Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and Adenovirus (Ad5) immune mice

Abstract Background Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP. Results In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice. Conclusions While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8+ T cells and a higher number of IFNγ secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naïve animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies should undertake similarly stringent analyses of alternative Ad serotypes to establish their effectiveness as replacements for Ad5.</p

Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

<div><p>Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c⁺ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.</p></div

Innate Immune genes.

<p>Ad5-gag induced gene expression in livers of C57BL/6 mice (fold over WT_Mock, 6 hpi). The numbers represent Mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. n = 4 for all Mock injected groups, n = 6 for all Ad5-HIV-gag injected groups.</p>a<p>Significant differences as compared to WT_Mock;</p>b<p>significant differences in transcriptional activation in ERAP1-KO_Ad5-HIV-gag group as compared to WT_Ad5-HIV-gag group (also indicated by boldface font).</p>*<p>denotes significant differences between WT_Mock and ERAP1-KO_Mock (baseline levels).</p

Mice lacking ERAP1 exhibit dramatically enhanced activation of B cells, CD8⁺ and CD8⁻ T cells in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were harvested at 6 hpi, processed, stained for expression of surface markers, and analyzed by FACS as described in Materials and Methods. CD69 activation of (<b>A</b>) CD8⁺ CD3⁺ T cells, (<b>B</b>) CD8<b>⁻</b> CD3⁺ T cells, and (<b>C</b>) B cells are shown. Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4 for all groups of mice. *, ** - indicate values, statistically different from those in mock-injected mice, p<0.05, p<0.001, respectively.</p

Mice lacking ERAP1 exhibit drastically increased activation of NK and NKT cells in the liver in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Liver lymphocytes were prepared at 6 hpi, processed, stained for expression of surface markers (intracellular staining was performed for IFNγ), and analyzed by FACS as described in Materials and Methods. (<b>A, B</b>) CD69 activation and (<b>C, D</b>) IFNγ release by NK cells (<b>A, C</b>) and NKT cells (<b>B, D</b>) are shown. Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4 for all groups of mice. *, ** - indicate values, statistically different from those in mock-injected mice, p<0.05, p<0.001, respectively.</p

Dendritic cells and macrophages derived from ERAP1-KO mice exhibit dramatically enhanced phagocytosis capabilities in response to innate stimuli.

<p>(<b>A</b>) C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intramuscularly injected with 2×10¹⁰ vp/mouse of Ad-HIV-Gag. Splenocytes were harvested at 12 hpi, processed, incubated with FITC⁺ latex beads and analyzed by FACS as described in Materials and Methods. (<b>B</b>) C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were prepared at 6 hpi, processed, incubated with FITC⁺ latex beads and stained for expression of surface markers, and analyzed by FACS as described in Materials and Methods. Phagocytosis abilities of macrophages and (<b>C</b>) Dendritic cells are shown. Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4 for all groups of mice. ** - indicate values, statistically different from those in mock-injected mice, p<0.001.</p

Increased number of licensed NK cells in ERAP1-KO mice.

<p>WT C57BL/6 and ERAP1-KO mice (n = 4 for mock and n = 7 for rEA injected groups of mice) were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were harvested at 12 hpi, processed, stained for expression of Ly49 receptors on NK cells, and FACS analysis was performed as described in materials and methods. (<b>A</b>) Representative figures of percentages of Ly49A and Ly49G expressing NK cells (CD3−, NK1.1+). (<b>B</b>) Representative figures of percentages of Ly49C, Ly49I, and Ly49D expressing NK cells. (<b>C</b>) Frequency of Ly49C/I expressing NK cells. (<b>D</b>) Frequency of Ly49D expressing NK cells. (<b>E</b>) Frequency of Ly49C/I positive and Ly49D negative NK cells. (<b>F</b>) Frequency of Ly49D positive and Ly49C/I negative NK cells. The bars represent mean ± SEM. Data was collected in LSR-II and analyzed by FlowJo software. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. p<0.05 was deemed a statistically significant difference.</p

ERAP1-KO mice exhibit dramatically enhanced capabilities to respond to rEA and release pro-inflammatory cytokines.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. n = 4 for all groups of mice. Plasma samples were collected at 6 hpi and were analyzed using a multiplexed bead array based quantitative system. Bars represent mean ± SEM. Statistical analysis was completed using two-tailed homoscedastic Student’s t-tests; *, ** - indicate values, statistically different between WT_rEA and ERAP1-KO_rEA groups, p<0.05, p<0.001, respectively.</p

Signaling pathways.

<p>Ad5-HIV-gag induced gene expression in livers of C57BL/6 mice (fold over WT_Mock, 6 hpi). The numbers represent Mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. n = 4 for all Mock injected groups, n = 6 for all Ad5-HIV-gag injected groups.</p>a<p>Significant differences as compared to WT_Mock;</p>b<p>significant differences in transcriptional activation in ERAP1-KO_Ad5-HIV-gag group as compared to WT_Ad5-HIV-gag group (also indicated by boldface font).</p

Mice lacking ERAP1 exhibit increased activation of NK cells in the spleen in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were prepared at 12 hpi, processed, stained for expression of surface markers and analyzed by FACS as described in Materials and Methods. CD69 activation NK cells is shown, Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4–7 for all groups of mice. *** - indicate values, statistically different from those in mock-injected mice, p<0.0001.</p