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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Nathaniel J Schuldt, Yasser A Aldhamen, Sarah Godbehere-Roosa, Sergey S Seregin, Youssef A Kousa, Andrea Amalfitano
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-209) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

NS Carried out animal injections, performed all ELISA, ELISpot and in vivo CTL assays, assisted with Ad5-CSP design and development, and drafted the manuscript. YA performed all other flow cytometry experiments. SG designed and developed Ad4-CSP and assisted with animal work. SS assisted with design and development of Ad4-CSP. YK designed and developed Ad5-CSP. AA conceived of the study and participated in design and coordination. All authors read and approved the final manuscript.

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.
Zusatzmaterial
Additional file 1: Ad4-CSP/Ad4-CSP and Ad5-CSP/Ad5-CSP vaccinated animals have no significant cross stimulation of splenocytes. Splenocytes were collected 14 days post final vaccination and were stimulated with either heat inactivated Ad4-Null or heat inactivated Ad5-Null. Animals treated with Ad5-CSP/Ad5-CSP were not significantly different from naïve animals when stimulated with heat inactivated Ad4-CSP as measured by IFNγ secretion by ELISpot. Likewise, animals treated with Ad4-CSP/Ad4-CSP were not significantly different from naïve animals when stimulated with heat inactivated Ad5-CSP as measured by IFNγ secretion by ELISpot. Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P < 0.05, P < 0.01, P < 0.001. (PDF 99 kb) (PDF 99 KB)
12936_2012_2222_MOESM1_ESM.pdf
Additional file 2: Sub-isotype analysis of IgG antibody from plasma of mice vaccinated with heterologous and homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP. Plasma was collected 14 days post final vaccination. The amount of CSP specific subisotype IgG1 (A), IgG2a (B), IgG2b (C), and IgG3 (D) were analysed by ELISA. Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P < 0.05, P < 0.01, P < 0.001. (PDF 114 kb) (PDF 114 KB)
12936_2012_2222_MOESM2_ESM.pdf
Additional file 3: Th1 to Th2 ratio (IgG2a/IgG1) of plasma from vaccinated Ad naïve animals. Plasma was collected 14 days post final vaccination. The amount of CSP specific IgG subisotypes was measured by ELISA. Th1 to Th2 ratio was determined by dividing O.D. values from IgG2a and IgG1. Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, * denotes significance over naïve, P < 0.05. (PDF 49 kb) (PDF 49 KB)
12936_2012_2222_MOESM3_ESM.pdf
Additional file 4: CD8 + T cell activation in Ad5 immune animals vaccinated with heterologous or homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP. Splenocytes were collected from vaccinated animals 14 days post the final vaccination. Cells were stained with CD8-Alexa flour700, CD3-APC-Cy7, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC and analysed by flow cytometry for INFγ secreting CD3+ CD8+ T cells (A), TNFα secreting CD3+ CD8+ T cells (B), and granzyme B+ CD3+ CD8+ T cells (C). Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P < 0.05, P < 0.01, P < 0.001. (PDF 111 kb) (PDF 112 KB)
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