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Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization.

Jiang G, Charoenvit Y, Moreno A, Baraceros MF, Banania G, Richie N, Abot S, Ganeshan H, Fallarme V, Patterson NB, Geall A, Weiss WR, Strobert E, Caro-Aquilar I, Lanar DE, Saul A, Martin LB, Gowda K, Morrissette CR, Kaslow DC, Carucci DJ, Galinski MR, Doolan DL - Malar. J. (2007)

Bottom Line: Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone.CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost.Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910-7500, USA. jiang_george@bah.com

ABSTRACT
The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

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Intracellular IFN-γ and IL-2 expression by CD4+ or CD8+ T cells. PBMC from monkey R immunized with CSLAM/PBS, collected at preimmunization and 4 weeks post virus boost time points, were assayed against PfSSP2/TRAP, or PfLSA1 peptide pools by intracellular cytokine staining. Data represent the percentage of CD4+ or CD8+ IFN-γ and IL-2 producing T cells. Note differences in the y-axis scales between CD4+ and CD8+ responses.
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Figure 8: Intracellular IFN-γ and IL-2 expression by CD4+ or CD8+ T cells. PBMC from monkey R immunized with CSLAM/PBS, collected at preimmunization and 4 weeks post virus boost time points, were assayed against PfSSP2/TRAP, or PfLSA1 peptide pools by intracellular cytokine staining. Data represent the percentage of CD4+ or CD8+ IFN-γ and IL-2 producing T cells. Note differences in the y-axis scales between CD4+ and CD8+ responses.

Mentions: The ELIspot assay as reported here measures the total number of cytokine secreting cells within a bulk cell population and does not allow discrimination between the responding cell phenotype(s). Therefore, to determine the phenotypes of IFN-γ-producing cells, the intracellular expression of IFN-γ and IL-2 for CD4+ and CD8+ T cell subsets, in CSLAM immunized monkeys was measured. Based on cell availability, PBMC from three monkeys (ROa7, RQk6, RBsG), collected at preimmunization and four weeks post virus boost time points, were analysed against peptide pools derived from PfCSP, PfSSP2/TRAP and PfLSA1. Figure 8 shows the result of this analysis from the monkey RBsG, for PfSSP2/TRAP and PfLSA1. Peptide pools PfSSP2/TRAP-1011 (residues 396–495), PfSSP2/TRAP-1213 (residues 484–562), PfLSA1–1415 (residues 1–84) and PfLSA-1516 (residues 44–143) preferentially induced CD4+ IL-2 responses, with low level CD4+IFN-γ responses. All four pools also induced low level CD8+ IFN-γ responses, and two of them induced low level CD8+ IL-2 responses (Figure 8). These data indicate that the CSLAM DNA vaccines preferentially induce CD4+ T cell cytokine responses, rather than CD8+ T cell responses, and that IL-2 responses predominate over IFN-γ responses. The data reported above describing the correlation of summed IFN-γ responses to synthetic peptide overlaps and IFN-γ responses to the recombinant protein also suggested that the IFN-γ responses were mediated by CD4+ T cells, rather than CD8+ T cells. Taken together, these data indicate that CD4 T cells, rather than CD8+ T cells, are primarily responsible for the antigen-specific IFN-γ responses detected by ELIspot, at least under the conditions evaluated herein.


Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization.

Jiang G, Charoenvit Y, Moreno A, Baraceros MF, Banania G, Richie N, Abot S, Ganeshan H, Fallarme V, Patterson NB, Geall A, Weiss WR, Strobert E, Caro-Aquilar I, Lanar DE, Saul A, Martin LB, Gowda K, Morrissette CR, Kaslow DC, Carucci DJ, Galinski MR, Doolan DL - Malar. J. (2007)

Intracellular IFN-γ and IL-2 expression by CD4+ or CD8+ T cells. PBMC from monkey R immunized with CSLAM/PBS, collected at preimmunization and 4 weeks post virus boost time points, were assayed against PfSSP2/TRAP, or PfLSA1 peptide pools by intracellular cytokine staining. Data represent the percentage of CD4+ or CD8+ IFN-γ and IL-2 producing T cells. Note differences in the y-axis scales between CD4+ and CD8+ responses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2147027&req=5

Figure 8: Intracellular IFN-γ and IL-2 expression by CD4+ or CD8+ T cells. PBMC from monkey R immunized with CSLAM/PBS, collected at preimmunization and 4 weeks post virus boost time points, were assayed against PfSSP2/TRAP, or PfLSA1 peptide pools by intracellular cytokine staining. Data represent the percentage of CD4+ or CD8+ IFN-γ and IL-2 producing T cells. Note differences in the y-axis scales between CD4+ and CD8+ responses.
Mentions: The ELIspot assay as reported here measures the total number of cytokine secreting cells within a bulk cell population and does not allow discrimination between the responding cell phenotype(s). Therefore, to determine the phenotypes of IFN-γ-producing cells, the intracellular expression of IFN-γ and IL-2 for CD4+ and CD8+ T cell subsets, in CSLAM immunized monkeys was measured. Based on cell availability, PBMC from three monkeys (ROa7, RQk6, RBsG), collected at preimmunization and four weeks post virus boost time points, were analysed against peptide pools derived from PfCSP, PfSSP2/TRAP and PfLSA1. Figure 8 shows the result of this analysis from the monkey RBsG, for PfSSP2/TRAP and PfLSA1. Peptide pools PfSSP2/TRAP-1011 (residues 396–495), PfSSP2/TRAP-1213 (residues 484–562), PfLSA1–1415 (residues 1–84) and PfLSA-1516 (residues 44–143) preferentially induced CD4+ IL-2 responses, with low level CD4+IFN-γ responses. All four pools also induced low level CD8+ IFN-γ responses, and two of them induced low level CD8+ IL-2 responses (Figure 8). These data indicate that the CSLAM DNA vaccines preferentially induce CD4+ T cell cytokine responses, rather than CD8+ T cell responses, and that IL-2 responses predominate over IFN-γ responses. The data reported above describing the correlation of summed IFN-γ responses to synthetic peptide overlaps and IFN-γ responses to the recombinant protein also suggested that the IFN-γ responses were mediated by CD4+ T cells, rather than CD8+ T cells. Taken together, these data indicate that CD4 T cells, rather than CD8+ T cells, are primarily responsible for the antigen-specific IFN-γ responses detected by ELIspot, at least under the conditions evaluated herein.

Bottom Line: Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone.CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost.Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910-7500, USA. jiang_george@bah.com

ABSTRACT
The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

Show MeSH
Related in: MedlinePlus