Sublingual (SL) delivery, a non-invasive 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+ T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naïve 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 naïve mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.
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.
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 × 109 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 × 1010 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.
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 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 multi-functional 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 pre-clinical studies must be carefully evaluated for successful development of novel Ad5-based vaccines.
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 ∼39,000) 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.
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
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