Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine

Jason S. Richardson is with the Public Health Agency of Canada, Michel K. Yao is with the Public Health Agency of Canada, Kaylie N. Tran is with the Public Health Agency of Canada and University of Manitoba, Maria A. Croyle is with UT Austin, James E. Strong is with the Public Health Agency of Canada and University of Manitoba, Heinz Feldmann is with the Public Health Agency of Canada and University of Manitoba, Gary P. Kobinger is with the Public Health Agency of Canada and University of Manitoba.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.Financial support was received from the following sources: The Public Health Agency of Canada and the Chemical, Biological, Radiological or Nuclear Research and Technology Initiative (grant #CRTI-06-0218RD awarded to GPK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Pharmac

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-γ+ 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-γ+ 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-γ+ 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 ∼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-γ+ 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

T and B cell responses following immunization.

<p>B10.BR mice were vaccinated I.M. with Ad-CMVZGP (1×10⁵, 1×10⁶ and 1×10⁷ IFU/mouse) or Ad-CAGoptZGP (1×10⁴, 1×10⁵ and 1×10⁶ IFU/mouse) and splenocytes were harvested 8 days later for A. IFN-γ CD8+ T cells frequency analysis or B. Neutralizing antibody (NAB) titers. Four to five mice were analyzed per group and the experiment was repeated twice. Levels of NAB to ZEBOV-EGFP were evaluated 25 days post-vaccination. Error bars represent the standard deviation of the data. n.d. refers to assays that were not done for Ad-CAGoptZGP at that IFU/mouse. * p = 0.0454; ** p = 0.1.</p

T cell frequency analysis at day 6 post-immunization.

<p>Groups of 4 B10.BR mice were vaccinated I.M. with 1×10⁸ IFU/mouse of Ad-CMVZGP or Ad-CAGoptZGP and splenocytes were harvested 6 days later, re-stimulated with the peptide TELRTFSI, and production of IFN-γ, TNF-α, and Il-2 from CD8+ T cells was monitored by FACS. Error bars represent the standard deviation of the data. The experiment was repeated once and showed similar results. * p<0.001; ** p<0.001; *** p<0.01; **** p<0.01.</p

Survival and weight loss of mice challenged with ZEBOV 28 days post-vaccination.

<p>Eight to nine B10.BR mice were used in each group.</p>1<p>not applicable.</p

Western blot expression analysis of Ad-CMVZGP or Ad-CAGoptZGP.

<p>Proteins isolated from infected HEK 293 cells were separated by a 10% SDS PAGE then transferred to PVDF membrane. A mouse monoclonal anti-ZGP was used as the primary antibody and a goat anti-mouse horseradish peroxidase (HRP) conjugated antibody as the secondary antibody. 24, 48 and 72 hours indicate the time of total protein harvest post-infection. Band density corresponding to each lane is shown as determined by densitometry of the bands. The control represents untreated HEK 293 cells. An M.O.I. of 5 was used to infect the cells with either Ad-CMVZGP or Ad-CAGoptZGP for each time point. The preparation of Ad-CAGoptZGP or Ad-CMVZGP used had a non-infectious to infectious ratio of 73∶1or 19∶1 respectively.</p

Pre-Existing Immunity Against Adenovirus Does not Compromise the Strength of the Cellular and Humoral Immune Response Against Ebola Glycoprotein After Intranasal Immunization.

<p>Naïve mice (n = 10) were vaccinated with a single dose of 1×10¹⁰ particles of adenovirus expressing the Ebola Zaire glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×10¹⁰ particles of adenovirus serotype 5 expressing beta-galactosidase (AdlacZ) 30 days prior to vaccination were also immunized in the same manner. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1∶320. Animals given a single dose of (AdlacZ) served as negative controls (AdlacZ Control). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). The TELRTFSI peptide, specific for the Ebola Zaire glycoprotein (0.4 µg/well), was incubated with 1×10⁶ splenocytes and cells analyzed by flow cytometry. (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). In both panels, error bars represent the standard deviation of the data.</p

Intranasal Immunization with Recombinant Adenovirus Expressing Ebola Glycoprotein Affords Protection Against Lethal Challenge Even in the Presence of Pre-Existing Immunity.

<p>Naïve mice (n = 10) were vaccinated with a single dose of 1×10¹⁰ particles of adenovirus expressing the Ebola glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×10¹⁰ particles of adenovirus 5 expressing beta-galactosidase (AdlacZ) were also vaccinated in the same manner. Twenty-eight days later, mice were challenged with 200 LD₅₀ of mouse-adapted Ebola virus (Zaire strain). Data represent survival (panel A) and loss of body weight (panel B) over time and is reported as average body weight from each treatment group. Mock - age matched, untreated, unchallenged mice included in this data set to indicate normal weight variation over time. NOTE: Data for naïve mice immunized by the oral route (P.O.) and those with pre-existing immunity and vaccinated by the I.M. route (I.M.+PEI) were not included in this figure for visual clarity. All naive mice immunized orally survived challenge while none in the I.M.+PEI group survived.</p

Oral Vaccination with PEGylated Adenovirus Improves the B-cell Mediated but Not the T-cell Mediated Immune Response Against Ebola Glycoprotein.

<p>Naïve mice and those with pre-existing immunity were vaccinated with 1×10¹⁰ particles of either unmodified (Vaccine) or PEGylated (PEG-Vaccine) Ad5-ZGP by oral gavage. Pre-existing immunity (PEI) was induced by I.M. injection with 5×10¹⁰ particles of adenovirus expressing beta-galactosidase (AdlacZ). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). (C) Profile of anti-Ebola-specific IgG antibodies. (D) Profile of anti-Ebola-specific IgA antibodies. In all panels, error bars represent the standard deviation of the data.</p

Pre-Existing Immunity Against Adenovirus Does Not Compromise the Systemic or Mucosal Cellular Response Generated Against Ebola Glycoprotein After Intranasal Immunization.

<p>The frequency of IFN-γ positive mononuclear cells was analyzed from the spleen (panels A and C), the lung via bronchioalveolar lavage (BAL) and the intestine via the mesenteric lymph nodes (MLN) and Peyer's patches (PP) (panels B and D) 45 days post-immunization by ELISPOT. Samples were obtained from naïve animals (Panels A and B) and those with pre-existing immunity to adenovirus serotype 5 (Panels C and D). Cells were plated at 1×10⁵ or 1×10⁴ cells per well, stimulated with the TELRTFSI peptide and expression of IFN-γ detected with an anti-mouse IFN-γ antibody. Cells isolated from BAL, MLN or PP of four mice were pooled and samples tested in duplicate. In each panel, the number of spot-forming cells (SFC) per million mononuclear cells (MNCs) is shown on the y-axis. Please note - the scale of this axis differs between panels to accent the differences between treatment groups. Error bars represent the standard deviation of the data. Animals immunized with an irrelevant adenoviral vector (AdlacZ) served as negative controls. Positive results obtained from this group are indicative of artificial cellular stimulation that may have occurred during processing and culturing of samples.</p