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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 lymphocyte proliferation in cultured splenocytes from mice immunized with recombinant proteins. Splenocytes from immunized mice were isolated and cultured for 48 h. The cells were stimulated with the recombinant protein alone or in fusion with DnaK. Cells were further incubated with BrdU and DNA synthesis was quantified by BrdU immunodetection kit (M&M). ConA and culture medium were employed as positive and negative stimulation controls, respectively (not shown). The data are from two independent experiments.
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fig5: Analysis of lymphocyte proliferation in cultured splenocytes from mice immunized with recombinant proteins. Splenocytes from immunized mice were isolated and cultured for 48 h. The cells were stimulated with the recombinant protein alone or in fusion with DnaK. Cells were further incubated with BrdU and DNA synthesis was quantified by BrdU immunodetection kit (M&M). ConA and culture medium were employed as positive and negative stimulation controls, respectively (not shown). The data are from two independent experiments.

Mentions: To evaluate the effect of proteins in fusion with DnaK on the cellular immune response of the recombinant protein alone, mice were immunized with the recombinant proteins, individually or in fusion with DnaK. Spleens from PBS-injected mice were employed as controls. Lymphocyte proliferation showed an increased, boosted effect when animals were immunized with fusion proteins compared with leptospiral proteins alone (Figure 5). In all cases, but remarkably with DnaK-rLIC12730, an improvement was observed when animals were primed and stimulated with proteins in fusion with DnaK (Figure 5), having a stimulation index of 8.72. DnaK-Lsa21, DnaK-rLIC10494, and DnaK-Lp95 C-terminus showed stimulation index of 4.89, 2.25, and 4.09, respectively. High proliferation level was obtained when cells were treated with ConA, employed as positive control of the experiment (not shown). Addition of proteins, either alone or in fusion with DnaK to lymphocytes from animals that have not been primed with either proteins or the corresponding DnaK fusions, produced nonsignificant levels of proliferation (data 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 lymphocyte proliferation in cultured splenocytes from mice immunized with recombinant proteins. Splenocytes from immunized mice were isolated and cultured for 48 h. The cells were stimulated with the recombinant protein alone or in fusion with DnaK. Cells were further incubated with BrdU and DNA synthesis was quantified by BrdU immunodetection kit (M&M). ConA and culture medium were employed as positive and negative stimulation controls, respectively (not shown). The data are from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Analysis of lymphocyte proliferation in cultured splenocytes from mice immunized with recombinant proteins. Splenocytes from immunized mice were isolated and cultured for 48 h. The cells were stimulated with the recombinant protein alone or in fusion with DnaK. Cells were further incubated with BrdU and DNA synthesis was quantified by BrdU immunodetection kit (M&M). ConA and culture medium were employed as positive and negative stimulation controls, respectively (not shown). The data are from two independent experiments.
Mentions: To evaluate the effect of proteins in fusion with DnaK on the cellular immune response of the recombinant protein alone, mice were immunized with the recombinant proteins, individually or in fusion with DnaK. Spleens from PBS-injected mice were employed as controls. Lymphocyte proliferation showed an increased, boosted effect when animals were immunized with fusion proteins compared with leptospiral proteins alone (Figure 5). In all cases, but remarkably with DnaK-rLIC12730, an improvement was observed when animals were primed and stimulated with proteins in fusion with DnaK (Figure 5), having a stimulation index of 8.72. DnaK-Lsa21, DnaK-rLIC10494, and DnaK-Lp95 C-terminus showed stimulation index of 4.89, 2.25, and 4.09, respectively. High proliferation level was obtained when cells were treated with ConA, employed as positive control of the experiment (not shown). Addition of proteins, either alone or in fusion with DnaK to lymphocytes from animals that have not been primed with either proteins or the corresponding DnaK fusions, produced nonsignificant levels of proliferation (data 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