Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species

<div><p>Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.</p> </div

Enhanced filovirus specific cellular immune response of a rAd26/rAd35 prime-boost regimen.

<p>Filovirus specific cellular immune response was detected by ELISPOT after stimulation with peptide pools spanning the entire glycoprotein. Splenocytes were isolated from groups of mice at two or eight weeks post boost vaccination with 10¹⁰ vp of with vectors coding for Ebola Zaire (A and B), Marburg Angola (C and D), or as control for prime only with a no-antigen coding control vector. The black filled circles represent the group of Ad35.Ebo(Z) or Ad35.Mar(A) primed and Ad26.Ebo(Z) or Ad26.Mar(A) boosted animals; black open circles represent the Ad35.Ebo(Z) or Ad35.Mar(A) primed animals which received the no-antigen coding control vector Ad26.empty as boost. The black filled squares represent the group of Ad26.Ebo(Z) or Ad26.Mar(A) primed and Ad35.Ebo(Z) or Ad35.Mar(A) boosted animals; black open squares represent the Ad26.Ebo(Z) or Ad26.Mar(A) primed animals which received the no-antigen coding control vector Ad35.empty as boost. The dark grey circles represent the control animal group receiving Ad35.empty for prime and Ad26.empty as boost and the light grey circles the animal group receiving Ad26.empty for prime and Ad35.empty as boost. The bar denote the geometric mean of the responsive cells of eight mice (five for the control groups).</p

Cross reactivity of identified murine T cell epitopes.

<p>The Sequence of reactive peptides identified is given and peptides with homologous sequences within the Ebola and Marburg strains are displayed for comparison. Underlined sequences are the predicted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044115#pone.0044115-Honeyman1" target="_blank">[65]</a> MHC class 1 9mer binding sequence using the ANN algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044115#pone.0044115-Honeyman1" target="_blank">[65]</a>, the binding strength to the specific MHC class 1 is given in the last column and was calculated using the T cell epitope prediction tool of the IEDB database (<a href="http://www.immuneepitope.org/" target="_blank">http://www.immuneepitope.org/</a>). Amino acids in bold label the anchoring amino acids of the 9mer according to Rammensee et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044115#pone.0044115-Rammensee1" target="_blank">[66]</a>.</p

Dose dependent cellular immune response of filovirus rAd26 and rAd35 vectors.

<p>The cellular immune response was detected by ELISPOT of isolated splenocytes from mice either infected with 10⁷ to 10¹⁰ vp of rAd26 or rAd35 coding for the glycoprotein Ebola Zaire (A), Ebola Sudan Gulu (C), Ebola Ivory Coast (E), Marburg Angola (C), or Marburg Ravn (D). For each individual experiment three mice each were immunized with rAd26 or rAd35 without a transgene in the E1 region. Displayed is the total number of strain specific filovirus glycoprotein responsive splenocytes in spot forming colonies per million cells (SFC/10⁶ cells). The bar represents the geometric mean of five mice and the grey balls an individual mouse.</p

Enhanced filovirus specific humoral immune response to a rAd26/rAd35 prime-boost regimen.

<p>The filovirus specific humoral immune response was detected by ELISA. Serum was isolated from groups of mice at two or eight weeks post boost vaccination with 10¹⁰ vp of with vectors coding for Ebola Zaire (A and B), Marburg Angola (C and D), or as control for prime only with a no-antigen coding control vector. The black filled circles represent the group of Ad35.Ebo(Z) or Ad35.Mar(A) primed and Ad26.Ebo(Z) or Ad26.Mar(A) boosted animals; black open circles represent the Ad35.Ebo(Z) or Ad35.Mar(A) primed animals which received the no-antigen coding control vector Ad26.empty as boost. The black filled squares represent the group of Ad26.Ebo(Z) or Ad26.Mar(A) primed and Ad35.Ebo(Z) or Ad35.Mar(A) boosted animals; black open squares represent the Ad26.Ebo(Z) or Ad26.Mar(A) primed animals which received the no-antigen coding control vector Ad35.empty as boost. The dark grey circles represent the control animal group receiving Ad35.empty for prime and Ad26.empty as boost and the light grey circles the animal group receiving Ad26.empty for prime and Ad35.empty as boost. The bar denotes the mean of the log transformed sample titers of eight mice (five for the control groups). The p values given are calculated by Anova and a Post-hoc Tukey test.</p

A Phase I, Open-Label Trial, Evaluating the Safety and Immunogenicity of Candidate Tuberculosis Vaccines AERAS-402 and MVA85A, Administered by Prime-Boost Regime in BCG-Vaccinated Healthy Adults

<div><p>Background</p><p>MVA85A and AERAS-402 are two clinically advanced viral vectored TB vaccine candidates expressing <i>Mycobacterium tuberculosis</i> antigens designed to boost BCG-induced immunity. Clinical trials with candidate malaria vaccines have demonstrated that adenoviral vector based priming immunisation, followed by MVA vector boost, induced high levels of immunity. We present the safety and immunogenicity results of the first clinical trial to evaluate this immunisation strategy in TB.</p><p>Methods</p><p>In this phase 1, open-label trial, 40 healthy previously BCG-vaccinated participants were enrolled into three treatment groups and vaccinated with 1 or 2 doses of AERAS-402 followed by MVA85A; or 3 doses of AERAS-402.</p><p>Results</p><p>Most related adverse events (AEs) were mild and there were no vaccine related serious AEs. Boosting AERAS-402 with MVA85A significantly increased Ag85A-specific T-cell responses from day of vaccination. Two priming doses of AERAS-402 followed by MVA85A boost, resulted in a significantly higher AUC post-peak Ag85A response compared to three doses of AERAS-402 and historical data with MVA85A vaccination alone. The frequency of CD8+ T-cells producing IFN-γ, TNF-α and IL-2 was highest in the group receiving two priming doses of AERAS-402 followed by MVA85A.</p><p>Conclusions</p><p>Vaccination with AERAS-402 followed by MVA85A was safe and increased the durability of antigen specific T-cell responses and the frequency and polyfunctionality of CD8+ T-cells, which may be important in protection against TB. Further clinical trials with adenoviral prime-MVA85A boost regimens are merited to optimise vaccination intervals, dose and route of immunisation and to evaluate this strategy in the target population in TB high burden countries.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01683773" target="_blank">NCT01683773</a>.</p></div

Study Timeline and Sampling Schedule.

<p>NHPs were boosted with AERAS-402 fifteen and twenty-seven weeks after the prime with BCG or AFRO-1, Animals in group 1 were primed with BCG, animals in group 2 with the recombinant BCG (AFRO-1) which combines endosomal escape, TB10.4 expression and over-expression of Ag85A and Ag85B. Animals in both groups were boosted with the non-replicating adenovirus 35 AERAS-402 which expresses the Ag85A, Ag85B and TB10.4 fusion protein. Animals in group 3 received the diluent (control group).</p

Prime with BCG or AFRO-1 induces a different IFN-γ production profile in response to <i>Mtb</i> antigen stimulation.

<p>The median of IFN-γ production (measured by ELISA) in whole blood cultures for each group in response to different <i>Mtb</i> antigen stimulation was assessed. Stronger IFN-γ production was seen in animals primed with AFRO-1 in response to Ag85A and Ag85B, as compared to animals primed with BCG one week after the first boost with AERAS-402.</p

Demographics of enrolled subjects.

<p>Abbreviations: BCG, Bacillus Calmette–Guérin; BMI, Body mass index.</p><p>Demographics of enrolled subjects.</p

Prime with AFRO-1 induces proliferation of Ag85B-specific T cells in CD4⁺ and CD8alpha/alpha⁺ T cells.

<p>The median of the proliferation index (% of blasts in response to antigen stimulation - % of blasts in negative control) in response to <i>Mtb</i> antigens was determined by flow cytometric analysis. Differential expansion of T cell subsets was gauged by gating on T cell subsets, i.e. CD4⁺, CD8alpha/beta⁺ and CD8alpha/alpha⁺. Animals primed with AFRO-1 showed stronger proliferation in response to Ag85B stimulation within CD4⁺ T cells (A) and CD8alpha/alpha⁺ T cells (B) as compared to animals primed with BCG one week after the first boost with AERAS-402. No difference was detectable between animals primed with AFRO-1 or BCG in the CD8alpha/beta⁺ T cell compartment (C).</p