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