specific formulations for pill form administration by swallowing or by suppository.
Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see, e.g., U.S. Pat. No. 5,466,468). Fluid forms to the extent that easy syringability exists may be preferred. Forms that are stable under the conditions of manufacture and storage are within the bounds of this invention. In various embodiments, forms are preserved against the contaminating action of microorganisms, such as bacteria, molds and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. It may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
In one embodiment, for parenteral administration in an aqueous solution, the solution can be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage may occur depending on the condition of the subject being treated.
Carriers of formulation can comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. GP, NP, VP40, VP35, VP30, VP24, and L.
In certain embodiments, the viral vectors of the invention may be administered in conjunction with one or more immunostimulants, such as an adjuvant. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an antigen. One type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories); Merck Adjuvant 65 (Merck and Company, Inc.) AS-2 (SmithKline Beecham); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, IFN-γ, TNFα, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13, and others, like growth factors, may also be used as adjuvants.
Within certain embodiments of the invention, the adjuvant composition can be one that induces an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient may support an immune response that includes Th1- and/or Th2-type responses. Within certain embodiments, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. Thus, various embodiments of the invention relate to therapies raising an immune response against a target antigen, for example SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ. ID. NO.:6 using cytokines, e.g., IFN-γ, TNFα, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13 supplied concurrently with a replication defective viral vector treatment. In some embodiments, a cytokine or a nucleic acid encoding a cytokine, is administered together with a replication defective viral described herein. In some embodiments, cytokine administration is performed prior or subsequent to viral vector administration. In some embodiments, a replication defective viral vector capable of raising an immune response against a target antigen, for example, SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ. ID. NO.:6 further comprises a sequence encoding a cytokine.
Certain illustrative adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL® adjuvants are commercially available. CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described. Immunostimulatory DNA sequences are also described. Another adjuvant for use in the present invention comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7; escin; digitonin; or gypsophila or chenopodium quinoa saponins. Other formulations may include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escin, or digitonin.
In certain embodiments, the compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. The delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described.
In certain embodiments, Liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the Compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions of the present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see, Lasic, Trends Biotechnol 1998 July; 16(7):307-21; Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit. Rev Ther Drug Carrier Syst. 1995; 12(2-3):233-61; U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, the use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.
In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described by Couvreur et al., Crit. Rev Ther Drug Carrier Syst. 1988; 5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2; 50(1-3):31-40; and U.S. Pat. No. 5,145,684.
Methods
Compositions and methods of the invention, in various embodiments, take advantage of human cytolytic T-cells (CTLs), such as those that recognize GP, NP, VP40, VP35, VP30, VP24, and/or L epitopes which bind to selected MHC molecules, e.g., HLA-A2, A3, and A24. Individuals expressing MHC molecules of certain serotypes, e.g., HLA-A2, A3, and A24 may be selected for therapy using the methods and compositions of the invention. For example, individuals expressing MHC molecules of certain serotypes, e.g., HLA-A2, A3, and A24, may be selected for a therapy including raising an immune response against GP, NP, VP40, VP35, VP30, VP24, and/or L, using the methods and compositions described herein.
In various embodiments, these T-cells can be generated by in vitro cultures using antigen-presenting cells pulsed with the epitope of interest to stimulate peripheral blood mononuclear cells. In addition, T-cell lines can also be generated after stimulation with GP, NP, VP40, VP35, VP30, VP24, and/or L latex beads, GP, NP, VP40, VP35, VP30, VP24, and/or L protein-pulsed plastic adherent peripheral blood mononuclear cells, or DCs sensitized with GP, NP, VP40, VP35, VP30, VP24, and/or L RNA. T-cells can also be generated from patients immunized with a vaccine vector encoding GP, NP, VP40, VP35, VP30, VP24, and/or L immunogen. HLA A2-presented peptides from GP, NP, VP40, VP35, VP30, VP24, and/or L can further be found in patients infected with Ebola. In various embodiments, the invention relates to an HLA A2 restricted epitope of GP, NP, VP40, VP35, VP30, VP24, and/or L, with ability to stimulate CTLs from patients immunized with vaccine-GP, NP, VP40, VP35, VP30, VP24, and/or L.
Methods of Treatment
The adenovirus vectors of the present invention can be used in a number of vaccine settings for generating an immune response against one or more target antigens as described herein. The present invention provides methods of generating an immune response against any target antigen, such as those described elsewhere herein. The adenovirus vectors are of particular importance because of the unexpected finding that they can be used to generate immune responses in subjects who have preexisting immunity to Ad and can be used in vaccination regimens that include multiple rounds of immunization using the adenovirus vectors, regimens not possible using previous generation adenovirus vectors.
Generally, generating an immune response comprises an induction of a humoral response and/or a cell-mediated response. It may desirable to increase an immune response against a target antigen of interest. Generating an immune response may involve a decrease in the activity and/or number of certain cells of the immune system or a decrease in the level and/or activity of certain cytokines or other effector molecules. A variety of methods for detecting alterations in an immune response (e.g., cell numbers, cytokine expression, cell activity) are known in the art and are useful in the context of the instant invention. Illustrative methods useful in this context include intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T-cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays.
Generating an immune response can comprise an increase in target antigen-specific CTL activity of between 1.5 and 5 fold in a subject administered the adenovirus vectors of the invention as compared to a control. In another embodiment, generating an immune response comprises an increase in target-specific CTL activity of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in a subject administered the adenovirus vectors as compared to a control.
Generating an immune response can comprise an increase in target antigen-specific HTL activity, such as proliferation of helper T-cells, of between 1.5 and 5 fold in a subject administered the adenovirus vectors of the invention that comprise nucleic acid encoding the target antigen as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific HTL activity of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19, 20, or more fold as compared to a control. In this context, HTL activity may comprise an increase as described above, or decrease, in production of a particular cytokine, such as interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, tumor necrosis factor-α (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), or other cytokine. In this regard, generating an immune response may comprise a shift from a Th2 type response to a Th1 type response or in certain embodiments a shift from a Th1 type response to a Th2 type response. In other embodiments, generating an immune response may comprise the stimulation of a predominantly Th1 or a Th2 type response.
Generating an immune response can comprise an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the adenovirus vectors of the present invention as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific antibody production of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in a subject administered the adenovirus vector as compared to a control.
Thus the present invention provides methods for generating an immune response against a target antigen of interest comprising administering to the individual an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) a nucleic acid encoding the target antigen; and readministering the adenovirus vector at least once to the individual; thereby generating an immune response against the target antigen. In certain embodiments, the present invention provides methods wherein the vector administered is not a gutted vector. In particular embodiments, the target antigen may be a wild-type protein, a fragment, a variant, or a variant fragment thereof. In some embodiments, the target antigen comprises GP, NP, VP40, VP35, VP30, VP24, L, a fragment, a variant, or a variant fragment thereof.
In a further embodiment, the present invention provides methods for generating an immune response against a target antigen in an individual, wherein the individual has preexisting immunity to Ad, by administering to the individual an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) a nucleic acid encoding the target antigen; and readministering the adenovirus vector at least once to the individual; thereby generating an immune response against the target antigen. In particular embodiments, the target antigen may be a wild-type protein, a fragment, a variant, or a variant fragment thereof. In some embodiments, the target antigen comprises GP, NP, VP40, VP35, VP30, VP24, L, a fragment, a variant, or a variant fragment thereof.
With regard to preexisting immunity to Ad, this can be determined using methods known in the art, such as antibody-based assays to test for the presence of Ad antibodies. Further, in certain embodiments, the methods of the present invention include first determining that an individual has preexisting immunity to Ad then administering the E2b deleted adenovirus vectors of the invention as described herein.
One embodiment of the invention provides a method of generating an immune response against one or more target antigens in an individual comprising administering to the individual a first adenovirus vector comprising a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and a nucleic acid encoding at least one target antigen; administering to the individual a second adenovirus vector comprising a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and a nucleic acid encoding at least one target antigen, wherein the at least one target antigen of the second adenovirus vector is the same or different from the at least one target antigen of the first adenovirus vector. In particular embodiments, the target antigen may be a wild-type protein, a fragment, a variant, or a variant fragment thereof. In some embodiments, the target antigen comprises GP, NP, VP40, VP35, VP30, VP24, L, a fragment, a variant, or a variant fragment thereof.
Thus, the present invention contemplates multiple immunizations with the same E2b deleted adenovirus vector or multiple immunizations with different E2b deleted adenovirus vectors. In each case, the adenovirus vectors may comprise nucleic acid sequences that encode one or more target antigens as described elsewhere herein. In certain embodiments, the methods comprise multiple immunizations with an E2b deleted adenovirus encoding one target antigen, and re-administration of the same adenovirus vector multiple times, thereby inducing an immune response against the target antigen. In some embodiments, the target antigen comprises GP, NP, VP40, VP35, VP30, VP24, L, a fragment, a variant, or a variant fragment thereof.
In a further embodiment, the methods comprise immunization with a first adenovirus vector that encodes one or more target antigens, and then administration with a second adenovirus vector that encodes one or more target antigens that may be the same or different from those antigens encoded by the first adenovirus vector. In this regard, one of the encoded target antigens may be different or all of the encoded antigens may be different, or some may be the same and some may be different. Further, in certain embodiments, the methods include administering the first adenovirus vector multiple times and administering the second adenovirus multiple times. In this regard, the methods comprise administering the first adenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times and administering the second adenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times. The order of administration may comprise administering the first adenovirus one or multiple times in a row followed by administering the second adenovirus vector one or multiple times in a row. In certain embodiments, the methods include alternating administration of the first and the second adenovirus vectors as one administration each, two administrations each, three administrations each, and so on. In certain embodiments, the first and the second adenovirus vectors are administered simultaneously. In other embodiments, the first and the second adenovirus vectors are administered sequentially. In some embodiments, the target antigen comprises GP, NP, VP40, VP35, VP30, VP24, L, a fragment, a variant, or a variant fragment thereof.
As would be readily understood by the skilled artisan, more than two adenovirus vectors may be used in the methods of the present invention. Three, 4, 5, 6, 7, 8, 9, 10 or more different adenovirus vectors may be used in the methods of the invention. In certain embodiments, the methods comprise administering more than one E2b deleted adenovirus vector at a time. In this regard, immune responses against multiple target antigens of interest can be generated by administering multiple different adenovirus vectors simultaneously, each comprising nucleic acid sequences encoding one or more target antigens.
Methods are also provided for treating or ameliorating the symptoms of Ebola. The methods of treatment comprise administering the adenovirus vectors one or more times to individuals suffering from an Ebola infection or at risk from suffering from an Ebola infection as described herein. As such, the present invention provides methods for vaccinating against Ebola in individuals who are at risk of being infected with such a virus. Individuals at risk may be individuals who may be exposed to Ebola at some time or have been previously exposed but do not yet have symptoms of Ebola infection or being particularly susceptible to an Ebola infection. Individuals suffering from an Ebola infection may be determined to express and/or present a target antigen, which may be use to guide the therapies herein. For example, an example can be found to express and/or present a target antigen and an adenovirus vector encoding the target antigen, a variant, a fragment or a variant fragment thereof may be administered subsequently.
The present invention contemplates the use of adenovirus vectors for the in vivo delivery of nucleic acids encoding a target antigen, or a fragment, a variant, or a variant fragment thereof. Once injected into a subject, the nucleic acid sequence is expressed resulting in an immune response against the antigen encoded by the sequence. The adenovirus vector vaccine can be administered in an “effective amount”, that is, an amount of adenovirus vector that is effective in a selected route or routes of administration to elicit an immune response as described elsewhere herein. An effective amount can induce an immune response effective to facilitate protection or treatment of the host against the target Ebola. The amount of vector in each vaccine dose is selected as an amount which induces an immune, immunoprotective or other immunotherapeutic response without significant adverse effects generally associated with typical vaccines. Once vaccinated, subjects may be monitored to determine the efficacy of the vaccine treatment. Monitoring the efficacy of vaccination may be performed by any method known to a person of ordinary skill in the art. In some embodiments, blood or fluid samples may be assayed to detect levels of antibodies. In other embodiments, ELISpot assays may be performed to detect a cell-mediated immune response from circulating blood cells or from lymphoid tissue cells.
Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, may vary from individual to individual, and from disease to disease, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), in pill form (e.g., swallowing, suppository for vaginal or rectal delivery). In certain embodiments, between 1 and 10 doses may be administered over a 52 week period. In certain embodiments, 6 doses are administered, at intervals of 1 month, and further booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. As such, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may be administered over a 1 year period or over shorter or longer periods, such as over 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 week periods. Doses may be administered at 1, 2, 3, 4, 5, or 6 week intervals or longer intervals.
A vaccine can be infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours. For example, the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs. More generally, the dosage of an administered vaccine construct may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or, the construct may be administered twice per week for 4-6 weeks. The dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule. Compositions of the present invention can be administered to a patient in conjunction with (e.g., before, simultaneously, or following) any number of relevant treatment modalities.
A suitable dose is an amount of an adenovirus vector that, when administered as described above, is capable of promoting a target antigen immune response as described elsewhere herein. In certain embodiments, the immune response is at least 10-50% above the basal (i.e., untreated) level. In certain embodiments, the immune response is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 125, 150, 200, 250, 300, 400, 500 or more over the basal level. Such response can be monitored by measuring the target antigen(s) antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing patient infected cells in vitro, or other methods known in the art for monitoring immune responses. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome of the disease in question in vaccinated patients as compared to non-vaccinated patients. In some embodiments, the improved clinical outcome comprises treating disease, reducing the symptoms of a disease, changing the progression of a disease, or extending life.
In general, an appropriate dosage and treatment regimen provides the adenovirus vectors in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome for the particular disease being treated in treated patients as compared to non-treated patients. The monitoring data can be evaluated over time. The progression of a disease over time can be altered. Such improvements in clinical outcome would be readily recognized by a treating physician. Increases in preexisting immune responses to a target protein can generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
While one advantage of the present invention is the capability to administer multiple vaccinations with the same or different adenovirus vectors, particularly in individuals with preexisting immunity to Ad, the adenoviral vaccines of this invention may also be administered as part of a prime and boost regimen. A mixed modality priming and booster inoculation scheme may result in an enhanced immune response. Thus, one aspect of this invention is a method of priming a subject with a plasmid vaccine, such as a plasmid vector comprising a target antigen of interest, by administering the plasmid vaccine at least one time, allowing a predetermined length of time to pass, and then boosting by administering the adenovirus vector. Multiple primings, e.g., 1-4, may be employed, although more may be used. The length of time between priming and boost may typically vary from about four months to a year, but other time frames may be used. In certain embodiments, subjects may be primed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times with plasmid vaccines, and then boosted 4 months later with the adenovirus vector.
Patient Selection
Various embodiments of the invention relate to compositions and methods for raising an immune response against one or more SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ. ID. NO.:6 antigens in selected patient populations. Any of the compositions provided herein may be administered to an individual. “Individual” may be used interchangeably with “subject” or “patient.” An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep. In embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In some cases, the compositions provided herein are administered to a cell ex vivo. In some cases, the compositions provided herein are administered to an individual as a method of treating an Ebola infection.
In some cases, a subject does not have an Ebola infection. In some cases, the treatment of the present invention is administered before an Ebola infection. A subject may have an undetected Ebola infection. A subject may have a low Ebola infection burden. A subject may also have a high Ebola infection burden.
In some embodiments, patients may be required to have received and, optionally, progressed through other therapies. In some cases, individual's refusal to accept such therapies may allow the patient to be included in a therapy eligible pool with methods and compositions of the invention. In some embodiments, individuals to receive therapy using the methods and compositions of the invention may be required to have an estimated life expectancy of at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 21, or 24 months. The patient pool to receive a therapy using the methods and compositions of the invention may be limited by age. For example, individuals who are older than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 50, 60, or more years old can be eligible for therapy with methods and compositions of the invention. For another example, individuals who are younger than 75, 70, 65, 60, 55, 50, 40, 35, 30, 25, 20, or fewer years old can be eligible for therapy with methods and compositions of the invention.
In some embodiments, patients receiving therapy using the methods and compositions of the invention are limited to individuals with adequate hematologic function, for example with one or more of a WBC count of at least 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more per microliter, a hemoglobin level of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or higher g/dL, a platelet count of at least 50,000; 60,000; 70,000; 75,000; 90,000; 100,000; 110,000; 120,000; 130,000; 140,000; 150,000 or more per microliter; with a PT-INR value of less than or equal to 0.8, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2.0, 2.5, 3.0, or higher, a PTT value of less than or equal to 1.2, 1.4, 1.5, 1.6, 1.8, 2.0×ULN or more. In various embodiments, hematologic function indicator limits are chosen differently for individuals in different gender and age groups, for example 0-5, 5-10, 10-15, 15-18, 18-21, 21-30, 30-40, 40-50, 50-60, 60-70, 70-80 or older than 80.
In various embodiments, samples, for example serum or urine samples, from the individuals or candidate individuals for a therapy using the methods and compositions of the invention may be collected. Samples may be collected before, during, and/or after the therapy for example, within 2, 4, 6, 8, 10 weeks prior to the start of the therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks from the start of the therapy, within 2, 4, 6, 8, 10 weeks prior to the start of the therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 9 weeks, or 12 weeks from the start of the therapy, in 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 9 weeks, or 12 weeks intervals during the therapy, in 1 month, 3 month, 6 month, 1 year, 2 year intervals after the therapy, within 1 month, 3 months, 6 months, 1 year, 2 years, or longer after the therapy, for a duration of 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer. The samples may be tested for any of the hematologic, renal, or hepatic function indicators described herein as well as suitable others known in the art, for example a ß-HCG for women with childbearing potential. In that regard, hematologic and biochemical tests, including cell blood counts with differential, PT, INR and PTT, tests measuring Na, K, Cl, CO2, BUN, creatinine, Ca, total protein, albumin, total bilirubin, alkaline phosphatase, AST, ALT and glucose are within the bounds of the invention. In some embodiments, the presence or the amount of HIV antibody, Hepatitis BsAg, or Hepatitis C antibody are determined in a sample from individuals or candidate individuals for a therapy using the methods and compositions of the invention. Biological markers, such as antibodies to SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, or the neutralizing antibodies to Ad5 vector can be tested in a sample, such as serum, from individuals or candidate individuals for a therapy using the methods and compositions of the invention. In some cases, one or more samples, such as a blood sample can be collected and archived from an individuals or candidate individuals for a therapy using the methods and compositions of the invention. Collected samples can be assayed for immunologic evaluation. Individuals or candidate individuals for a therapy using the methods and compositions of the invention can be evaluated in imaging studies, for example using CT scans or MRI of the chest, abdomen, or pelvis. Imaging studies can be performed before, during, or after therapy using the methods and compositions of the invention, during, and/or after the therapy, for example, within 2, 4, 6, 8, 10 weeks prior to the start of the therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks from the start of the therapy, within 2, 4, 6, 8, 10 weeks prior to the start of the therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 9 weeks, or 12 weeks from the start of the therapy, in 1 week, 10 day, 2 week, 3 week, 4 week, 6 week, 8 week, 9 week, or 12 week intervals during the therapy, in 1 month, 3 month, 6 month, 1 year, 2 year intervals after the therapy, within 1 month, 3 months, 6 months, 1 year, 2 years, or longer after the therapy, for a duration of 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer.
Dosages and Administration
Compositions and methods of the invention contemplate various dosage and administration regimens during therapy. Patients may receive one or more replication defective adenovirus or adenovirus vector, for example Ad5 [E1-, E2B-]-SEQ. ID. NO.:1, Ad5 [E1-, E2b-]-SEQ. ID. NO.:2, Ad5 [E1-, E2b-]-SEQ. ID. NO.:4, Ad5 [E1-, E2b-]-SEQ. ID. NO.:5, Ad5 [E1-, E2b-]-SEQ. ID. NO.:6 that is capable of raising an immune response in an individual against a target antigen described herein, for example SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ ID. NO.6. In various embodiments, the replication defective adenovirus is administered at a dose that suitable for affecting such immune response. In some cases, the replication defective adenovirus is administered at a dose that is greater than or equal to 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 1.5×1012, 2×1012, 3×1012, or more virus particles (VP) per immunization. In some cases, the replication defective adenovirus is administered at a dose that is less than or equal to 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 1.5×1012, 2×1012, 3×1012, or more virus particles per immunization. In various embodiments, a desired dose described herein is administered in a suitable volume of formulation buffer, for example a volume of about 0.1-10 mL, 0.2-8 mL, 0.3-7 mL, 0.4-6 mL, 0.5-5 mL, 0.6-4 mL, 0.7-3 mL, 0.8-2 mL, 0.9-1.5 mL, 0.95-1.2 mL, or 1.0-1.1 mL. Those of skill in the art appreciate that the volume may fall within any range bounded by any of these values (e.g., about 0.5 mL to about 1.1 mL). Administration of virus particles can be through a variety of suitable paths for delivery, for example it can be by injection (e.g., intracutaneously, intramuscularly, intravenously or subcutaneously), intranasally (e.g., by aspiration), in pill form (e.g., swallowing, suppository for vaginal or rectal delivery. In some embodiments, a subcutaneous delivery may be preferred and can offer greater access to dendritic cells.
Administration of virus particles to an individual may be repeated. Repeated deliveries of virus particles may follow a schedule or alternatively, may be performed on an as needed basis. For example, the individual's immunity against a target antigen, for example SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ. ID. NO.:6 may be tested and replenished as necessary with additional deliveries. In some embodiments, schedules for delivery include administrations of virus particles at regular intervals. Joint delivery regimens may be designed comprising one or more of a period with a schedule and/or a period of need based administration assessed prior to administration. For example, a therapy regimen may include an administration, such as subcutaneous administration once every three weeks then another immunotherapy treatment every three months until removed from therapy for any reason including death. Another example regimen comprises three administrations every three weeks then another set of three immunotherapy treatments every three months. Another example regimen comprises a first period with a first number of administrations at a first frequency, a second period with a second number of administrations at a second frequency, a third period with a third number of administrations at a third frequency, etc., and optionally one or more periods with undetermined number of administrations on an as needed basis. The number of administrations in each period can be independently selected and can for example be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. The frequency of the administration in each period can also be independently selected, can for example be about every day, every other day, every third day, twice a week, once a week, once every other week, every three weeks, every month, every six weeks, every other month, every third month, every fourth month, every fifth month, every sixth month, once a year etc. The therapy can take a total period of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36 months or more. The scheduled interval between immunizations may be modified so that the interval between immunizations is revised by up to a fifth, a fourth, a third, or half of the interval. For example, for a 3-week interval schedule, an immunization may be repeated between 20 and 28 days (3 weeks −1 day to 3 weeks +7 days). For the first 3 immunizations, if the second and/or third immunization is delayed, the subsequent immunizations may be shifted allowing a minimum amount of buffer between immunizations. For example, for a three week interval schedule, if an immunization is delayed, the subsequent immunization may be scheduled to occur no earlier than 17, 18, 19, or 20 days after the previous immunization.
Compositions of the invention, such as Ad5 [E1-, E2B-]-SEQ. ID. NO.:1, Ad5 [E1-, E2B-]-SEQ. ID. NO.:2, Ad5 [E1-, E2B-]-SEQ. ID. NO.:4, Ad5 [E1-, E2B-]-SEQ. ID. NO.:5, and Ad5 [E1-, E2B-]-SEQ. ID. NO.:6 vectors and virus particles produced using these vectors, can be provided in various states, for example, at room temperature, on ice, or frozen. Compositions may be provided in a container of a suitable size, for example a vial of 2 mL vial. In one embodiment, 12 ml vial with 1.0 mL of extractable vaccine contains 5×1011 total virus particles/mL. Storage conditions including temperature and humidity may vary. For example, compositions for use in therapy may be stored at room temperature, 4° C., −20° C., or lower.
In various embodiments, general evaluations are performed on the individuals receiving treatment according to the methods and compositions of the invention. One or more of any tests may be performed as needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. A different set of tests may be performed concurrent with immunization vs. at time points without immunization.
General evaluations may include one or more of medical history, ECOG Performance Score, Karnofsky performance status, and complete physical examination with weight by the attending physician. Any other treatments, medications, biologics, or blood products that the patient is receiving or has received since the last visit may be recorded. Patients may be followed at the clinic for a suitable period, for example approximately 30 minutes, following receipt of vaccine to monitor for any adverse reactions. Local and systemic reactogenicity after each dose of vaccine will may be assessed daily for a selected time, for example for 3 days (on the day of immunization and 2 days thereafter). Diary cards may be used to report symptoms and a ruler may be used to measure local reactogenicity. Immunization injection sites may be assessed. CT scans or MRI of the chest, abdomen, and pelvis may be performed.
In various embodiments, hematological and biochemical evaluations are performed on the individuals receiving treatment according to the methods and compositions of the invention. One or more of any tests may be performed as needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. A different set of tests may be performed concurrent with immunization vs. at time points without immunization. Hematological and biochemical evaluations may include one or more of blood test for chemistry and hematology, CBC with differential, Na, K, Cl, CO2, BUN, creatinine, Ca, total protein, albumin, total bilirubin, alkaline phosphatase, AST, ALT, glucose, and ANA.
In various embodiments, biological markers are evaluated on individuals receiving treatment according to the methods and compositions of the invention. One or more of any tests may be performed as needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. A different set of tests may be performed concurrent with immunization vs. at time points without immunization.
Biological marker evaluations may include one or more of measuring antibodies to SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6 or the Ad5 vector, from a serum sample of adequate volume, for example about 5 mL. Biomarkers may be reviewed if determined and available.
In various embodiments, an immunological assessment is performed on individuals receiving treatment according to the methods and compositions of the invention. One or more of any tests may be performed as needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. A different set of tests may be performed concurrent with immunization vs. at time points without immunization.
Peripheral blood, for example about 90 mL may be drawn prior to each immunization and at a time after at least some of the immunizations, to determine whether there is an effect on the immune response at specific time points during the study and/or after a specific number of immunizations. Immunological assessment may include one or more of assaying peripheral blood mononuclear cells (PBMC) for T-cell responses to SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ. ID. NO.:6 using ELISpot, proliferation assays, multi-parameter flow cytometric analysis, and cytotoxicity assays. Serum from each blood draw may be archived and sent and determined.
In various embodiments, an Ebola infection assessment or Ebola replication assay is performed on individuals receiving treatment according to the methods and compositions of the invention. One or more of any tests may be performed as needed or in a scheduled basis, such as prior to treatment, on weeks 0, 3, 6 etc. A different set of tests may be performed concurrent with immunization vs. at time points without immunization. Ebola infection may include one or more Ebola immonospecific tests prior to treatment, at a time after at least some of the immunizations and at approximately every week to three months following the completion of a selected number, for example 2, 3, or 4, of first treatments and for example until removal from treatment.
Immune responses against a target antigen described herein, such as SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:5, or SEQ ID. NO.6 may be evaluated from a sample, such as a peripheral blood sample of an individual using one or more suitable tests for immune response, such as ELISpot, cytokine flow cytometry, or antibody response. A positive immune response can be determined by measuring a T-cell response. A T-cell response can be considered positive if the mean number of spots adjusted for background in six wells with antigen exceeds the number of spots in six control wells by 10 and the difference between single values of the six wells containing antigen and the six control wells is statistically significant at a level of p≤0.05 using the Student's t-test. Immunogenicity assays may occur prior to each immunization and at scheduled time points during the period of the treatment. For example, a time point for an immunogenicity assay at around week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 24, 30, 36, or 48 of a treatment may be scheduled even without a scheduled immunization at this time. In some cases, an individual may be considered evaluable for immune response if they receive at least a minimum number of immunizations, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or more immunizations.
Determination of Clinical Response
In some embodiments, disease progression or clinical response determination is made according to the RECIST 1.1 criteria among patients with measurable/evaluable disease. In some embodiments, therapies using the methods and compositions of the invention affect a Complete Response (CR; disappearance of all antigens or symptoms target sites or disappearance of all non-target sites and normalization of antigens or symptoms level for non-target sites) in an individual receiving the therapy. In some embodiments, therapies using the methods and compositions of the invention affect a Partial Response (PR; at least a 30% decrease in the sum of the LD of target sites, taking as reference the baseline sum LD for target sites) in an individual receiving the therapy.
In some embodiments, therapies using the methods and compositions of the invention affect a Stable Disease (SD; neither sufficient reduction of antigens or symptoms to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started for target sites) in an individual receiving the therapy. In some embodiments, therapies using the methods and compositions of the invention affect an Incomplete Response/Stable Disease (SD; persistence of one or more non-target sites) or/and maintenance of antigens or symptoms above the normal limits for non-target sites) in an individual receiving the therapy. In some embodiments, therapies using the methods and compositions of the invention affect a Progressive Disease (PD; at least a 20% increase in the sum of the LD of antigens or symptoms, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new antigens or symptoms or persistence of one or more non-target antigens or symptoms or/and maintenance of antigens level above the normal limits for in an individual receiving the therapy.
Kits
The compositions, immunotherapy or vaccines may be supplied in the form of a kit. The kits of the present disclosure may further comprise instructions regarding the dosage and or administration including treatment regimen information.
In some embodiments, kits comprise the compositions and methods for providing combination multi-targeted Ebola immunotherapy. In some embodiments, kits comprise the compositions and methods for the combination multi-targeted treatment of an Ebola infection. In some embodiment's kits may further comprise components useful in administering the kit components and instructions on how to prepare the components. In some embodiments, the kit can further comprise software for conducting monitoring patient before and after treatment with appropriate laboratory tests, or communicating results and patient data with medical staff. The components comprising the kit may be in dry or liquid form. If they are in dry form, the kit may include a solution to solubilize the dried material. The kit may also include transfer factor in liquid or dry form. If the transfer factor is in dry form, the kit will include a solution to solubilize the transfer factor. The kit may also include containers for mixing and preparing the components. The kit may also include instrument for assisting with the administration such for example needles, tubing, applicator, inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. The kits or drug delivery systems of the present invention also will typically include a means for containing compositions of the present disclosure in close confinement for commercial sale and distribution.
EXAMPLES Example 1: Multiple Injections of Ad5Null Adenovirus Vector Produces Anti-Adenovirus AntibodiesThis example shows that multiple injections of Ad5-null results in the production of anti-adenovirus antibodies in the injected subjects.
It was demonstrated that the Ad5-null adenovirus vector that does not contain any heterologous nucleic acid sequences, generated a neutralizing immune response in mice. In one experiment, female Balb/c mice aged 5-7 weeks were immunized with Ad5-null viral particles at 14 day intervals. To determine the presence of anti-adenovirus antibodies, an enzyme linked immunosorbent assay (ELISA) was used. For this ELISA, 109 viral particles were coated onto microtiter wells in 100 μL of 0.05M carbonate/bicarbonate buffer, pH 9.6, and incubated overnight at room temperature. For a standard immunoglobulin G (IgG) reference curve, 200 ng, 100 ng, 50 ng, 25 ng, and 0 ng of purified mouse IgG were coated onto microtiter wells as described above. After incubation, all wells were washed 3 times with 250 μL of 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), pH 7.4. After washing, 250 μL of BSA/PBS was added to all and incubated for 30 minutes at room temperature to block unbound sites. After incubation, all wells were washed 3 times with 250 μL of BSA/PBS. After washing, 200 μL of a 1/100 serum dilution in BSA/PBS was added to wells and incubated for 1 hour at room temperature. For a positive control, 200 μL of a 1/10000 dilution of anti-adenovirus antiserum in BSA/PBS was added to wells. Control wells contained BSA/PBS only. After incubation, all wells were washed 3 times with 250 μL of BSA/PBS. After washing, 200 μL of a 1/10000 dilution of peroxidase conjugated γ-chain specific goat anti-mouse IgG (Sigma Chemicals) in BSA/PBS were added to each well and incubated for 1 hour at room temperature. After incubation, all wells were washed 3 times with 250 μL of BSA/PBS. After washing, 200 μL of developing reagent (0.5 mg/mL 1,2-phenylene-diamine in 0.2M potassium phosphate buffer, pH 5.0, containing 0.06% hydrogen peroxide) was added to each well and incubated for 30-40 minutes at room temperature. After incubation, the color reaction was stopped by addition of 50 μL 5N HCl to each well. All wells were then read in a microwell plate reader at 492 nm. After readings were obtained, the optical density readings of unknown samples were correlated with the standard IgG curve to obtain the ngs of IgG bound per well. This was performed using the INSTAT statistical package.
ELISA to Detect Antibodies Against EAs
ELISA plates will be coated with 100 ng of GP, NP, VP40, VP35, VP30, VP24, or L in 0.05 M carbonate-bicarbonate buffer pH 9.6 and incubated overnight at room temperature. Plates were washed three times with phosphate buffered saline containing 1% Tween-20 (PBS-T) and then blocked with PBS containing 1% BSA for 60 min at room temperature. After an additional three washes, serum diluted 1/50 in PBS-T will be added to the wells and the plates will be incubated for 1 hour at room temperature. Peroxidase labeled goat anti-mouse immunoglobulin (Ig) G (γ-chain specific) (Sigma-Aldrich) antibody at a 1:5000 dilution will be added to the wells after washings and plates were incubated for 1 hour. Plates will be washed three times and 1,2-phenylene-diamine substrate solution will be added to each well. The reaction will be stopped by adding 10% phosphoric acid. Absorbance will be measured at 492 nm on a SpectraMax 190 ELISA reader. The nanogram equivalents of IgG bound to GP, NP, VP40, VP35, VP30, VP24, or L, per well will be obtained by reference to a standard curve generated using purified mouse IgG and developed at the same time as the GP, NP, VP40, VP35, VP30, VP24, or L ELISA. The results were analyzed and quantitated using SoftMax Pro 6.3 software.
Significant levels (P<0.001) of anti-adenovirus IgG antibody were detected in mice 2 weeks after a first injection with 1010 Ad-5-null (
To determine the presence of neutralizing antibody to Ad, the following assay was utilized. A HEK-293T-cell line was cultured in 200 μL of culture medium consisting of DMEM containing 10% fetal calf serum (DMEM/FCS) in microwell tissue culture plates at a cell concentration of 2×103 cells per well for 24 hours at 37° C. in 5% CO2. After incubation, 100 μL of culture medium was removed from triplicate wells and mixed with 20 μL of DMEM/FCS containing viral particles (VP). After mixing, the 120 μL mixture was added back to the respective microwells. In another set of triplicate wells, 100 μL of culture medium was removed and mixed with 20 μL of heat inactivated (56° C. for 1 h) Ad immune mouse serum previously incubated with VP for one hour at room temperature. After mixing, the 120 μL mixture was added back to the respective wells. In triplicate cell control wells, 20 μL of DMEM/FCS was added to control for total culture medium volume. Triplicate medium-only control wells contained 220 μL of DMEM/FCS. The tissue culture plate was incubated for an additional 3 days at 37° C. in 5% CO2. After incubation, 40 μL of PROMEGA cell viability reagent (Owen's reagent) was added to all wells and incubated for 75 minutes at 37° C. in 5% CO2. In this assay, the Owen's reagent (MTS tetrazolium compound) is bioreduced by viable cells into a colored formazan product that is soluble in tissue culture medium. The quantity of formazan product as measured by absorbance at 490 nm is directly proportional to the number of living cells in culture. After incubation, 150 μL was removed from each well and transferred to another microwell plate for optical density readings. Optical density readings at 492 nm were subsequently obtained using a microwell plate reader.
To detect the presence of neutralizing antibodies to Ad, groups of 5 mice each were injected once, twice, or three times with 1010 Ad5-null at two week intervals. Two weeks after the final injection of virus, mice were bled, pooled, and assessed for neutralizing antibody as described above using 4×107 VP incubated with or without heat inactivated sera. Cells cultured alone served as a control group. Normal mice and mice injected one time with Ad5null did not exhibit significant levels of neutralizing antibody (
This example shows that the Ad5 [E1-, E2b-] vector platform induces CMI responses against the Ebola associated antigens GP, NP, VP40, VP35, VP30, VP24, and/or L in the presence of pre-existing Ad5 immunity in mice.
Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L will constructed and produced. Briefly, the transgenes will be sub-cloned into the E1 region of the Ad5 [E1-, E2b-] vector using a homologous recombination-based approach. The replication deficient virus will be propagated in the E.C7 packaging cell line, CsCl2 purified, and titered. Viral infectious titer will be determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration will be determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.
Characterization of Ad5 CEA Vectors
Initial studies were performed to confirm GP, NP, VP40, VP35, VP30, VP24, and/or L gene expression of two Ad5-GP, NP, VP40, VP35, VP30, VP24, and/or L vector platforms. It was first determined that the antigens could be expressed on cells transfected with the vaccine vector platforms. A549 cells were obtained from ATCC and transfected with Ad5 [E1-]-GP, Ad5 [E1-]-NP, Ad5 [E1-]-VP40, Ad5 [E1-]-VP35, Ad5 [E1-]-VP30, [E1-]-VP24, and/or [E1-]-L; or Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L. Western blot analysis revealed that cells transfected with the vector platforms expressed the indicated antigens.
Methods
A549 cells were inoculated at a MOI of 555 VPs/cell with Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L. Cells were incubated for 48 hours at 37° C. in 5% CO2 After 48 hours cells were harvested and washed with PBS and freeze/thawed three times. The whole cell lysate was heated for 70° C. for 10 min prior to loading on the gel. Recombinant GP, NP, VP40, VP35, VP30, VP24, and/or L control was loaded at 30 ng/lane and the prepared lysate at 20 μL/lane. Sample loading buffer was included as an additional negative control and the positive controls were Magic Mark CP Western markers and the recombinant GP, NP, VP40, VP35, VP30, VP24, and/or L. The gel was transferred to a nitrocellulose membrane and blocked with SuperBlock Blocking solution for 60 min. The membrane was probed with mouse monoclonal anti-GP, NP, VP40, VP35, VP30, VP24, or L primary antibody (1:1000) and a secondary anti-mouse HRP (1:2500) conjugated antibody. The membrane was washed three times then incubated with SuperSignal chemiluminescent reagent and banding was visualized by exposing X-ray film to the membrane followed by development.
Induction of Ad5 Immunity in Mice
To assess the levels of Ad5 immunity that could be induced, groups of Ad5 naive C57Bl/6 mice will be injected subcutaneously with the Ad5 vector platform (VP). Twenty eight to forty two days later, serum samples will be collected and assessed for endpoint Ad5 NAb titers. Undetectable Ad5 NAb titers (endpoint Ad5 NAb titer <1/25) may be observed in normal control mice. Ad5 NAb (endpoint titers of 1/25 to 1/50) may be detectable after one injection but may dramatically increase after three injections of 1010 Ad5. Therefore, in additional Ad5 immune studies, mice will be injected twice with 1010 Ad5 VP to render the animals Ad5 immune.
Immunization of Ad5 Immune Mice with Ad5 [E1-]-EAs or Ad5 [E1-, E2b-]-EAs.
These experiments will be designed to determine and compare the immunization induction potential of Ad5 [E1-]-EA and Ad5 [E1-, E2b-]-EA vaccines in Ad5 immune mice. Groups of female C57Bl/6 mice, 4 to 8 weeks old, will be immunized 2 times at 2 week intervals with 1010 Ad5-null VP. Two weeks following the last Ad5-null immunization, the mice will be immunized 3 times at weekly intervals with 1010 VP of Ad5 [E1-]-EA or Ad5 [E1-, E2b-]-EA. Two weeks following the last immunization, mice will be euthanized and their spleens and sera harvested for analyses.
CMI responses will be assessed by ELISpot assays performed on splenocytes exposed to intact EA antigen. Splenocytes from Ad5 immune C57Bl/6 mice that were immunized subcutaneously with Ad5 E1-]-EA or Ad5 [E1-, E2b-]-EA will be harvested and assessed for the number of IFN-γ and IL-2 secreting cells. Significantly elevated numbers of both IFN-γ and IL-2 secreting cells may be observed in spleens assayed from mice immunized with Ad5 [E1-, E2b-]-EA as compared to immunized Ad5 [E1-]-EA mice. Immunization of Ad5 immune mice with Ad5 [E1-, E2b-]-CEA may be shown to induce significantly higher CMI responses.
Lack of Adverse Liver Effects in Immunized Mice
Toxicity studies will be performed on serum from Ad5 immune female C57Bl/6 mice immunized with Ad5 [E1-]-EAs, Ad5 [E1-, E2b-]-EAs as described above. Ad5 naive or Ad5 immune mice injected with buffer alone will serve as controls. Three days after the third immunization, aspartate aminotransferase (AST) levels will be assessed on the blood samples to determine liver toxicity due to the treatment. AST levels may not be elevated over controls following immunization with either vector. Alanine aminotransferase (ALT) levels will also be assessed and similar results may be observed.
Ad5 [E1-, E2b-]-CEA Immunotherapy in Ad5 Immune Ebola Infected Mice
Based upon the successful immunological results observed above, studies in which Ebola infections were established in mice and then treated were performed as described below. After Ebola infection, the mice will be treated with the novel Ad5 [E1-, E2b-]-EA vector platform. To determine if Ad5 immune Ebola infected mice could be treated with the Ad5 [E1-, E2b-]-EA vector, C57Bl/6 mice will be injected two times subcutaneously with 1010 Ad5 [E1-]-null VP at 14 day intervals to render the mice Ad5 immune. Two weeks after the last injection, two groups of 7 C57Bl/6 mice will be injected subcutaneously with 106 EA expressing cells. Seven days later, when Ebola infection may be palpable, one group of mice will be treated by distal subcutaneous injection with 1010 VP of Ad5 [E1-, E2b-]-EA on days 7, 13 and 19. A group of injection buffer only treated C57Bl/6 mice will serve as untreated controls. All mice will be monitored for Ebola infection regression over a 21 day period and Ebola virus infection titer will be determined. At the termination of the study, spleens will be collected from mice and the CEA specific CMI response will be determined by ELISpot assay.
Analysis of CMI Responses by ELISpot Assay.
An ELISpot assay for IFN-γ secreting lymphocytes will be performed. Briefly, isolated PBMCs (2×105 cells/well) from individual patient samples will be incubated 36-40 h with a EA peptide pool to stimulate IFN-γ producing T-cells. CMI responses to Ad5 will be determined after exposure of patient PBMC to Ad5-null (empty vector). Cells will be stimulated with concanavalin A (Con A) at a concentration of 0.25 μg/well served as positive controls. Colored spot-forming cells (SFC) will be counted using an Immunospot ELISpot plate reader and responses will be considered to be positive if 50 SFC were detected/106 cells after subtraction of the negative control and SFC were ≥2-fold higher than those in the negative control wells.
Determination of Ad5 Neutralizing Antibody (NAb) Titers.
Endpoint Ad5 NAb titers will be determined. Briefly, dilutions of heat inactivated test sera in 100 μL of DMEM containing 10% fetal calf serum will be mixed with 4×107 VP of Ad5 [E1-]-null and incubated for 60 minutes at room temperature. The samples will be added to microwells containing HEK293 cells cultured in DMEM containing 10% heat inactivated calf serum at 2×103 cells/well for 24 hours at 37° C. in 5% CO2. The mixture will be incubated for an additional 72 hours at 37° C. in 5% CO2. An MTS tetrazolium bioreduction assay will be used to measure cell killing and endpoint Ad5 NAb titers. Endpoint titers with a value less than 1:25 will be assigned a value of 0.
Statistics.
Statistical analyses comparing immune responses will be performed employing the Mann-Whitney test (PRISM, Graph Pad). Survival comparisons will be performed employing Kaplan-Meier plots (PRISM, Graph Pad). Ad5 NAb titer and EA-specific CMI will be analyzed as continuous variables. The association of Ad5 NAb titer with change in EA-specific CMI will be tested with the Spearman correlation coefficient. The association of Ad5 NAb titer with survival will be tested with the Wald test of the proportional hazards model.
A secondary objective will be to evaluate EA specific immune responses following immunization treatments with the product.
Dendritic cells will be generated from the peripheral blood mononuclear cells (PBMCs) of a Ebola infected subject; using PBMCs from this patient post-vaccination, individual T-cell lines specific for EAs, will be attempted to be established. Briefly, PBMCs will be isolated using lymphocyte separation medium gradient, resuspended in AIM-V medium (2×107 cells) and allowed to adhere in a 6-well plate for 2 hours. Adherent cells will be cultured for 5 days in AIM-V medium containing 100 ng/mL of recombinant human (rh) GM-CSF and 20 ng/ml of rhIL-4 The culture medium will be replenished every 3 days.
Example 3: GLP Production of Clinical Grade Multi-Targeted VaccineThis example shows the production of clinical-grade multi-target vaccine using good laboratory practice (GLP) standards. Previously, the Ad5 [E1-, E2b-]-CEA(6D) product was produced using a 5 L Cell Bioreactor under GLP conditions in accordance with good manufacturing practice standards. This example may show that the Ad5 [E1-, E2b-]-EA products GP, NP, VP40, VP35, VP30, VP24, and L can be produced in a 5 L Cell Bioreactor using a similar approaches.
Briefly, vials of the E.C7 manufacturing cell line will be thawed, transferred into a T225 flasks, and initially cultured at 37° C. in 5% CO2 in DMEM containing 10% FBS/4 mM L-glutamine. After expansion, the E.C7 cells will be expanded using 10-layered CellSTACKS (CS-10) and transitioned to FreeStyle serum-free medium (SFM). The E.C7 cells will be cultured in SFM for 24 hours at 37° C. in 5% CO2 to a target density of 5×105 cells/mL in the Cell Bioreactor. The E.C7 cells will then be infected with Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L, respectively, and cultured for 48 hours.
Mid-stream processing will be performed in an identical manner as that used to prepare clinical grade Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L product. 30 minutes before harvest, Benzonase nuclease will be added to the culture to promote better cell pelleting for concentration. After pelleting by centrifugation, the supernatant will be discarded and the pellets re-suspended in Lysis Buffer containing 1% Polysorbate-20 for 90 min at room temperature. The lysate will then be treated with Benzonase and the reaction quenched by addition of 5 M NaCl. The slurry will be centrifuged and the pellet discarded. The lysate will be clarified by filtration and subjected to a two-column ion exchange procedure.
To purify the vaccine products, a two-column anion exchange procedure will be performed. A first column will be packed with Q Sepharose XL resin, sanitized, and equilibrated with loading buffer. The clarified lysate will be loaded onto the column and washed with loading buffer. The vaccine product will be eluted and the main elution peak (eluate) containing Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L carried forward to the next step. A second column will be packed with Source 15Q resin, sanitized, and equilibrated with loading buffer. The eluate from the first anion exchange column will be loaded onto the second column and the vaccine product eluted with a gradient starting at 100% Buffer A (20 mM Tris, 1 mM MgCl2, pH 8.0) running to 50% Buffer B (20 mM Tris, 1 mM MgCl2, 2 M NaCl, pH 8.0). The elution peak containing Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and/or [E1-, E2b-]-L will be collected and stored overnight at 2-8° C. The peak elution fraction will be processed through a tangential flow filtration (TFF) system for concentration and diafiltration against formulation buffer (20 mM Tris, 25 mM NaCl, 2.5% (v/v) glycerol, pH 8.0). After processing, the final vaccine product will be sterile filtered, dispensed into aliquots, and stored at ≤−60° C. A highly purified product approaching 100% purity is typically produced and similar results for these products are predicted.
The concentration and total number of VP product produced will be determined spectrophotometrically. Product purity will be assessed by HPLC. Infectious activity will be determined by performing an Ad5 hexon-staining assay for infectious particles using kits.
Western blots will be performed using lysates from vector transfected cells to verify EA expression. Quality control tests will be performed to determine that the final vaccine products are mycoplasma-free, have no microbial bioburden, and exhibit endotoxin levels less than 2.5 endotoxin units (EU) per mL. To confirm immunogenicity, the individual vectors will be tested in mice.
Example 7: Immunogenicity of Single and Multi-Targeted GP, NP, VP40, VP35, VP30, VP24, L Viral VectorsFemale C57BL/6 mice will be injected s.c. with 1010 VP of Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, or [E1-, E2b-]-L, or a combination of 1010 VP of two or more viruses at a ratio of 1:1:1. Control mice will be injected with 3×1010 VP of Ad-null (no transgene insert). Doses will be administered in 25 μl of injection buffer (20 mM HEPES with 3% sucrose) and mice will be vaccinated three times at 14-day intervals. Fourteen days after the final injection spleens and sera will be collected. Sera will be frozen at −20° C. Splenocyte suspensions will be generated by gently crushing the spleens through a 70 μM nylon cell strainer (BD Falcon, San Jose, Calif.). Red cells will be removed by the addition of red cell lysis buffer (Sigma-Aldrich, St. Louis, Mo.) and the splenocytes will be washed twice and resuspended in R10 (RPMI 1640 supplemented with L-glutamine (2 mM), HEPES (20 mM), penicillin 100 U/mL and streptomycin 100 μg/ml, and 10% fetal bovine serum. Splenocytes will be assayed for cytokine production by ELISPOT and flow cytometry.
Immunogenicity Studies:
Two weeks after the last immunization, CMI activity will be determined employing ELISpot assays for IFN-γ secreting cells (SFC) after exposure of splenocytes to EA peptide pools.
Briefly, CMI responses against EAs as assessed by ELISpot assays for IFN-γ secreting splenocytes (SFC) will be detected in multi-targeted immunized mice but not control mice (injected with Ad5-Null empty vector). Specificity of the ELISpot assay responses will be confirmed by lack of reactivity to irrelevant SIV-nef or SIV-vif peptide antigens. A positive control will include cells exposed to concanavalin A (Con A).
ELISPOT Assay
GP, NP, VP40, VP35, VP30, VP24, or L-specific IFN-γ- or IL-2-secreting T cells will be determined by ELISPOT assay from freshly isolated mouse splenocytes. Briefly, 2×105 splenocytes will be stimulated with 0.2 μg/well of overlapping 15-mer peptides in a single pool derived from GP, NP, VP40, VP35, VP30, VP24, or L. Cells will be stimulated with Con A at a concentration of 0.0625 μg/per well as a positive control and overlapping 15-mer complete peptides pools derived from SIV-Nef and SIV-Vif will be used as irrelevant peptide controls. The numbers of SFCs will be determined using an Immunospot ELISpot plate reader and results will be reported as the number of SFCs per 106 splenocytes.
To determine the level of complement dependent cellular cytotoxicity (CDCC), a CDCC test will be performed using EA target cells.
Intracellular Cytokine Stimulation
Splenocytes will be prepared. Stimulation assays will be performed using 1×106 live splenocytes per well in 96-well U-bottom plates. Pools of overlapping peptides spanning the entire coding sequences of GP, NP, VP40, VP35, VP30, VP24, and L will be synthesized as 15-mers with 11-amino acid overlaps and lyophilized peptide pools will be dissolved in DMSO. Similarly constructed peptide pools corresponding to SIV-Vif and SIV-Nef will serve as off-target controls. Splenocytes in R10 media (RPMI 1640, 10% fetal bovine serum, and antibiotics) will be stimulated by the addition of peptide pools at 2 μg/mL/peptide for 6 h at 37° C. and 5% CO2, with protein transport inhibitor (GolgiStop) added 2 h into the incubation. Stimulated splenocytes will then be stained for lymphocyte surface markers CD8α and CD4, fixed, permeabilized, and then stained for the intracellular accumulation of IFN-γ and TNFα. Antibodies against mouse CD8α, CD4, IFN-γ, and TNFα will be used and staining was performed in the presence of anti-CD16/CD32. Flow cytometry will be performed and analyzed in BD Accuri C6 Software.
Complement-Dependent Cytotoxicity Assay (CDC)
EA cells will be cultured overnight at a density of 2×104 cells per well in 96-well tissue culture microplates. Pooled heat inactivated mouse sera will be added at a 1:50 dilution and incubated at 37° C. for 1 hour. Rabbit serum will then be added at a 1:50 dilution as a source of complement and cells will be incubated an additional 2.5 hours at 37° C. Cell culture supernatants will be assayed using Promega Cytotox 96 non-radioactive cytotoxicity assay, according to the manufacturer's instructions. Percent lysis of EA cells will be calculated by the formula % lysis=(experimental−target spontaneous)/(target maximum−target spontaneous)×100%.
Anti-Ebola Immunotherapy Studies:
Studies will be conducted to test the anti-Ebola capability of Ad5 [E1-, E2b-]-based multi-vaccines in immunotherapy studies in mice with established Ebola infections. In this study the anti-Ebola activity of the individual components of the Ad5 [E1-, E2b-]-based multi-vaccine will be assessed.
Groups of C57Bl/6 mice will be injected subcutaneously in the right flank with 5×105 EA expressing cells. Mice will be treated by 3 subcutaneous injections with 1×1010 VP each of Ad5 [E1-, E2b-]-null (no transgene, e.g., empty vector), Ad5 [E1-, E2B-]-GP, Ad5 [E1-, E2B-]-NP, Ad5 [E1-, E2B-]-VP40, Ad5 [E1-, E2B-]-VP35, Ad5 [E1-, E2b-]-VP30, [E1-, E2b-]-VP24, and [E1-, E2b-]-L, respectively.
Example 8: Use of Ebola Virus GP and NP as Vaccine TargetsEarlier generation recombinant Ad5 based vector (Ad5 [E1-]) vaccines containing Ebola virus gene components, including GP and/or NP have been constructed, produced, and tested in a laboratory setting. Promising pre-clinical protective effects have been obtained in mice and non-human primates (NHP) using these vaccines but the effectiveness was negated when animals exhibited pre-existing or Ad5 vector induced immunity to adenovirus.
In this example, GP and NP components of the Ebola virus will be used in a vaccine. Since the Zaire and Sudan strains are responsible for the most species-specific case fatalities, virus components from these two strains will be initially used. GP will be employed because it is a surface glycoprotein that can be targeted. GP genes from both isolate strains of Ebola will be used in order to induce broadly reactive immune responses against Ebola. Since NP associates with VP35, VP30, and RNA-dependent RNA polymerase to the functional transcriptase-replicase complex, it will be used in the vaccine to induce immune responses that interfere and/or prevent virus replication. In this manner, a broadly reactive vaccine based upon an Ad5 [E1-, E2b-] platform that will induce humoral and cell-mediated immune (CMI) responses against Ebola will be developed.
Use of Recombinant Ad5 [E1-, E2b-]-Based Vectors as Vaccines.
The vaccine will be delivered directly by subcutaneous injection for exposure of defined Ebola antigens to antigen-presenting cells (APC) that induce potent immune responses. Amplification of Ad5 [E1-, E2b-]-based vector Ebola vaccines.
Four Ad5 [E1-, E2b-]-based vaccines were constructed (
For the Sudan strain vaccine, the nucleotide sequences of GP and NP of a Sudan strain of Ebola virus (Sudan Ebola virus isolate EboSud-682 2012, complete genome GenBank: KC545392.1) were optimized to human codon usage and cloned into the Ad5 [E1-, E2b-] vector under the regulation of CMV promoter. Viral particles were rescued by transfecting E.C7 cells that stably express adenoviral E1 and E2b genes with linearized Ad5 [E1-, E2b-] plasmid constructs, producing an Ad5 [E1-, E2b-]-GPES product and an Ad5 [E1-, E2b-]-NPES product.
Production, Purification, and Testing of Recombinant Ad5 [E1-, E2b-]-Based Vaccines.
Additional vaccines will be prepared for later studies with NHP. The E.C7 cell line allows Ad5 [E1-, E2b-] vectors to grow with high and reproducible yields. The Ad5 [E1-, E2b-] vectors will be manufactured and produced by release from E.C7 human cells using Triton X-100, purification on CsCl gradients or ion exchange chromatography, and dialysis against 20 mM HEPES (pH 7.4) containing 5% sucrose. The purified recombinant Ad5 [E1-, E2b-]-based virus vaccines will be aliquoted and frozen in a dry ice-ethanol bath. The infectivity of the virus particles is measured using a plaque assay. Virus particle (VP) concentrations will be calculated from the absorbance at 260 nm and used to determine the amounts of virus for in vivo immunizations. Western blots from transfected A549 cell lysates will verify antigen transgene expression. An example of Ebola GP (Zaire strain) expression in a cell lysate of E.C7 cells after 24 hours transfection with Ad5 [E1-, E2b-]-GPEZ is shown in
Dose Escalation Immunogenicity Study.
These studies will be performed using a clinically relevant Ad5 immune mouse model. BALB/c mice will be made Ad5-immune by two subcutaneous injections with 1010 VPs of Ad5-null at two-week intervals. Two weeks after the second injection, sera will be tested for Ad5 neutralizing Ab (NAb) to verify that the mice are Ad5-immune. Mice and monkeys treated in this manner achieve Ad5 NAb titers of 1/100 to 1/200 and in a clinical trial the mean pre existing Ad5 NAb titer among all patients was 1:189±1:71 (mean±SEM). Immune responses that are induced after immunization with increasing doses of Ad5 [E1-, E2b-]-GPEZ/NPEZ and Ad5 [E1-, E2b-]-GPES/NPES (an equally combined mixture) will be assessed. Groups of Ad5 immune mice (n=5/group) will be immunized twice by subcutaneous route every two weeks using escalating doses of 4×108, 4×109, or 4×1010 VPs (a vaccine mixture containing equal amounts of Ad5 [E1-, E2b-]-GPEZ/NPEZ and Ad5 [E1-, E2b-]-GPES/NPES). Control mice (n=5) will be injected with 4×1010 VPs of Ad5 [E1-, E2b-]-null on the same schedule. Fourteen days after the final immunization, antibody (Ab) and cell-mediated immune (CMI) responses will be assessed. For CMI evaluation, splenocytes from individual mice will be assessed using previously described ELISpot assays to measure the number of interferon-γ (IFN-γ) and IL-2-secreting lymphoid cells after exposure to GP or NP peptide pools. Serum samples will be assessed for Ab activity using purified commercially available.
Using the highest immunogenic dose, a study employing one or two subcutaneous immunizations will be performed to determine if one or two doses are required for effective vaccination. Groups of mice (n=5/group) will be immunized one time or twice every two weeks using the most effective dose of vaccine. Control mice (n=5) will be injected with Ad5 [E1-, E2b-]-null (VP quantity same as vaccine) on the same schedule. Fourteen days after the final immunization, Ab and CMI responses will be assessed as described above. These studies will allow determination of whether or not one or two immunizations are required to induce significant immune responses.
Immunogenicity Studies in Ad5 Immune Mice by ELISA, ELISpot, and Flow Cytometry.
For these studies, the most effective dose and frequency of vaccination determined above will be used. In a proof-of-concept study in monkeys with influenza vaccine, it was observed that Ab responses, as assessed by hemagglutination inhibition (HAI) assays, might require up to 30 days to develop maximum levels (
Based upon the above studies in which the highest CMI responses are observed, flow cytometry studies will also be performed to characterize T-cell immune responses induced by immunization with the vaccines. Ad5-immune BALB/c mice (n=5) will be immunized with the vaccine using the most effective dose and frequency of vaccination. Control mice (n=5) will be injected with Ad5 [E1-, E2b-]-null (VP quantity same as vaccine) on the same schedule. Based upon the optimal time after vaccination, spleens will be harvested and CD4+ and CD8+ T cells will be determined by flow cytometry for IFN-γ and/or TNF-α expressing cells after exposure to GP or NP peptide pools. Briefly, splenocytes from immunized and control mice will be harvested and incubated for 5 hrs with 0.5 μg/ml of GP or NP peptide pools, in the presence of GolgiStop, a protein transport inhibitor. The CD4+ or CD8+ T cells will then be fixed, permeabilized, stained for IFN-γ or TNF-α, and analyzed by flow cytometry.
Protection of Vaccinated Ad5 Immune Mice Against Virus Challenge.
In a proof-of-principle test, a challenge study will be performed in mice. Two groups of BALB/c mice (n=15/group) will be made Ad5-immune as described above. Using the most effective dose and frequency for vaccination, one group will be immunized with the vaccine (vaccine mixture containing equal amounts of Ad5 [E1-, E2b-]-GPEZ/NPEZ and Ad5 [E1-, E2b-]-GPES/NPES). A control group will be injected with Ad5 [E1-, E2b-]-null (VP quantity same as vaccine) on the same schedule. When induced virus neutralizing titers are achieved, the mice will be challenged by inoculations with a lethal mouse adapted strain of Ebola. Weight loss and time to death for the endpoints will be monitored. Mice will be observed daily for clinical signs of morbidity for over 21 days after challenge.
Phase 2 Studies
In phase 2 studies, the immunogenicity of the Ebola vaccines in NHP will be evaluated. The appropriate immunizing dose, boosting schedule, and live Ebola challenge potential will be determined. In addition, the Ad5 [E1-, E2b-]-based platform containing fused GP and NP genes from the Zaire and Sudan strains of Ebola virus will be evaluated. In this manner two recombinant vectors will be produced, an Ad5 [E1-, E2b-]-GPEZ/NPEZ product and an Ad5 [E1-, E2b-]-GPES/NPES product. By introducing multiple genes into one recombinant Ad5 [E1-, E2b-]-based vector, the number of viral particles that would be required will be significantly cut down compared to producing and combining each of 4 individual recombinant vectors.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
Claims
1. A composition comprising an adenovirus 5 vector comprising a nucleotide sequence encoding a first antigen, wherein the first antigen is a glycoprotein (GP), wherein the adenovirus 5 vector comprises a deletion in an E1 gene region and a deletion in an E2b gene region, and wherein the GP is from an Ebola selected from the group consisting of Zaire ebolavirus (EBOV), Sudan virus (SUDV), Tai Forest ebolavirus (TAFV), Bundibugyo ebolavirus (BDBV), Reston virus (RESTV), and any combination thereof.
2. The composition of claim 1, wherein the adenovirus 5 vector further comprises a deletion in an E3 gene region, an E4 gene region, or any combination thereof.
3. The composition of claim 1, wherein the adenovirus 5 vector is a replication defective adenovirus 5 vector.
4. The composition of claim 1, further comprising an immunogenic component, wherein the immunogenic component comprises a cytokine selected from the group consisting of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13.
5. The composition of claim 4, wherein the immunogenic component is IL-7.
6. The composition of claim 3, wherein the replication defective adenovirus vector is capable of generating an immune response to the GP following one round, two rounds, or multiple rounds of immunization with the same type of replication defective adenovirus vector.
7. The composition of claim 1, wherein the adenovirus 5 vector is capable of inducing an immune response against cells expressing the Ebola virus antigen in a human that is at least 2 fold over basal, as measured as an Ebola virus antigen specific antibody response, as a neutralizing Ebola virus antigen specific antibody response, as Ebola virus antigen specific cell-mediated immunity (CMI), as Ebola virus antigen specific IFN-γ secretion, as Ebola virus antigen specific IL-2 secretion, by ELISpot assay, or by any combination thereof.
8. The composition of claim 7, wherein the immune response is measured as an Ebola virus antigen specific antibody response.
9. The composition of claim 1, further comprising a nucleotide encoding an immunological fusion partner.
10. The composition of claim 8, wherein the immunological fusion partner comprises a Mycobacterium tuberculosis-derived Ra12 fragment, protein D fragment, NS1, or LYTA.
11. The composition of claim 1, wherein the adenovirus 5 vector comprises a nucleotide encoding a second antigen.
12. The composition of claim 11, wherein the adenovirus 5 vector comprises a nucleotide encoding a third antigen.
13. The composition of claim 11, wherein the second antigen is selected from NP, VP40, VP35, VP30, VP24, and L.
14. The composition of claim 12, wherein the third antigen is selected from NP, VP40, VP35, VP30, VP24, and L.
15. The composition of claim 1 in a single dose form comprising 5.0×1011 virus particles (VPs).
16. A method of generating an immune response against an Ebola virus antigen in a human comprising:
- (a) a first phase of treatment comprising administering to the human a first composition comprising the composition of claim 1; and
- (b) a subsequent second phase of treatment comprising administering to the human a second composition comprising a second replication defective adenovirus vector encoding an Ebola virus antigen that induces an immune response against cells expressing the Ebola virus antigen in the human.
17. The method of claim 16, wherein the first phase is at least 2 weeks.
18. The method of claim 16, wherein the second phase is at least 2 months.
19. The method of claim 16, wherein the second phase starts 3-16 weeks after first phase ends.
20. The method of claim 16, wherein in the first phase two administrations of the composition are at least 18 days apart.
21. The method of claim 20, wherein in the first phase two administrations of the composition are about 21 days apart.
22. The method of claim 20, wherein in the first phase two administrations of the composition are at most 24 days apart.
23. The method of claim 16, wherein in the second phase two administrations of the replication defective adenovirus are at least 10 weeks apart.
24. The method of claim 23, wherein in the second phase two administrations of the replication defective adenovirus are about 13 weeks apart.
25. The method of claim 23, wherein in the second phase two administrations of the replication defective adenovirus are at most 16 weeks apart.
| 5145684 | September 8, 1992 | Liversidge et al. |
| 5466468 | November 14, 1995 | Schneider et al. |
| 5552157 | September 3, 1996 | Yagi et al. |
| 5565213 | October 15, 1996 | Nakamori et al. |
| 5567434 | October 22, 1996 | Szoka, Jr. |
| 5738868 | April 14, 1998 | Shinkarenko |
| 5795587 | August 18, 1998 | Gao et al. |
| 6045802 | April 4, 2000 | Schlom et al. |
| 6057158 | May 2, 2000 | Chamberlain et al. |
| 6063622 | May 16, 2000 | Chamberlain et al. |
| 6083750 | July 4, 2000 | Chamberlain et al. |
| 6348450 | February 19, 2002 | Tang et al. |
| 6451596 | September 17, 2002 | Chamberlain et al. |
| 6544947 | April 8, 2003 | Holaday et al. |
| 6706693 | March 16, 2004 | Tang et al. |
| 6716823 | April 6, 2004 | Tang et al. |
| 6756038 | June 29, 2004 | Schlom et al. |
| 7211569 | May 1, 2007 | Neeper et al. |
| 7410758 | August 12, 2008 | Sastry et al. |
| 7488482 | February 10, 2009 | Balloul et al. |
| 7547681 | June 16, 2009 | Scholler et al. |
| 7662586 | February 16, 2010 | Monaci et al. |
| 7723096 | May 25, 2010 | Schlom et al. |
| 7771715 | August 10, 2010 | Schlom et al. |
| 7786278 | August 31, 2010 | Parrington et al. |
| 7999071 | August 16, 2011 | Schlom et al. |
| 8012468 | September 6, 2011 | Kim et al. |
| 8017590 | September 13, 2011 | Berinstein et al. |
| 8188244 | May 29, 2012 | La Monica et al. |
| 8207314 | June 26, 2012 | Berinstein et al. |
| 8298549 | October 30, 2012 | Balint et al. |
| 8609395 | December 17, 2013 | Schlom et al. |
| 9248177 | February 2, 2016 | Tang et al. |
| 9605276 | March 28, 2017 | Jones et al. |
| 20040091995 | May 13, 2004 | Schlom et al. |
| 20040265274 | December 30, 2004 | Wei et al. |
| 20050037439 | February 17, 2005 | Bourner et al. |
| 20060104986 | May 18, 2006 | Duke et al. |
| 20070104685 | May 10, 2007 | La Monica et al. |
| 20100055069 | March 4, 2010 | Rooke et al. |
| 20100183673 | July 22, 2010 | Balint et al. |
| 20100209386 | August 19, 2010 | Schlom et al. |
| 20100285065 | November 11, 2010 | Parrington et al. |
| 20130224144 | August 29, 2013 | Balint et al. |
| 20130251741 | September 26, 2013 | Pietersz et al. |
| 20140212434 | July 31, 2014 | Blaney et al. |
| 20140377294 | December 25, 2014 | Fueyo-Margareto et al. |
| 20150086541 | March 26, 2015 | Aguilar-Cordova et al. |
| 20150182621 | July 2, 2015 | Wu et al. |
| 20150352198 | December 10, 2015 | Berinstein et al. |
| 20150374790 | December 31, 2015 | Liu et al. |
| 20170065693 | March 9, 2017 | Balint et al. |
| 20170065706 | March 9, 2017 | Balint et al. |
| 20170165341 | June 15, 2017 | Jones et al. |
| 1017810 | May 2004 | EP |
| 1447414 | June 2007 | EP |
| 1227837 | May 2008 | EP |
| 1015035 | January 2009 | EP |
| 2465520 | June 2012 | EP |
| WO-9614876 | May 1996 | WO |
| WO-0034494 | June 2000 | WO |
| WO-0208436 | January 2002 | WO |
| WO-03008649 | January 2003 | WO |
| WO-2005012527 | February 2005 | WO |
| WO-2005051991 | June 2005 | WO |
| WO-2005058937 | June 2005 | WO |
| WO-2005058950 | June 2005 | WO |
| WO-2006033672 | March 2006 | WO |
| WO-2006044923 | April 2006 | WO |
| WO-2006033672 | June 2006 | WO |
| WO-2007008780 | January 2007 | WO |
| WO-2007008780 | March 2007 | WO |
| WO-2007092792 | August 2007 | WO |
| WO-2009006479 | January 2009 | WO |
| WO-2009006479 | March 2009 | WO |
| WO-2010033841 | March 2010 | WO |
| WO-2010065626 | June 2010 | WO |
| WO-2010121180 | October 2010 | WO |
| WO-2011032119 | March 2011 | WO |
| WO-2011115914 | September 2011 | WO |
| WO-2012019127 | February 2012 | WO |
| WO-2012109404 | August 2012 | WO |
| WO-2012174220 | December 2012 | WO |
| WO-2013025972 | February 2013 | WO |
| WO 2014/031178 | February 2014 | WO |
| WO-2014031178 | February 2014 | WO |
| WO-2014043518 | March 2014 | WO |
| WO-2014160747 | October 2014 | WO |
| WO-2015031778 | March 2015 | WO |
| WO-2015061416 | April 2015 | WO |
| WO-2015103602 | July 2015 | WO |
| WO-2015127027 | August 2015 | WO |
| WO-2015157639 | October 2015 | WO |
| WO-2016007499 | January 2016 | WO |
| WO-2016112188 | July 2016 | WO |
| WO-2016112195 | July 2016 | WO |
| WO-2016172249 | October 2016 | WO |
- Audet, J., et al., Nov. 2014, Molecular characterization of the monoclonal antibodies composing ZMAb: A protective cocktail against Ebola virus, Sci. Reports 4(6881):1-8 (DOI:10.1038/srep06881).
- Dube, D., et al., Apr. 2009, The primed ebolavirus glycoprotein (19-kilodalton GP1,2): Sequence residues critical for host binding, J. Virol. 83(7):2883-2891.
- Amalfitano, A. Use of multiply deleted adenovirus vectors to probe adenovirus vector performance and toxicities. Curr Opin Mol Ther. Aug. 2003;5(4):362-6.
- Amalfitano, et al. Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy. Curr Gene Ther 2:111-133 (2002).
- Amara, et al. A new generation of HIV vaccines. Trends Mol Med 8;489-95 (2002).
- Appledorn, et al. (2008) Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo. J Immunol. 181:2134-2144.
- Appledorn, et al. (2008) Wild-type adenoviruses from groups A-F evoke unique innate immune responses, of which HAd3 and SAd23 are partially complement dependent. Gene Ther. 15:885-901.
- Balint, et al. Extended evaluation of a phase ½ trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA (6D) vaccine in late-stage colorectal cancer. Cancer Immunology, Immunotherapy 64.8 (2015): 977-987.
- Bangari, et al. (2006) Development of nonhuman adenoviruses as vaccine vectors. Vaccine 24:849-862.
- Bangari, et al. Current strategies and future directions for eluding adenoviral vector immunity. Curr Gene Ther. Apr. 2006; 6(2):215-226.
- Barjot, et al. Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses. J Gene Med 4;480-9 (2002).
- Barouch, et al. (2011) International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine 29:5203-5209.
- Barouch, et al. Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum Gene Ther. 16:149-156 (2005).
- Barratt-Boyes, et al. Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors. J Gen Virol 87:.Pt 1 139-149 (2006).
- Bewig, et al. (2000) Accelerated titering of adenoviruses. BioTechniques 28:871-873.
- Brave, et al. Vaccine delivery methods using viral vectors. Mol Pharm 4:.1 18-32 (2007).
- Campos, et al. (2007) Current advances and future challenges in adenoviral vector biology and targeting. Curr Gene Ther 7:189-204.
- Chamberlain, et al. Packaging cell lines for generating replication-defective and gutted adenoviral vectors. Methods Mol Med 76;153-66 (2003).
- Co-pending U.S. Appl. No. 15/564,413, filed Oct. 4, 2017.
- Dellorusso, et al. Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin. Proc Natl Acad Sci U S A. Oct. 1, 2002;99(20):12979-84. Epub Sep. 23, 2002.
- Ding, et al. Long-term efficacy after [E1-, polymerase-] adenovirus-mediated transfer of human acid-alpha-glucosidase gene into glycogen storage disease type II knockout mice. Hum Gene Ther 12;955-65 (2001).
- Eo, et al. Prime-boost immunization with DNA vaccine: mucosal route of administration changes the rules. J Immunol 166;5473-9 (2001).
- Evans, et al. Development of stable liquid formulations for adenovirus-based vaccines. J Pharm Sci. Oct. 2004;93(10):2458-75.
- Everett, et al. Liver toxicities typically induced by first-generation adenoviral vectors can be reduced by use of E1, E2b-deleted adenoviral vectors. Hum Gene Ther, 2003. 14(18): p. 1715-1726.
- Everett, et al. Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors. Virology. Jul. 20, 2004; 325(1):96-105.
- Gabaglia CR, Sercarz EE, Diaz-De-Durana Y, Hitt M, Graham FL, Gauldie J, and Braciak TA. Life-long systemic protection in mice vaccinated with L. major and adenovirus IL-12 vector requires active infection, macrophages and intact lymph nodes.Vaccine 23:.2 247-257 (2004).
- Gabitzsch, et al. (2009) Novel adenovirus type 5 vaccine platform induces cellular immunity aginst HIV-Gag, Pol, Nef despite the presence of Ad5 immunity. Vaccine 27:6394-6398.
- Gabitzsch, et al. (2010) Anti-tumor immunity despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA. Cancer Immunol Immunother 59:1131-1135.
- Gabitzsch, et al. (2011) Induction and Comparison of SIV immunity in Ad5 Naïve and Ad5 Immune Non-human Primates using an Ad5 [E1-, E2b-] based vaccine. Vaccine 29:8101-8107.
- Gabitzsch, et al. (2011) New Recombinant Ad5 Vector Overcomes Ad5 Immunity Allowing for Multiple Safe, Homologous Immunizations. J Clin Cell Immunol. S4:001. doi:10.4172/2155-9899.S4-001.
- Gabitzsch, et al. (2012) Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform. Vaccine 2012; 30:7265-7270.
- Gabitzsch, et al. The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic. Oncotarget. Oct. 13, 2015; 6(31): 31344-31359.
- Gao et al. Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization. J Virol, 2006. 80(4): p. 1959-1964.
- Garnett, et al. TRICOM vector based cancer vaccines. Curr Pharm Des. 2006;12(3):351-61.
- Gomez-Roman, et al. Adenoviruses as vectors for HIV vaccines. AIDS Rev 5;178-85 (2003).
- Gulley, et al. (2008) Pilot study of vaccination with recombinant CEA-MUC-1-TRICOM poxviral-based vaccines in patients with metastatic carcinoma. Clin Cancer Res. 14:3060-3069.
- Haglund, et al. Robust recall and long-term memory T-cell responses induced by prime-boost regimens with heterologous live viral vectors expressing human immunodeficiency virus type 1 Gag and Env proteins. J Virol 76;7506-17 (2002).
- Harris, et al. (2002) Acute Regression of Advanced and Retardation of Early Aortic Atheroma in Immunocompetent Apolipoprotein-E (Apoe) Deficient Mice by Administration of a Second Generation [E1(-), E3(-), Polymerase(-)] Adenovirus Vector Expressing Human Apoe. Human Molecular Genetics 11:43-58.
- Hartigan-O'Connor, et al. Developments in gene therapy for muscular dystrophy. Microsc Res Tech 48;223-38 (2000).
- Hartigan-O'Connor, et al. Efficient rescue of gutted adenovirus genomes allows rapid production of concentrated stocks without negative selection. Hum Gene Ther. Mar. 1, 2002;13(4):519-31.
- Hartigan-O'Connor, et al. Generation and growth of gutted adenoviral vectors. Methods Enzymol 346;224-46 (2002).
- Hartigan-O'Connor, et al. Immune evasion by muscle-specific gene expression in dystrophic muscle. Mol Ther. Dec. 2001;4(6):525-33.
- Hartman, et al. (2008) Adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications. Virus Res 132:1-14.
- Hartman, et al. Adenoviral infection induces a multi-faceted innate cellular immune response that is mediated by the toll-like receptor pathway in A549 cells. Virology. Feb. 20, 2007;358(2):357-72. Epub Oct. 5, 2006.
- Hartman, et al. Adenovirus infection triggers a rapid, MyD88-regulated transcriptome response critical to acute-phase and adaptive immune responses in vivo. J Virol. Feb. 2007;81(4):1796-812. Epub Nov. 22, 2006.
- Harui, et al. 2004. Vaccination with helper-dependent adenovirus enhances the generation of transgene-specific CTL. Gene Ther 11:1617-1626.
- Hauser, et al. Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors. Mol Ther 2;16-25 (2000).
- Hirschowitz, et al. 2000. Murine dendritic cells infected with adenovirus vectors show signs of activation. Gene Ther 7:1112-1120.
- Hodges, et al. (2000) Multiply deleted [E1, polymerase-, and pTP-] adenovirus vector persists despite deletion of the preterminal protein. J Gene Med 2:250-259.
- Hodges, et al. Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications. J Virol. Jul. 2001;75(13):5913-20.
- Hoelscher, et al. Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet, 2006. 367(9509): p. 475-481.
- Huang Chun-Ming et al. A differential proteome in tumors suppressed by an adenovirus-based skin patch vaccine encoding human carcinoembryonic antigen. Proteomics, 5(4); 1013-1023 (Mar. 2005).
- Jonuleit, et al. 2000. Efficient transduction of mature CD83+ dendritic cells using recombinant adenovirus suppressed T cell stimulatory capacity. Gene Ther 7:249-254.
- Joshi, et al. (2009) Adenovirus DNA polymerase is recognized by human CD8+T cells. J Gen Virol 90:84-94.
- Kaufman, et al. (2004) Phase II randomized study of vaccine treatment of advanced prostate cancer (E7897): A trial of the Eastern Cooperative Oncology Group. J Clin Oncol 22:2122-2132.
- Kawano, et al. (2007) MUC1 oncoprotein regulates Bcr-Abl stability and pathogenesis in chronic myelogenous leukemia cells. Cancer Res. 67:11576-11584.
- Khanam, et al. An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes. BMC Biotechnol 7:.1-11 (2007).
- Kiang et al. Fully deleted Ad persistently expressing GAA accomplishes long-term skeletal muscle glycogen correction in tolerant and nontolerant GSD-II mice. Mol Ther, 2006. 13(1):127.
- Kiang, et al. Multiple innate inflammatory responses induced after systemic adenovirus vector delivery depend on a functional complement system. Mol Ther. Oct. 2006;14(4):588-98. Epub Jun. 2, 2006.
- Kirk, et al. Gene-modified dendritic cells for use in tumor vaccines. Hum Gene Ther 11;797-806 (2000).
- Kong, et al. Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines. J Virol. 77:12764-72 (2003).
- Lemiale et al., Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol., 77 (2003): 10078-87.
- Letvin, et al. Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys. J Virol. 78;7490-7 (2004).
- Lozier, et al. Toxicity of a first-generation adenoviral vector in rhesus macaques. Hum Gene Ther 13;113-24 (2002).
- Lubaroff, et al. Clinical protocol: phase I study of an adenovirus/prostate-specific antigen vaccine in men with metastatic prostate cancer. Hum Gene Ther. 17:220-229 (2006).
- Luebke, et al. (2001) A Modified Adenovirus Can Transfect Cochlear Hair Cells In Vivo Without Compromising Cochlear Function. Gene Ther. 8:789-794.
- Maione, et al. An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus. Proc Natl Acad Sci U S A. May 22, 2001;98(11):5986-91. Epub May 15, 2001.
- Maione, et al. Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector. Hum Gene Ther. Apr. 10, 2000;11(6):859-68.
- McDermott, et al. Cytotoxic T-Lymphocyte Escape Does Not Always Explain the Transient Control of Simian Immunodeficiency Virus SIVmac239 Viremia in Adenovirus-Boosted and DNA-Primed Mamu-A*01-Positive Rhesus Macaques. J Virol. 79:15556-66 (2005).
- Miller, et al. 2000. Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication. Hum Gene Ther 11:53-65.
- Mohebtash, et al. A pilot study of MUC-1/CEA/TRICOM poxviral-based vaccine in patients with metastatic breast and ovarian cancer. Clin Cancer Res. Nov. 15, 2011;17(22):7164-73. doi: 10.1158/1078-0432.CCR-11-0649. Epub Nov. 8, 2011.
- Moore, et al. Progress in DNA-based heterologous prime-boost immunization strategies for malaria. Immunol Rev. 199:126-143 (2004).
- Moore, et al. Effects of antigen and genetic adjuvants on immune responses to human immunodeficiency virus DNA vaccines in mice. J Virol 76;243-50 (2002).
- Morelli, et al. 2000. Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway. J Virol 74:9617-9628.
- Morral, et al. Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons. Hum Gene Ther 13;143-54 (2002).
- Morse, et al. (2005) Phase I study of immunization with dendritic cells modified with recombinant fowlpox encoding carcinoembryonic antigen and the triad of costimulatory molecules CD54, CD58, and CD80 in patients with advanced malignancies. Clin Cancer Res 11:3017-3024.
- Morse, et al. (2013) Novel Adenoviral Vector Induces T Cell Responses Despite Anti-Adenoviral Neutralizing Antibodies in Colorectal Cancer Patients. Cancer Immunol Immunother. 62:1293-1301.
- Morse, et al. Effect of the vaccine Ad5 [E1-, E2b-]-CEA(6D) on CEA-directed CMI responses in patients with advanced CEA-expressing malignancies in a phase I/II clinical trial. Etubics Corporation, Seattle, WA. Poster. 2012. http://www.etubics.com/pdf/ASCO%202012.pdf.
- Nazir, et al. Innate immune response to adenovirus. J Investig Med. Sep. 2005;53(6):292-304.
- Nemunaitis, et al. Pilot trial of intravenous infusion of a replication-selective adenovirus (ONYX-015) in combination with chemotherapy or IL-2 treatment in refractory cancer patients. Cancer Gene Ther. 10:341-352 (2003).
- Nwanegbo, et al. (2004) Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clin Diagn Lab Immunol 11:351-357.
- Oh, et al. Dendritic cells transduced with recombinant adenoviruses induce more efficient anti-tumor immunity than dendritic cells pulsed with peptide. Vaccine, 24; 2860-2868 (2006).
- Ojima et al. Successful cancer vaccine therapy for carcinoembryonic antigen (CEA)-expressing colon cancer using genetically modified dendritic cells that express CEA and T helper-type 1 cytokines in CEA transgenic mice, International Journal of Cancer 120(3), 585-593 (2006).
- Osada, et al. (2009) Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity. Cancer Gene Ther 16:673-682.
- Osada, et al. Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity. Cancer Gene Ther.vol. 16, Issue No. 9, pp. 673-682 (Sep. 2009).
- Perkins, et al. Boosting with an adenovirus-based vaccine improves protective efficacy against Venezuelan equine encephalitis virus following DNA vaccination. Vaccine. 2006; 24:3440-5.
- Etubics press release. Etubics and Duke Cancer Institute report positive phase I/II results for colorectal cancer immunotherapy. Etubics corporation. Seattle (May 16, 2012). URL:<http://etubics.com/etubics-and-duke-cancer-institute-report-positive-phase-iii-results-for-colorectal-cancer-immunotherapy/.>.
- Phillpotts, et al. Intranasal immunization with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus. Vaccine 23:1615-1623. (2005).
- Qualikene, et al. Protease-deleted adenovirus vectors and complementing cell lines: potential applications of single-round replication mutants for vaccination and gene therapy. Human Gene Therapy Jun. 10, 2000;11(9):1341-53.
- Ramlau, et al. (2008) A phase II study of Tg4010 (Mva-Muc1-II2) in association with chemotherapy in patients with stage III/IV Non-small cell lung cancer. J Thorac Oncol. 3:735-744.
- Reddy, et al. Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector. Mol Ther. Jan. 2002;5(1):63-73.
- Rice, et al. An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression. Cancer Gene Therapy 22, 454-462 (Sep. 2015).
- Roberts, et al. Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature, 2006. 441(7090):239-43.
- Sandig, et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc Natl Acad Sci U S A. Feb. 1, 2000;97(3):1002-7.
- Santosuosso, et al. Mucosal luminal manipulation of T cell geography switches on protective efficacy by otherwise ineffective parenteral genetic immunization. J Immunol 178:.4 2387-395 (2007).
- Scott, et al. Gutted adenoviral vectors for gene transfer to muscle. Methods Mol Biol 219;19-28 (2003).
- Scott et al. Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin. Neuromuscul. Disord. 12(Suppl 1):S23-9 (2002).
- Seregin, et al. (2009) Overcoming pre-existing Adenovirus immunity by genetic engineering of Adenovirus-based vectors. Expert Opin Biol Ther 9(12): 1521-1531.
- Shiver, et al. Recent advances in the development of HIV-1 vaccines using replication-incompetent adenovirus vectors. Annu Rev Med.55;355-72 (2004).
- Shiver, et al. 2002. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415:331-335.
- Morse, et al. Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo. Int J Cancer 126(12):2893-2903 (2010). First published online Oct. 23, 2009. URL:<https://doi.org/10.1002/ijc.24995>.
- Steel, et al. Interleukin-15 and its Receptor Augment Dendritic Cell Vaccination Against the neu Oncogene Through the Induction of Antibodies Partially Independent of CD4-help. Cancer Res. Feb. 1, 2010; 70(3): 1072.
- Sullivan, et al. Development of a preventive vaccine for Ebola virus infection in primates. Nature 408;605-9 (2000).
- Sumida, et al. Neutralizing antibodies and CD8+T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors. J Virol. Mar. 2004;78(6):2666-73.
- Tatsis, et al. (2004) Adenoviruses as vaccine vectors. Molecular Ther 10:616-629.
- Tatsis, et al. A CD46-binding Chimpanzee Adenovirus Vector as a Vaccine Carrier. Mol Ther (2007).
- Thomas, et al. Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases. Proc Natl Acad Sci U S A 97;7482-7 (2000).
- Thorner, et al. Immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity. J Virol. Dec. 2006;80(24):12009-16. Epub Oct. 11, 2006.
- Tillman, et al. 2000. Adenoviral vectors targeted to CD40 enhance the efficacy of dendritic cell-based vaccination against human papillomavirus 16-induced tumor cells in a murine model. Cancer Res 60:5456-5463.
- Tsang, et al. (2004) A human cytotoxic T-lymphocyte epitope and its agonist epitope from the nonvariable number of tandem repeat sequence of MUC-1. Clin Cancer Res. 10:2139-2149.
- Van Cutsem, et al. (2007) Open-label Phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol 25:1658-1664.
- Van Kampen, et al. Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. Vaccine, 2005. 23(8): p. 1029-1036.
- Varnavski, et al. Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive and pre-immunized mice. Gene Ther 12:.5 427-436.(2005).
- Vaxgen I. VaxGen Announces Initial Results of its Phase III AIDS Vaccine Trial. http://www.corporate-ir.net/ireye/ir_site.zhtml?ticker=VXGN&script=410&layout=6&item_id=385014. Accessed Jul. 14, 2003.
- Vergati, et al. (2010) Strategies for cancer vaccine development. J Biomed Biotechnol 2010. pii: 596432.
- Wang, et al. Episomal segregation of the adenovirus enhancer sequence by conditional genome rearrangement abrogates late viral gene expression. J Virol. 2000; 74:11296-303.
- Ward, et al. E. coli expression and purification of human and cynomolgus IL-15. Protein Expr Purif. Nov. 2009;68(1):42-8. doi: 10.1016/j.pep.2009.05.004. Epub May 10, 2009.
- Weaver, et al. Comparison of replication-competent, first generation, and helper-dependent adenoviral vaccines. PLoS One. 2009;4(3):e5059. doi: 10.1371/journal.pone.0005059. Epub Mar. 31, 2009.
- Wieking, et al. (2012) A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors. Cancer Gene Ther. 2012; 19:667-674.
- Zhao, et al. Enhanced cellular immunity to SIV Gag following co-administration of adenoviruses encoding wild-type or mutant HIV Tat and SIV Gag. Virology 342:.1 1-12 (2005).
- Zhi, et al. Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus. Hum Gene Ther 17:.5 500-06 (2006).
- Zhu, et al. (2000) Specific cytolytic T-cell responses to human CEA from patients immunized with recombinant avipox-CEA vaccine. Clin. Cancer Res. 6:24-33.
- EP16735409.1 Extended European Search Report dated Jun. 20, 2018.
- Barouch, et al. Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J Virol. Aug. 2003;77(16):8729-35.
- Berinstein, Neil L. Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review. Journal of Clinical Oncology, J Clin Oncol. Apr. 15, 2002;20(8):2197-207.
- Casimiro, et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol. Jun. 2003;77(11):6305-13.
- Chandran, et al. Recent trends in drug delivery systems: liposomal drug delivery system—preparation and characterisation. Indian J Exp Biol. Aug. 1997;35(8):801-9.
- Chirmule, et al. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther.Sep. 1999;6(9):1574-83.
- Couvreur, P. Polyalkylcyanoacrylates as colloidal drug carriers. Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20.
- Gabitzsch, et al. A preliminary and comparative evaluation of a novel Ad5 [E1-, E2b-] recombinant-based vaccine used to induce cell mediated immune responses. Immunol Lett. Jan. 29, 2009;122(1):44-51. doi: 10.1016/j.imlet.2008.11.003. Epub Dec. 13, 2008.
- Gaynor, et al. Cis-acting induction of adenovirus transcription. Cell. Jul. 1983;33(3):683-93.
- Hoenen, et al. Current ebola vaccines. Expert Opin Biol Ther. Jul. 2012;12(7):859-72.
- International Application No. PCT/US2016/012482 International Search Report and Written Opinion dated Mar. 21, 2016.
- Jones, et al. Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine. Vaccine. Sep. 16, 2011; 29(40): 7020-7026.
- Lasic, DD. Novel applications of liposomes. Trends Biotechnol. Jul. 1998;16(7):307-21.
- Lauer, et al. Natural variation among human adenoviruses: genome sequence and annotation of human adenovirus serotype 1. J Gen Virol. Sep. 2004;85(Pt 9):2615-25.
- Ledgerwood, J. et al. A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults. Vaccine. Dec. 16, 2010;29(2):304-13.
- Leza, et al. Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element. J Virol. Aug. 1988;62(8):3003-13.
- Margalit, R. Liposome-mediated drug targeting in topical and regional therapies. Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61.
- McCoy, et al. Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors. J Virol. Jun. 2007;81(12):6594-604. Epub Apr. 11, 2007.
- McMichael, et al. The quest for an AIDS vaccine: is the CD8+T-cell approach feasible? Nat Rev Immunol. Apr. 2002;2(4):283-91.
- Miralles, et al. The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system. J Biol Chem. Jun. 25, 1989;264(18):10763-72.
- Nevins, Jr. Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell. Oct. 1981;26(2 Pt 2):213-20.
- Schaack, et al. E1A and E1B proteins inhibit inflammation induced by adenovirus. Proc Natl Acad Sci U S A. Mar. 2, 2004;101(9):3124-9. Epub Feb. 19, 2004.
- Schaack. Induction and inhibition of innate inflammatory responses by adenovirus early region proteins. Viral Immunol. 2005;18(1):79-88.
- Takakura, et al. [Drug delivery systems in gene therapy]. Nihon Rinsho. Mar. 1998;56(3):691-5.
- Yang, et al. Overcoming immunity to a viral vaccine by DNA priming before vector boosting. J Virol. Jan. 2003; 77(1): 799-803.
- Yang, et al. Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J Virol. Oct. 1996;70(10):7209-12.
- Zambaux, et al. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. J Control Release. Jan. 2, 1998;50(1−3):31-40.
- Zur Muhlen, et al. Solid lipid nanoparticles (SLN) for controlled drug delivery—drug release and release mechanism. Eur J Pharm Biopharm. Mar. 1998;45(2):149-55.
- Li et al., “Progress in the Production of Adenovirus Vector”, The Chinese Journal of Process Engineering, 2004, vol. 4, No. 5, pp. 475-480. (English Abstract).
- Official Action (with English translation) for Chinese Patent Application No. 201680014847.2 dated Jan. 21, 2020, 12 pages.
- Notice of Intention to Grant for European Patent Application No. 16735409.1 dated Oct. 11, 2019, 7 pages.
- Notice of Intention to Grant for European Patent Application No. 16735409.1 dated Jan. 9, 2020, 7 pages.
- Official Action (with English translation) for South Korean Patent Application No. 10-2017-7022208 dated Apr. 2, 2020, 13 pages.
Type: Grant
Filed: Jan 7, 2016
Date of Patent: Jun 30, 2020
Patent Publication Number: 20170368161
Assignee: Etubics Corporation (Seattle, WA)
Inventors: Frank R. Jones (Seattle, WA), Elizabeth Gabitzsch (Seattle, WA)
Primary Examiner: Jeffrey S Parkin
Application Number: 15/542,003
