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DNA-protein immunization using Leishmania peroxidoxin-1 induces a strong CD4+ T cell response and partially protects mice from cutaneous leishmaniasis: role of fusion murine granulocyte-macrophage colony-stimulating factor DNA adjuvant.

Bayih AG, Daifalla NS, Gedamu L - PLoS Negl Trop Dis (2014)

Bottom Line: To date, no universally effective and safe vaccine has been developed for general human use.The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines.To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: To date, no universally effective and safe vaccine has been developed for general human use. Leishmania donovani Peroxidoxin-1 (LdPxn-1) is a member of the antioxidant family of proteins and is predominantly expressed in the amastigote stage of the parasite. The aim of this study was to evaluate the immunogenicity and protective efficacy of LdPxn-1 in BALB/c mice in heterologous DNA-Protein immunization regimen in the presence of fusion murine granulocyte-macrophage colony-stimulating factor (mGMCSF) DNA adjuvant.

Methodology and principal findings: A fusion DNA of LdPxn1 and mGMCSF was cloned into a modified pcDNA vector. To confirm the expression in mammalian system, Chinese hamster ovary cells were transfected with the plasmid vector containing LdPxn1 gene. BALB/c mice were immunized twice with pcDNA-mGMCSF-LdPxn-1 or pcDNA-LdPxn1 DNA and boosted once with recombinant LdPxn-1 protein. Three weeks after the last immunization, mice were infected with Leishmania major promastigotes. The result showed that immunization with pcDNA-mGMCSF-LdPxn1 elicited a mixed Th-1/Th-2 immune response with significantly higher production of IFN-γ than controls. Intracellular cytokine staining of antigen-stimulated spleen cells showed that immunization with this antigen elicited significantly higher proportion of CD4+ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines. Moreover, a significant reduction in the footpad swelling was seen in mice immunized with pcDNA-mGMCSF-LdPxn1 antigen. Expression study in CHO cells demonstrated that pcDNA-mGMCSF-LdPxn-1 was expressed in mammalian system.

Conclusion: The result demonstrates that immunization of BALB/c mice with a plasmid expressing LdPxn1 in the presence of mGMCSF adjuvant elicits a strong specific immune response with high level induction of multipotent CD4+ cells that mediate protection of the mice from Leishmania major infection. To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1.

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Related in: MedlinePlus

Antigen-specific cytokine expressing CD4+ T cells at the time of challenge.Integrated median fluorescent intensity (iMFI) of IFN-γ (A), TNF-α (B), and IL-2 (C) as well as the percentage of multipotent CD4+ T cells that simultaneously express IFN-γ +, TNF-α +, and IL-2 + (D). Integrated MFI was calculated as a product of the percentage of the cytokine producing CD4+ T cells and the median fluorescent intensity of the cytokine. Mice immunized twice with pcDNA-LdPxn1 or pcDNA-mGMCSF-LdPxn1 were boosted with rLdPxn1 in the presence of CpG ODN. The control mice received three doses of pcDNA, pcDNA-mGMCSF, or CpG ODN alone. Asterisks indicate statistically significant difference between cells from antigen immunized mice and controls (p<0.05).
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pntd-0003391-g004: Antigen-specific cytokine expressing CD4+ T cells at the time of challenge.Integrated median fluorescent intensity (iMFI) of IFN-γ (A), TNF-α (B), and IL-2 (C) as well as the percentage of multipotent CD4+ T cells that simultaneously express IFN-γ +, TNF-α +, and IL-2 + (D). Integrated MFI was calculated as a product of the percentage of the cytokine producing CD4+ T cells and the median fluorescent intensity of the cytokine. Mice immunized twice with pcDNA-LdPxn1 or pcDNA-mGMCSF-LdPxn1 were boosted with rLdPxn1 in the presence of CpG ODN. The control mice received three doses of pcDNA, pcDNA-mGMCSF, or CpG ODN alone. Asterisks indicate statistically significant difference between cells from antigen immunized mice and controls (p<0.05).

Mentions: The result clearly shows that immunization with two doses of pcDNA-mGMCSF-LdPxn1 DNA followed by a single booster immunization with rLdPxn1 protein induced significantly higher CD4+ T cell response with high level expression of IFN-γ, TNF-α, and IL-2 (Fig. 4). However, no appreciable difference was seen between the immunized and control mice with respect to cytokine production by antigen-stimulated CD8+ T cells.


DNA-protein immunization using Leishmania peroxidoxin-1 induces a strong CD4+ T cell response and partially protects mice from cutaneous leishmaniasis: role of fusion murine granulocyte-macrophage colony-stimulating factor DNA adjuvant.

Bayih AG, Daifalla NS, Gedamu L - PLoS Negl Trop Dis (2014)

Antigen-specific cytokine expressing CD4+ T cells at the time of challenge.Integrated median fluorescent intensity (iMFI) of IFN-γ (A), TNF-α (B), and IL-2 (C) as well as the percentage of multipotent CD4+ T cells that simultaneously express IFN-γ +, TNF-α +, and IL-2 + (D). Integrated MFI was calculated as a product of the percentage of the cytokine producing CD4+ T cells and the median fluorescent intensity of the cytokine. Mice immunized twice with pcDNA-LdPxn1 or pcDNA-mGMCSF-LdPxn1 were boosted with rLdPxn1 in the presence of CpG ODN. The control mice received three doses of pcDNA, pcDNA-mGMCSF, or CpG ODN alone. Asterisks indicate statistically significant difference between cells from antigen immunized mice and controls (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263403&req=5

pntd-0003391-g004: Antigen-specific cytokine expressing CD4+ T cells at the time of challenge.Integrated median fluorescent intensity (iMFI) of IFN-γ (A), TNF-α (B), and IL-2 (C) as well as the percentage of multipotent CD4+ T cells that simultaneously express IFN-γ +, TNF-α +, and IL-2 + (D). Integrated MFI was calculated as a product of the percentage of the cytokine producing CD4+ T cells and the median fluorescent intensity of the cytokine. Mice immunized twice with pcDNA-LdPxn1 or pcDNA-mGMCSF-LdPxn1 were boosted with rLdPxn1 in the presence of CpG ODN. The control mice received three doses of pcDNA, pcDNA-mGMCSF, or CpG ODN alone. Asterisks indicate statistically significant difference between cells from antigen immunized mice and controls (p<0.05).
Mentions: The result clearly shows that immunization with two doses of pcDNA-mGMCSF-LdPxn1 DNA followed by a single booster immunization with rLdPxn1 protein induced significantly higher CD4+ T cell response with high level expression of IFN-γ, TNF-α, and IL-2 (Fig. 4). However, no appreciable difference was seen between the immunized and control mice with respect to cytokine production by antigen-stimulated CD8+ T cells.

Bottom Line: To date, no universally effective and safe vaccine has been developed for general human use.The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines.To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: To date, no universally effective and safe vaccine has been developed for general human use. Leishmania donovani Peroxidoxin-1 (LdPxn-1) is a member of the antioxidant family of proteins and is predominantly expressed in the amastigote stage of the parasite. The aim of this study was to evaluate the immunogenicity and protective efficacy of LdPxn-1 in BALB/c mice in heterologous DNA-Protein immunization regimen in the presence of fusion murine granulocyte-macrophage colony-stimulating factor (mGMCSF) DNA adjuvant.

Methodology and principal findings: A fusion DNA of LdPxn1 and mGMCSF was cloned into a modified pcDNA vector. To confirm the expression in mammalian system, Chinese hamster ovary cells were transfected with the plasmid vector containing LdPxn1 gene. BALB/c mice were immunized twice with pcDNA-mGMCSF-LdPxn-1 or pcDNA-LdPxn1 DNA and boosted once with recombinant LdPxn-1 protein. Three weeks after the last immunization, mice were infected with Leishmania major promastigotes. The result showed that immunization with pcDNA-mGMCSF-LdPxn1 elicited a mixed Th-1/Th-2 immune response with significantly higher production of IFN-γ than controls. Intracellular cytokine staining of antigen-stimulated spleen cells showed that immunization with this antigen elicited significantly higher proportion of CD4+ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced significantly higher proportion of multipotent CD4+ cells that simultaneously express the three Th-1 cytokines. Moreover, a significant reduction in the footpad swelling was seen in mice immunized with pcDNA-mGMCSF-LdPxn1 antigen. Expression study in CHO cells demonstrated that pcDNA-mGMCSF-LdPxn-1 was expressed in mammalian system.

Conclusion: The result demonstrates that immunization of BALB/c mice with a plasmid expressing LdPxn1 in the presence of mGMCSF adjuvant elicits a strong specific immune response with high level induction of multipotent CD4+ cells that mediate protection of the mice from Leishmania major infection. To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1.

Show MeSH
Related in: MedlinePlus