Generation of Ad5 vectors with chimeric hexons derived from Ad2, Ad43 and Ad34.

<p>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.</p

Prime boost immunogenicity studies.

<p>BALB/c mice (n = 20 mice/group) were immunized with DNA or adenovectors as indicated. (A) Schematic of the experiment. (B) <i>Py</i>CSP-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 (<i>Py</i>CSP280–288), CD4⁺ T cell epitope with nested CD8+ T cell epitope (<i>Py</i>CSP280–296), the immunodominant CD4⁺ T cell epitope (<i>Py</i>CSP57–70), the subdominant CD8⁺ T cell epitope (<i>Py</i>CSP58–67), and CD4⁺ T cell epitope with nested CD8+ T cell epitope (<i>Py</i>CSP58–79) from <i>Py</i>CSP. Negative control values were subtracted from reported data. Error bars indicate the standard deviation of the mean from quadruplicate samples. (C) ELISA against <i>Py</i>CSP repeat peptide capture antigen. Error bars indicate the standard error of the mean.</p

Polyfunctional T cell responses following different vaccine regimens.

<p>Splenocytes from mice immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033920#pone-0033920-g006" target="_blank">Figure 6</a> (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 (<i>Py</i>CSP280–288). 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.</p

NAb directed at both HVRs and fiber are dominant <i>in vitro</i>.

<p>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.</p

Ad5L.H(43-43) is not neutralized efficiently by Ad5 NAb from mice.

<p>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).</p

Protective efficacy of adenovector prime-boost regimens.

<p>DNA dose = 100 µg.</p><p>Ad dose = 1×10⁹ pu.</p><p>Fischer's Exact Test, two-tailed versus Ad Null group.</p

Ad5<i>Py</i>CSP.H(43 m-43) induces T cells responses similar to Ad5<i>Py</i>CSP in naïve mice and is not inhibited by Ad5 NAb <i>in vivo</i>.

<p>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 Ad5<i>Py</i>CSP, or hexon-modified vector Ad5PyCSP.H(43 m-43) (n = 6 mice/group). <i>Py</i>CSP-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 (<i>Py</i>CSP57–70) from <i>Py</i>CSP 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 <i>Py</i>CSP 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.</p

A Glycolipid Adjuvant, 7DW8-5, Enhances CD8+ T Cell Responses Induced by an Adenovirus-Vectored Malaria Vaccine in Non-Human Primates

<div><p>A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of <i>Plasmodium falciparum</i>. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.</p> </div

Activity of 7DW8-5 on DCs and <i>i</i>NKT cells in rhesus macaques.

<p>(<b>A</b>) Percentage of circulating and activated monocytoid DCs upon 7DW8-5 and AdPfCA co-administration <i>in </i><i>vivo</i>. PBMCs were isolated 24 hours post prime and stained for circulating (left panel) and activated (right panel) DCs. Each column indicates the mean value for 5 animals per group; errors bars denote SEM. * = p < 0.05 and ** = p <0.01 when compared to control group receiving AdPfCA + 0 µg 7DW8-5. (<b>B</b>) AdPfCA administration with or without 7DW8-5 induces a transient decrease in the percentage of iNKT cells. PBMCs were isolated at baseline and up to 2 weeks post prime and stained for iNKT cells as described. Each point represents % iNKT cells from one animal; lines indicate mean % iNKT cells per dose group at the indicated time point.</p

Adjuvant effect of 7DW8-5 on AdPfCA in rhesus macaques.

<p>(A) Cellular immunogenicity upon 7DW8-5 and AdPfCA co-administration <i>in </i><i>vivo</i>. Five animals per group were vaccinated with AdPfCA alone (control) or in combination with one of four ascending doses of 7DW8-5. PBMCs were isolated before prime and boost and at eight weeks post prime and post boost, stimulated with <i>Pf</i>CSP- or <i>Pf</i>AMA1-specific peptides, and the number of IFN-γ-secreting cells was measured by ELISpot assay. Stimulation index was calculated as the number of spots detected in the respective peptide stimulated well divided by the number of spots in the media only well, and the bars displayed are the mean of 5 animals per dose group. All samples were run in duplicate. Asterisks represent statistical significance (p < 0.05) for the summation of <i>Pf</i>CSP- and <i>Pf</i>AMA1 T-cell responses for each respective dose group as compared to the control dose group (AdPfCA + 0 μg 7DW8-5), and error bars represent the standard error for the five animals per group. (B) Enhancement of malaria-specific CD8+ T-cell responses by 7DW8-5. PBMCs from four macaques that received AdPfCA + 100 µg 7DW8-5 were isolated at eight weeks post boost, depleted of CD4+ or CD8+ T cells, stimulated with <i>Pf</i>CSP-or <i>Pf</i>AMA1-specific peptides, and the relative number of IFN-γ secreting cells were determined by ELISpot assay. All samples were run in duplicate and subtracted for background levels measured in cells stimulated with culture medium containing 0.01% DMSO (negative control). Error bars represent the standard error between duplicated wells. Representative data from one animal are shown. (C) Humoral immunogenicity upon in vivo co-administration of 7DW8-5 and AdPfCA. Animals were vaccinated with AdPfCA alone or in combination with 7DW8-5 as described above. Sera were isolated at four weeks post prime and three weeks post boost and measured for levels of PfCSP- and PfAMA1-directed antibodies. Values presented are a mean of 5 animals per group at each respective time point. All samples were run in duplicate. </p