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Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Atzingen MV, Rodriguez D, Siqueira GH, Leite LC, Nascimento AL - Biomed Res Int (2014)

Bottom Line: Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity.Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins.The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil.

ABSTRACT
Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

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Related in: MedlinePlus

Analysis of cytokine profiles from spleen cells supernatant. Spleen cells from immunized mice were isolated and cultured in 24-well tissue culture plates. After 48 h, cell-free culture supernatants were collected, and the level of cytokines, IFN-γ and IL-10, was measured by ELISA. For statistical analysis, concentration values for the recombinant fusion proteins-immunized group stimulated with the same protein were compared with those immunized and stimulated with the corresponding protein alone by the two-tailed t-test.
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fig6: Analysis of cytokine profiles from spleen cells supernatant. Spleen cells from immunized mice were isolated and cultured in 24-well tissue culture plates. After 48 h, cell-free culture supernatants were collected, and the level of cytokines, IFN-γ and IL-10, was measured by ELISA. For statistical analysis, concentration values for the recombinant fusion proteins-immunized group stimulated with the same protein were compared with those immunized and stimulated with the corresponding protein alone by the two-tailed t-test.

Mentions: In order to assess secreted cytokines, supernatants of cultured spleen cells from recombinant proteins immunized mice were analyzed for the presence of IL-10, IL-4, IFN-γ, and TNF-α, selected to discriminate Th1 (IFN-γ and TNF-α) and Th2 (IL-10 and IL-4) immune responses [31, 32]. Recombinant proteins Lsa21 and Lp95 C-terminal were not capable to promote secretion of IL-10 and the amount detected were similar to the controls (Figure 6). Proteins rLIC10494 and rLIC12730, alone, induced small amounts of IL-10, statistically significant compared to the controls (cell culture medium). A booster effect was elicited by the presence of DnaK in the protein fusions, being statistically significant with the proteins Lsa21, rLIC10494, and C-terminus of Lp95. The presence of DnaK does not promote an enhancement on IFN-gamma level, and in the case of rLIC12730, a decrease in the amount of IFN-gamma detected. Measurements of the same parameters with spleen cells from control animals immunized with medium either stimulated or not with the recombinant proteins produced negligible results (not shown). Cytokines IL-4 and TNF-alfa evaluation resulted in very low values (not shown).


Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Atzingen MV, Rodriguez D, Siqueira GH, Leite LC, Nascimento AL - Biomed Res Int (2014)

Analysis of cytokine profiles from spleen cells supernatant. Spleen cells from immunized mice were isolated and cultured in 24-well tissue culture plates. After 48 h, cell-free culture supernatants were collected, and the level of cytokines, IFN-γ and IL-10, was measured by ELISA. For statistical analysis, concentration values for the recombinant fusion proteins-immunized group stimulated with the same protein were compared with those immunized and stimulated with the corresponding protein alone by the two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109591&req=5

fig6: Analysis of cytokine profiles from spleen cells supernatant. Spleen cells from immunized mice were isolated and cultured in 24-well tissue culture plates. After 48 h, cell-free culture supernatants were collected, and the level of cytokines, IFN-γ and IL-10, was measured by ELISA. For statistical analysis, concentration values for the recombinant fusion proteins-immunized group stimulated with the same protein were compared with those immunized and stimulated with the corresponding protein alone by the two-tailed t-test.
Mentions: In order to assess secreted cytokines, supernatants of cultured spleen cells from recombinant proteins immunized mice were analyzed for the presence of IL-10, IL-4, IFN-γ, and TNF-α, selected to discriminate Th1 (IFN-γ and TNF-α) and Th2 (IL-10 and IL-4) immune responses [31, 32]. Recombinant proteins Lsa21 and Lp95 C-terminal were not capable to promote secretion of IL-10 and the amount detected were similar to the controls (Figure 6). Proteins rLIC10494 and rLIC12730, alone, induced small amounts of IL-10, statistically significant compared to the controls (cell culture medium). A booster effect was elicited by the presence of DnaK in the protein fusions, being statistically significant with the proteins Lsa21, rLIC10494, and C-terminus of Lp95. The presence of DnaK does not promote an enhancement on IFN-gamma level, and in the case of rLIC12730, a decrease in the amount of IFN-gamma detected. Measurements of the same parameters with spleen cells from control animals immunized with medium either stimulated or not with the recombinant proteins produced negligible results (not shown). Cytokines IL-4 and TNF-alfa evaluation resulted in very low values (not shown).

Bottom Line: Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity.Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins.The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil.

ABSTRACT
Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

Show MeSH
Related in: MedlinePlus