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. 2012;7(4):e33920.
doi: 10.1371/journal.pone.0033920. Epub 2012 Apr 5.

Modification of Ad5 hexon hypervariable regions circumvents pre-existing Ad5 neutralizing antibodies and induces protective immune responses

Joseph T Bruder  1 , Elena Semenova, Ping Chen, Keith Limbach, Noelle B Patterson, Maureen E Stefaniak, Svetlana Konovalova, Charlie Thomas, Melissa Hamilton, C Richter King, Thomas L Richie, Denise L Doolan
Affiliations

Modification of Ad5 hexon hypervariable regions circumvents pre-existing Ad5 neutralizing antibodies and induces protective immune responses

Joseph T Bruder et al. PLoS One. 2012.

Erratum in

  • PLoS One. 2012;7(5): doi/10.1371/annotation/c110beed-3cac-48db-9039-ba4498d5db50

Abstract

The development of an effective malaria vaccine is a high global health priority. Vaccine vectors based on adenovirus type 5 are capable of generating robust and protective T cell and antibody responses in animal models and are currently being evaluated in clinical trials for HIV and malaria. They appear to be more effective in terms of inducing antigen-specific immune responses as compared with non-Ad5 serotype vectors. However, the high prevalence of neutralizing antibodies to Ad5 in the human population, particularly in the developing world, has the potential to limit the effectiveness of Ad5-based vaccines. We have generated novel Ad5-based vectors that precisely replace the hexon hypervariable regions with those derived from Ad43, a subgroup D serotype with low prevalence of neutralizing antibody in humans. We have demonstrated that these hexon-modified adenovectors are not neutralized efficiently by Ad5 neutralizing antibodies in vitro using sera from mice, rabbits and human volunteers. We have also generated hexon-modified adenovectors that express a rodent malaria parasite antigen, PyCSP, and demonstrated that they are as immunogenic as an unmodified vector. Furthermore, in contrast to the unmodified vector, the hexon-modified adenovectors induced robust T cell responses in mice with high levels of Ad5 neutralizing antibody. We also show that the hexon-modified vector can be combined with unmodified Ad5 vector in prime-boost regimens to induce protective responses in mice. Our data establish that these hexon-modified vectors are highly immunogenic even in the presence of pre-existing anti-adenovirus antibodies. These hexon-modified adenovectors may have advantages in sub-Saharan Africa where there is a high prevalence of Ad5 neutralizing antibody in the population.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of Ad5 vectors with chimeric hexons derived from Ad2, Ad43 and Ad34.
A schematic view of the Ad5 hexon protein and viable chimeric vectors is shown. The positions of surface loops (DE1 and FG1) within the hexon protein are indicated. The HVRs 1–9, indicated in black, are shown above the Ad5 hexon. Sequences from Ad5 are indicated in white, Ad2 in diagonal stripes, Ad34 in gray and Ad43 in black. Viable vectors produced were: Ad5H(5–5) containing the wild-type Ad5 hexon; Ad5H(2–2) containing the Ad2 DE1 and FG1 loops; Ad5H(5–43) containing the FG1 loop from Ad43; and Ad5H(5–34) containing the FG1 loop from Ad34.
Figure 2
Figure 2. Ad5 vectors containing hexon hypervariable regions from Ad43 express PyCSP transgene efficiently.
Generation of Ad5 vectors with hypervariable regions of Ad43. A. Schematic showing the DE1 and FG1 loops of the unmodified Ad5 vector and three chimeric vectors containing Ad43 HVR substitutions in the FG1 loop, DE1 loop and both loops as indicated. B. Western blot showing the expression of the PyCSP antigen following infection of A549 cells with 200 pu/cell of purified vectors. Mock represents an uninfected cell lysate and serves as a negative control. ScPyCSP represents purified PyCSP protein from Saccharomyces cerevisiae and serves as a positive control. Hexon designations are indicated above the associated well (H = hexon) and the serotype source of the DE1 and FG1 loops shown in parentheses (DE1–FG1). Immunoblots were probed with antibody specific for PyCSP.
Figure 3
Figure 3. Ad5L.H(43-43) is not neutralized efficiently by Ad5 NAb from mice.
Mice were immunized with two administrations (1×1010 pu each) of adenovector at an interval of one month. The serum from these mice obtained three weeks after the last immunization was pooled, diluted 1∶48 and then 3-fold serial dilutions were tested for neutralizing activity toward an unmodified Ad5L vector and the Ad5L.H(43 m-43) hexon-modified vector. Two different adenovectors were used to immunize mice and generate serum, an Ad5 vector (Ad5) and an Ad5 vector with an Ad35 fiber replacement (Ad5.F35).
Figure 4
Figure 4. NAb directed at both HVRs and fiber are dominant in vitro.
Sera from (A) rabbits immunized with two administrations of 1×1010 pu of AdNull or (B) human volunteers screened for the presence of neutralizing antibodies specific for Ad5 were analyzed for NAb titers against a panel of chimeric adenovectors expressing the luciferase reporter gene. Ad5L is an unmodified Ad5 vector; Ad5L.H(43 m-43) is a hexon-modified Ad5 vector containing HVRs 1–9 from Ad43; Ad5L.F35 is an Ad5 vector carrying the Ad35 fiber; Ad5L.H(43 m-43).F35 is a hexon-modified vector carrying the Ad35 fiber. The asterisk indicates serum samples that scored positive for Ad35-specific NAb.
Figure 5
Figure 5. Ad5PyCSP.H(43 m-43) induces T cells responses similar to Ad5PyCSP in naïve mice and is not inhibited by Ad5 NAb in vivo.
Naïve BALB/c mice or mice pre-immunized with two injections (1×1010 pu each) of Ad5 vector or an Ad5 vector containing the Ad35 fiber, were immunized with Ad5PyCSP, or hexon-modified vector Ad5PyCSP.H(43 m-43) (n = 6 mice/group). PyCSP-specific T cell responses were assessed by Intracellular cytokine staining (ICS) and IFNγ ELISpot four weeks after immunization. Targets were MHC-matched A20.2J cells pulsed with synthetic peptides representing the immunodominant CD8+ T cell epitope (PyCSP280–288) or CD4+ T cell epitope with nested subdominant CD8+ T cell epitope (PyCSP57–70) from PyCSP or a defined CD8+ T cell epitope for the influenza virus hemagglutinin antigen (HA 332–340). (A) Schematic of the experiment. (B) ICS analysis of CD8+ IFNγ+ T cell responses from immunized mice assayed individually. Error bars indicate the standard error of the mean, n = 6. (C) IFNγ ELISpot responses from pooled splenocytes (250,000 cells/well). Error bars indicate the standard deviation of the mean from quadruplicate samples. (D) ELISA against recombinant PyCSP protein capture antigen. Box-and-whisker plot with values for individual mice shown (n = 6). The mean is indicated by the red line. * indicates groups where titers from all mice were <8.
Figure 6
Figure 6. Prime boost immunogenicity studies.
BALB/c mice (n = 20 mice/group) were immunized with DNA or adenovectors as indicated. (A) Schematic of the experiment. (B) PyCSP-specific T cell responses were assessed from a subset of the mice (n = 6) by IFNγ ELISpot two weeks after immunization. Targets were MHC-matched A20.2J cells pulsed with synthetic peptides representing the immunodominant CD8+ T cell epitope (PyCSP280–288), CD4+ T cell epitope with nested CD8+ T cell epitope (PyCSP280–296), the immunodominant CD4+ T cell epitope (PyCSP57–70), the subdominant CD8+ T cell epitope (PyCSP58–67), and CD4+ T cell epitope with nested CD8+ T cell epitope (PyCSP58–79) from PyCSP. Negative control values were subtracted from reported data. Error bars indicate the standard deviation of the mean from quadruplicate samples. (C) ELISA against PyCSP repeat peptide capture antigen. Error bars indicate the standard error of the mean.
Figure 7
Figure 7. Polyfunctional T cell responses following different vaccine regimens.
Splenocytes from mice immunized as in Figure 6 (n = 6) were analyzed for polyfunctional CD8+ T cell responses following stimulation with A20.2J cells pulsed with a synthetic peptide representing the immunodominant CD8+ T cell epitope (PyCSP280–288). Responses were measured by ICS for IFNγ, IL-2 and TNFα. Black bars indicate the mean percentage of cells positive for each combination of cytokines. Responses from individual animals are shown by the colored dots. (g = IFNγ, 2 = IL2, t = TNFα). Pie charts indicate the fraction of the total response comprising cells expressing each of the seven possible combinations of IFNγ, IL-2 and TNFα.
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