ABSTRACT
The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW â¼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.
Subject(s)
Adenoviridae Infections/immunology , Adenoviridae/immunology , Ebola Vaccines/administration & dosage , Ebolavirus/immunology , Hemorrhagic Fever, Ebola/prevention & control , Immunity, Innate , Nasal Sprays , Adenoviridae/genetics , Animals , Cells, Cultured , Ebola Vaccines/chemical synthesis , Ebola Vaccines/immunology , Genetic Vectors/immunology , HEK293 Cells , HeLa Cells , Hemorrhagic Fever, Ebola/immunology , Humans , Immunization, Secondary , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Transgenes/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunologyABSTRACT
As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.
Subject(s)
Adenoviridae/immunology , Ebola Vaccines/administration & dosage , Ebolavirus/immunology , Hemorrhagic Fever, Ebola/prevention & control , Vaccines, Synthetic/administration & dosage , Adenoviridae/genetics , Animals , Cells, Cultured , Chlorocebus aethiops , HEK293 Cells , Hemorrhagic Fever, Ebola/immunology , Humans , Macaca fascicularis , Male , Vaccination/methods , Vaccines, Synthetic/genetics , Vero CellsABSTRACT
Pre-existing immunity (PEI) to human adenovirus serotype 5 (Ad5) worldwide is the primary limitation to routine clinical use of Ad5-based vectors in immunization platforms. Using systemic and mucosal PEI induction models in rodents (mice and guinea pigs), we assessed the influence of PEI on the type of adaptive immune response elicited by an Ad5-based vaccine for Ebola with respect to immunization route. Splenocytes isolated from vaccinated animals revealed that immunization by the same route in which PEI was induced significantly compromised Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ CD8+ T cells and ZGP-specific multifunctional CD8+ T cell populations. ZGP-specific IgG1 antibody levels were also significantly reduced and a sharp increase in serum anti-Ad5 neutralizing antibody (NAB) titers were noted following immunization. These immune parameters correlated with poor survival after lethal challenge with rodent-adapted Ebola Zaire virus (ZEBOV). Although the number of IFN-γ+ CD8+ T cells was reduced in animals given the vaccine by a different route from that used for PEI induction, the multifunctional CD8+ T cell response was not compromised. Survival rates in these groups were higher than when PEI was induced by the same route as immunization. These results suggest that antigen-specific multifunctional CD8(+) T cell and Th2 type antibody responses compromised by PEI to Ad5 are required for protection from Ebola. They also illustrate that methods for induction of PEI used in preclinical studies must be carefully evaluated for successful development of novel Ad5-based vaccines.
Subject(s)
Adenoviridae/immunology , Ebola Vaccines/immunology , Animals , Antibody Formation/immunology , CD8-Positive T-Lymphocytes/immunology , Ebolavirus/immunology , Guinea Pigs , Male , Mice , T-Lymphocytes/immunologyABSTRACT
Sublingual (SL) delivery, a noninvasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 108 infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ⺠T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naive mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naive mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.
Subject(s)
Antigens, Viral/administration & dosage , Ebola Vaccines/administration & dosage , Ebolavirus , Hemorrhagic Fever, Ebola/prevention & control , Adenoviridae/genetics , Administration, Sublingual , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, Viral/adverse effects , Antigens, Viral/therapeutic use , CD11c Antigen/metabolism , Cell Line , Ebola Vaccines/adverse effects , Ebola Vaccines/immunology , Guinea Pigs , Hemorrhagic Fever, Ebola/immunology , Hemorrhagic Fever, Ebola/mortality , Humans , Immunity, Cellular , Immunization, Secondary , Immunologic Memory , Male , Mice , Mouth Mucosa/cytology , Mouth Mucosa/immunology , Mouth Mucosa/metabolism , Mouth Mucosa/virology , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Survival Analysis , TransgenesABSTRACT
A novel, thin-film platform that preserves live viruses, bacteria, antibodies, and enzymes without refrigeration for extended periods of time is described. Studies with recombinant adenovirus in an optimized formulation that supports recovery of live virus through 16 freeze-thaw cycles revealed that production of an amorphous solid with a glass transition above room temperature and nitrogen-hydrogen bonding between virus and film components are critical determinants of stability. Administration of live influenza virus in the optimized film by the sublingual and buccal routes induced antibody-mediated immune responses as good as or better than those achieved by intramuscular injection. This work introduces the possibility of improving global access to a variety of medicines by offering a technology capable of reducing costs of production, distribution, and supply chain maintenance.
Subject(s)
Adenoviridae/immunology , Antibodies, Viral/biosynthesis , Immunization/methods , Influenza A Virus, H1N1 Subtype/immunology , Orthomyxoviridae Infections/prevention & control , Preservation, Biological/methods , Vaccines, Attenuated/pharmacology , Adenoviridae/genetics , Administration, Buccal , Administration, Sublingual , Animals , Antibodies, Neutralizing/biosynthesis , HEK293 Cells , Humans , Influenza A Virus, H1N1 Subtype/pathogenicity , Injections, Intramuscular , Male , Membranes, Artificial , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology , Temperature , Vaccine Potency , Vaccines, Attenuated/biosynthesisABSTRACT
The immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity. In vitro and in vivo assays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 10(11) adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND(50)) elicited strong virus- and transgene-specific T cell responses. The 0.05-ND(50) formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P = 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 ± 5.2% proliferation versus 7.7 ± 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND(50)) of antibody did not improve cellular and humoral responses in naïve animals, they did promote strong cellular (0.005 ND(50)) and humoral (0.0005 ND(50)) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naïve individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.