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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

Immunogenicity of rLdPxn1 in cutaneous and visceral leishmaniasis patient samples.(A) The level of LdPxn1-specific total IgG measured from plasma collected from cutaneous (n = 8) and visceral leishmaniasis patients (n = 10) as well as healthy individuals (n = 9) using antibody ELISA. The result was expressed as mean OD450nm and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. “HC” refers to “Healthy Controls”. (B) The level of IFN-γ and IL-10 in rLdPxn1-stimulated peripheral blood mononuclear cells isolated from venous blood of cutaneous leishmaniasis patients (n = 8). Positive and negative control cells were included by stimulation with phytohaemagglutinin (PHA) or adding medium only, respectively. The result represented as mean concentration of IFN-γ and IL-10 and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. Stimulation with PHA gave 1510.4±67.3 pg/ml IFN-γ and 380.3±77.7 pg/ml IL-10. Asterisks indicate statistically significant difference between: CL/VL patient samples and healthy controls (A) or rLdPxn1 stimulated and unstimulated PBMCs from CL patients (B) (p<0.05). The antibody ELISA assay was done in triplicate wells for each patient's serum sample.
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pntd-0003391-g009: Immunogenicity of rLdPxn1 in cutaneous and visceral leishmaniasis patient samples.(A) The level of LdPxn1-specific total IgG measured from plasma collected from cutaneous (n = 8) and visceral leishmaniasis patients (n = 10) as well as healthy individuals (n = 9) using antibody ELISA. The result was expressed as mean OD450nm and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. “HC” refers to “Healthy Controls”. (B) The level of IFN-γ and IL-10 in rLdPxn1-stimulated peripheral blood mononuclear cells isolated from venous blood of cutaneous leishmaniasis patients (n = 8). Positive and negative control cells were included by stimulation with phytohaemagglutinin (PHA) or adding medium only, respectively. The result represented as mean concentration of IFN-γ and IL-10 and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. Stimulation with PHA gave 1510.4±67.3 pg/ml IFN-γ and 380.3±77.7 pg/ml IL-10. Asterisks indicate statistically significant difference between: CL/VL patient samples and healthy controls (A) or rLdPxn1 stimulated and unstimulated PBMCs from CL patients (B) (p<0.05). The antibody ELISA assay was done in triplicate wells for each patient's serum sample.

Mentions: Fig. 9A shows the level of antigen-specific IgG response in the sera of cutaneous and visceral leishmaniasis patients as well as those of healthy controls. The level of human IgG specific to rLdPxn1 in both cutaneous and visceral leishmaniasis patient samples was significantly higher than that of healthy controls (p<0.05).


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)

Immunogenicity of rLdPxn1 in cutaneous and visceral leishmaniasis patient samples.(A) The level of LdPxn1-specific total IgG measured from plasma collected from cutaneous (n = 8) and visceral leishmaniasis patients (n = 10) as well as healthy individuals (n = 9) using antibody ELISA. The result was expressed as mean OD450nm and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. “HC” refers to “Healthy Controls”. (B) The level of IFN-γ and IL-10 in rLdPxn1-stimulated peripheral blood mononuclear cells isolated from venous blood of cutaneous leishmaniasis patients (n = 8). Positive and negative control cells were included by stimulation with phytohaemagglutinin (PHA) or adding medium only, respectively. The result represented as mean concentration of IFN-γ and IL-10 and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. Stimulation with PHA gave 1510.4±67.3 pg/ml IFN-γ and 380.3±77.7 pg/ml IL-10. Asterisks indicate statistically significant difference between: CL/VL patient samples and healthy controls (A) or rLdPxn1 stimulated and unstimulated PBMCs from CL patients (B) (p<0.05). The antibody ELISA assay was done in triplicate wells for each patient's serum sample.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003391-g009: Immunogenicity of rLdPxn1 in cutaneous and visceral leishmaniasis patient samples.(A) The level of LdPxn1-specific total IgG measured from plasma collected from cutaneous (n = 8) and visceral leishmaniasis patients (n = 10) as well as healthy individuals (n = 9) using antibody ELISA. The result was expressed as mean OD450nm and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. “HC” refers to “Healthy Controls”. (B) The level of IFN-γ and IL-10 in rLdPxn1-stimulated peripheral blood mononuclear cells isolated from venous blood of cutaneous leishmaniasis patients (n = 8). Positive and negative control cells were included by stimulation with phytohaemagglutinin (PHA) or adding medium only, respectively. The result represented as mean concentration of IFN-γ and IL-10 and standard error of the mean. Statistical comparison between groups was performed using Mann-Whitney U test. Stimulation with PHA gave 1510.4±67.3 pg/ml IFN-γ and 380.3±77.7 pg/ml IL-10. Asterisks indicate statistically significant difference between: CL/VL patient samples and healthy controls (A) or rLdPxn1 stimulated and unstimulated PBMCs from CL patients (B) (p<0.05). The antibody ELISA assay was done in triplicate wells for each patient's serum sample.
Mentions: Fig. 9A shows the level of antigen-specific IgG response in the sera of cutaneous and visceral leishmaniasis patients as well as those of healthy controls. The level of human IgG specific to rLdPxn1 in both cutaneous and visceral leishmaniasis patient samples was significantly higher than that of healthy controls (p<0.05).

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