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

Schuldt NJ, Aldhamen YA, Godbehere-Roosa S, Seregin SS, Kousa YA, Amalfitano A - Malar. J. (2012)

Bottom Line: 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 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics Program, Michigan State University, 2240 E Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.

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.

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Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell response. A prime injection of 1 × 1010 vp/mouse Ad4-CSP followed by a boost of 1 × 1010 vp/mouse of Ad5-CSP resulted in significantly more IFNγ secretion by splenocytes measured by ELISpot (A) and CD3+ CD8+ T cells measured by flow cytometry (B). Ad4-CSP/Ad5-CSP was the only treatment to stimulate a significantly higher percentage of TNFα production as compared to unvaccinated animals (C). Cells were stained with CD3-APC-Cy7, CD8-Alexa flour700, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC. 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.
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Figure 1: Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell response. A prime injection of 1 × 1010 vp/mouse Ad4-CSP followed by a boost of 1 × 1010 vp/mouse of Ad5-CSP resulted in significantly more IFNγ secretion by splenocytes measured by ELISpot (A) and CD3+ CD8+ T cells measured by flow cytometry (B). Ad4-CSP/Ad5-CSP was the only treatment to stimulate a significantly higher percentage of TNFα production as compared to unvaccinated animals (C). Cells were stained with CD3-APC-Cy7, CD8-Alexa flour700, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC. 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.

Mentions: rAds of serotype 4 and serotype 5 were engineered to express a codon optimized form of CSP using methods previously described[33,34]. Four vaccination regimens were utilized; 1. Ad5-CSP/Ad5-CSP, 2. Ad5-CSP/Ad4-CSP, 3. Ad4-CSP/Ad4-CSP, and 4. Ad4-CSP/Ad5-CSP, where the Ad serotype used in the priming vaccination is immediately followed by the serotype of the boosting vaccination in each vaccine regimen or group. Initially, (day 0) Ad naïve BALB/cJ mice were injected with either Ad4-CSP or Ad5-CSP (1x1010 vp/mouse) (n = 10). 14 days later 5 mice from each treatment group received a homologous boost (same Ad-CSP serotype vaccine) of 1x1010 vp/mouse, the other five mice from the same group received a heterologous boosting vaccination of 1x1010 vp/mouse with the alternative Ad-CSP serotype vaccine. 28 days after the priming vaccinations, splenocytes were harvested from the animals and stimulated with the CSP derived peptide (NYDNAGTNL) and the number of IFNγ secreting splenocytes were quantified by ELISpot. While every vaccine treatment resulted in a significant increase in the numbers of CSP responsive INFγ secreting splenocytes when compared to non-vaccinated animals, the Ad4-CSP/Ad5-CSP heterologous prime boosting vaccine treatment group induced significantly higher numbers of IFNγ secreting splenocytes than any other treatment group (Figure 1A). Of note, previous experiments have confirmed that simple administration of Ad vaccines does not significantly increase numbers of IFNγ secreted cells, for example when splenocytes derived from Ad vaccine treated animals are stimulated with control peptides[33-35]. These results were further supported by intracellular staining with antibodies against CD3, CD8, and IFNγ, as the percentage of CSP responsive CD3+ CD8+ IFNγ+ cells present in splenocytes derived from mice vaccinated with the Ad4-CSP/Ad5-CSP regimen were significantly higher when compared to splenocytes from animals treated with the other vaccination strategies (Figure 1B). Intracellular staining was also performed to enumerate the frequency of TNF and Granzyme B producing CD8+ T cells present in the spleens of the variously vaccinated animals. Again, the Ad4-CSP/Ad5-CSP experimental vaccination regimen appeared to confer the most robust immune responses against CSP, as it was the only treatment to induce significantly higher percentages of CSP responsive CD3+ CD8+ TNF+ cells as compared to non-vaccinated animals (Figure 1C). Interestingly, none of the vaccination strategies induced significantly higher percentages of CSP responsive CD3+ CD8+ Granzyme B+ cells as compared to non-vaccinated animals; however, animals from the Ad5-CSP/Ad4-CSP vaccination group had significantly lower percentages of CD3+, CD8+, Granzyme B+ T cells as compared to all other treatment groups (Figure 1D).


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

Schuldt NJ, Aldhamen YA, Godbehere-Roosa S, Seregin SS, Kousa YA, Amalfitano A - Malar. J. (2012)

Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell response. A prime injection of 1 × 1010 vp/mouse Ad4-CSP followed by a boost of 1 × 1010 vp/mouse of Ad5-CSP resulted in significantly more IFNγ secretion by splenocytes measured by ELISpot (A) and CD3+ CD8+ T cells measured by flow cytometry (B). Ad4-CSP/Ad5-CSP was the only treatment to stimulate a significantly higher percentage of TNFα production as compared to unvaccinated animals (C). Cells were stained with CD3-APC-Cy7, CD8-Alexa flour700, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell response. A prime injection of 1 × 1010 vp/mouse Ad4-CSP followed by a boost of 1 × 1010 vp/mouse of Ad5-CSP resulted in significantly more IFNγ secretion by splenocytes measured by ELISpot (A) and CD3+ CD8+ T cells measured by flow cytometry (B). Ad4-CSP/Ad5-CSP was the only treatment to stimulate a significantly higher percentage of TNFα production as compared to unvaccinated animals (C). Cells were stained with CD3-APC-Cy7, CD8-Alexa flour700, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC. 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.
Mentions: rAds of serotype 4 and serotype 5 were engineered to express a codon optimized form of CSP using methods previously described[33,34]. Four vaccination regimens were utilized; 1. Ad5-CSP/Ad5-CSP, 2. Ad5-CSP/Ad4-CSP, 3. Ad4-CSP/Ad4-CSP, and 4. Ad4-CSP/Ad5-CSP, where the Ad serotype used in the priming vaccination is immediately followed by the serotype of the boosting vaccination in each vaccine regimen or group. Initially, (day 0) Ad naïve BALB/cJ mice were injected with either Ad4-CSP or Ad5-CSP (1x1010 vp/mouse) (n = 10). 14 days later 5 mice from each treatment group received a homologous boost (same Ad-CSP serotype vaccine) of 1x1010 vp/mouse, the other five mice from the same group received a heterologous boosting vaccination of 1x1010 vp/mouse with the alternative Ad-CSP serotype vaccine. 28 days after the priming vaccinations, splenocytes were harvested from the animals and stimulated with the CSP derived peptide (NYDNAGTNL) and the number of IFNγ secreting splenocytes were quantified by ELISpot. While every vaccine treatment resulted in a significant increase in the numbers of CSP responsive INFγ secreting splenocytes when compared to non-vaccinated animals, the Ad4-CSP/Ad5-CSP heterologous prime boosting vaccine treatment group induced significantly higher numbers of IFNγ secreting splenocytes than any other treatment group (Figure 1A). Of note, previous experiments have confirmed that simple administration of Ad vaccines does not significantly increase numbers of IFNγ secreted cells, for example when splenocytes derived from Ad vaccine treated animals are stimulated with control peptides[33-35]. These results were further supported by intracellular staining with antibodies against CD3, CD8, and IFNγ, as the percentage of CSP responsive CD3+ CD8+ IFNγ+ cells present in splenocytes derived from mice vaccinated with the Ad4-CSP/Ad5-CSP regimen were significantly higher when compared to splenocytes from animals treated with the other vaccination strategies (Figure 1B). Intracellular staining was also performed to enumerate the frequency of TNF and Granzyme B producing CD8+ T cells present in the spleens of the variously vaccinated animals. Again, the Ad4-CSP/Ad5-CSP experimental vaccination regimen appeared to confer the most robust immune responses against CSP, as it was the only treatment to induce significantly higher percentages of CSP responsive CD3+ CD8+ TNF+ cells as compared to non-vaccinated animals (Figure 1C). Interestingly, none of the vaccination strategies induced significantly higher percentages of CSP responsive CD3+ CD8+ Granzyme B+ cells as compared to non-vaccinated animals; however, animals from the Ad5-CSP/Ad4-CSP vaccination group had significantly lower percentages of CD3+, CD8+, Granzyme B+ T cells as compared to all other treatment groups (Figure 1D).

Bottom Line: 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 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics Program, Michigan State University, 2240 E Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.

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.

Show MeSH
Related in: MedlinePlus