Thermostability and in vivo performance of AAV9 in a film matrix.

BACKGROUND: Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection. METHODS: AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions. Films were packaged in individual particle-free bags with foil overlays and stored at various temperatures under controlled humidity. Recovery of AAV9 from films was determined by serial dilution of rehydrated film in media and infection of HeLa RC32 cells. Luciferase expression was compared to that of films rehydrated immediately after drying. Biodistribution of vector was determined by in vivo imaging and quantitative real-time PCR. Residual moisture in films was determined by Karl Fischer titration. RESULTS: AAV9 embedded within a film matrix and stored at 4 °C for 5 months retained 100% of initial titer. High and low viscosity formulations maintained 90 and 85% of initial titer after 6 months at 25 °C respectively. AAV was not detected after 4 months in a Standard Control Formulation under the same conditions. Biodistribution and transgene expression of AAV stored in film at 25 or 4 °C were as robust as vector stored at -80 °C in a Standard Control Formulation. CONCLUSIONS: These results suggest that storage of AAV in a film matrix facilitates easy transport of vector to remote sites without compromising in vivo performance.

Adeno-associated viruses (AAVs) are small viruses that are used to deliver medicines and vaccines. Prior to administration, they are stored in freezers set to very low temperatures and must be discarded if they thaw during transportation to clinics. AAV was embedded in a film to protect the virus during transportation and storage. The virus remained stable for 6 months at room temperature and during shipment from Texas to North Carolina. The ability to store and transport AAV without the need for complex packaging and temperature control will increase global access to vaccines and other medicines that use AAVs for delivery.

Evaluation of intermolecular interactions required for thermostability of a recombinant adenovirus within a film matrix.

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7-1.17 × 10-4 M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10-9 M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process.

Immunization and Drug Metabolizing Enzymes: Focus on Hepatic Cytochrome P450 3A.

OBJECTIVE: Infectious disease emergencies like the 2013-2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism. METHODS: The impact of an adenovirus-based vaccine expressing Ebola glycoprotein (AdEBO) and H1N1 and H3N2 influenza viruses on hepatic CYP 3A4 and associated nuclear receptors was evaluated in human hepatocytes (HC-04 cells) and in mice. RESULTS: CYP3A activity was suppressed by 55% in mice 24 h after administration of mouse-adapted H1N1, while ˂10% activity remained in HC-04 cells after infection with H1N1 and H3N2 due to global suppression of cellular translation capacity, indicated by reduction (70%, H1N1, 56%, H3N2) of phosphorylated eukaryotic translation initiation factor 4e (eIF4E). AdEBO suppressed CYP3A activity in vivo (44%) and in vitro (26%) 24 hours after infection. CONCLUSION: As the clinical evaluation of vaccines for SARS-CoV-2 and other global pathogens rise, studies to evaluate the impact of new vaccines and emerging pathogens on CYP3A4 and other metabolic enzymes are warranted to avoid therapeutic failures that could further compromise the public health during infectious disease emergencies.

Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature.

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.

Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A.

Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrinß-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states.

A long-lasting, single-dose nasal vaccine for Ebola: a practical armament for an outbreak with significant global impact.

In response to the severity and scale of the 2014 Ebola outbreak, several experimental vaccines were granted fast-track status for clinical testing. Although they may provide long-lasting protection from Ebola, they are, in their current states, far from optimal for populations that need them the most. In this context, nasal immunization addresses the: immune response required at the mucosa where Ebola initiates infection; needs of a population in terms of cost and compliance; and potency of each platform as they contain viruses that naturally infect the respiratory tract. Understanding the attributes of nasal immunization and its application will lead to potent vaccines that can effectively end Ebola and other emerging infectious diseases in developing and industrialized countries.

Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

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.

A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.

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.

Evaluation of the HC-04 cell line as an in vitro model for mechanistic assessment of changes in hepatic cytochrome P450 3A during adenovirus infection.

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 µM) and phenobarbital (500 µM) increased activity by 230 and 124%, whereas ketoconazole (10 µM) and lipopolysaccharide (LPS) (10 µg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

Modeling pre-existing immunity to adenovirus in rodents: immunological requirements for successful development of a recombinant adenovirus serotype 5-based ebola vaccine.

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.

Emerging targets and novel approaches to Ebola virus prophylaxis and treatment.

Ebola is a highly virulent pathogen causing severe hemorrhagic fever with a high case fatality rate in humans and non-human primates (NHPs). Although safe and effective vaccines or other medicinal agents to block Ebola infection are currently unavailable, a significant effort has been put forth to identify several promising candidates for the treatment and prevention of Ebola hemorrhagic fever. Among these, recombinant adenovirus-based vectors have been identified as potent vaccine candidates, with some affording both pre- and post-exposure protection from the virus. Recently, Investigational New Drug (IND) applications have been approved by the US Food and Drug Administration (FDA) and phase I clinical trials have been initiated for two small-molecule therapeutics: anti-sense phosphorodiamidate morpholino oligomers (PMOs: AVI-6002, AVI-6003) and lipid nanoparticle/small interfering RNA (LNP/siRNA: TKM-Ebola). These potential alternatives to vector-based vaccines require multiple doses to achieve therapeutic efficacy, which is not ideal with regard to patient compliance and outbreak scenarios. These concerns have fueled a quest for even better vaccination and treatment strategies. Here, we summarize recent advances in vaccines or post-exposure therapeutics for prevention of Ebola hemorrhagic fever. The utility of novel pharmaceutical approaches to refine and overcome barriers associated with the most promising therapeutic platforms are also discussed.

A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs.

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⁺ 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.

Optimized adenovirus-antibody complexes stimulate strong cellular and humoral immune responses against an encoded antigen in naive mice and those with preexisting immunity.

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.

Species differences in the pharmacology and toxicology of PEGylated helper-dependent adenovirus.

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.

Recent advances in Ebolavirus vaccine development.

Ebolavirus is a highly infectious pathogen with a case fatality rate as high as 90%. Currently there is a lack of licensed Ebolavirus vaccines as well as pre- and post-exposure treatments. Recent increases in the frequency of natural human Ebolavirus infections and its potential use as a bioterrorism agent makes vaccine development a priority for many nations. Significant progress has been made in understanding the pathogenesis of Ebolavirus infection and several promising vaccine candidates were shown to be successful in protecting NHPs against lethal infection. These include replication-deficient adenovirus vectors, replication-competent VSV, HPIV-3 vectors and virus-like particle preparations. Recent advances in the generation of effective post-exposure immunization strategies highlight the possibility of developing a single dose vaccine that will confer full protection in humans following Ebolavirus exposure. Post-exposure protection is particularly important in outbreak and biodefense settings, as well as clinical and laboratory settings in the case of accidental exposure.

Development of a nasal adenovirus-based vaccine: Effect of concentration and formulation on adenovirus stability and infectious titer during actuation from two delivery devices.

A nasal adenovirus-based vaccine is under development. To determine if aggregation occurs during vaccination, infectious titer (limiting dilution) and capsid integrity (dynamic light scattering) were assessed after extrusion of a model vector from two intranasal delivery devices. Preparations of 2.5x10(12) and 1.25x10(11) virus particles (vp)/ml were studied. Virus aggregated ( approximately 10%) in the multi-dose vessel. Virus titer dropped by one log. Virus in the unit-dose device aggregated ( approximately 1%). Titer remained unchanged. Aggregation was concentration dependent. Formulations prevented aggregation during actuation, freeze-thaw and long-term storage. The device, formulation and dose may significantly influence aggregation and potency of any nasal adenovirus 5-based vaccine.

PEGylated Adenoviruses: From Mice to Monkeys.

Covalent modification with polyethylene glycol (PEG), a non-toxic polymer used in food, cosmetic and pharmaceutical preparations for over 60 years, can profoundly influence the pharmacokinetic, pharmacologic and toxciologic profile of protein and peptide-based therapeutics. This review summarizes the history of PEGylation and PEG chemistry and highlights the value of this technology in the context of the design and development of recombinant viruses for gene transfer, vaccination and diagnostic purposes. Specific emphasis is placed on the application of this technology to the adenovirus, the most potent viral vector with the most highly characterized toxicity profile to date, in several animal models.

Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

BACKGROUND: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. METHODOLOGY/PRINCIPAL FINDINGS: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. CONCLUSIONS/SIGNIFICANCE: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.

Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection.

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 x 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p

Nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice.

Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-gamma+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-gamma+ CD8+ T cells (3.9+/-1% naïve vs. 3.6+/-1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40+/-10 reciprocal dilution, both groups). The number of INF-gamma+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146+/-14, naïve vs. 120+/-16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by approximately 25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-gamma+ CD8+ T cells 10 days after administration (0.3+/-0.3% PEG vs. 1.7+/-0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.