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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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Analysis of IgG production induced in mice by recombinant proteins. The sera from mice immunized with fused-proteins were analyzed by ELISA (a) and Western blotting (b). In (a) wells were coated with DnaK or surface proteins, as depicted; in (b) blotted proteins are DnaK (lane 1), Lsa21 (lane 2), rLIC10494 (lane 3), Lp95 C-terminus region (lane 4), and rLIC12730 (lane 5). M: molecular mass protein marker. In both methods, proteins were probed with the respective antifusion protein serum (1 : 20,000 dilution) and the reactions were developed with HRP-conjugated anti-goat IgG (1 : 10,000) (ELISA) and HRP-conjugated anti-mouse IgG (1 : 5,000) (Western).
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fig3: Analysis of IgG production induced in mice by recombinant proteins. The sera from mice immunized with fused-proteins were analyzed by ELISA (a) and Western blotting (b). In (a) wells were coated with DnaK or surface proteins, as depicted; in (b) blotted proteins are DnaK (lane 1), Lsa21 (lane 2), rLIC10494 (lane 3), Lp95 C-terminus region (lane 4), and rLIC12730 (lane 5). M: molecular mass protein marker. In both methods, proteins were probed with the respective antifusion protein serum (1 : 20,000 dilution) and the reactions were developed with HRP-conjugated anti-goat IgG (1 : 10,000) (ELISA) and HRP-conjugated anti-mouse IgG (1 : 5,000) (Western).

Mentions: Total IgG antibody elicited in mice by fused-proteins was analyzed by ELISA (Figure 3(a)) and Western blotting (Figure 3(b)). The DnaK and the leptospiral proteins were used to individually coat ELISA microplates. The immune sera from mice injected with the fusion proteins, DnaK-Lsa21, DnaK-rLIC10494, DnaK-Lp95 C-terminal region, and DnaK-rLIC12730 were employed to probe the respective coated protein. The ELISA data show that mice responded with IgG antibodies against both proteins (Figure 3(a)). These results were strengthened by Western blotting data (Figure 3(b)), which show that both blotted proteins are individually recognized by their corresponding mice antisera injected with the fusion proteins, except for the proteins Lsa21 (DnaK-Lsa21) and Lp95 C-terminal region (DnaK-Lp95), where the place of the expected bands are indicated by symbols (*).


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 IgG production induced in mice by recombinant proteins. The sera from mice immunized with fused-proteins were analyzed by ELISA (a) and Western blotting (b). In (a) wells were coated with DnaK or surface proteins, as depicted; in (b) blotted proteins are DnaK (lane 1), Lsa21 (lane 2), rLIC10494 (lane 3), Lp95 C-terminus region (lane 4), and rLIC12730 (lane 5). M: molecular mass protein marker. In both methods, proteins were probed with the respective antifusion protein serum (1 : 20,000 dilution) and the reactions were developed with HRP-conjugated anti-goat IgG (1 : 10,000) (ELISA) and HRP-conjugated anti-mouse IgG (1 : 5,000) (Western).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Analysis of IgG production induced in mice by recombinant proteins. The sera from mice immunized with fused-proteins were analyzed by ELISA (a) and Western blotting (b). In (a) wells were coated with DnaK or surface proteins, as depicted; in (b) blotted proteins are DnaK (lane 1), Lsa21 (lane 2), rLIC10494 (lane 3), Lp95 C-terminus region (lane 4), and rLIC12730 (lane 5). M: molecular mass protein marker. In both methods, proteins were probed with the respective antifusion protein serum (1 : 20,000 dilution) and the reactions were developed with HRP-conjugated anti-goat IgG (1 : 10,000) (ELISA) and HRP-conjugated anti-mouse IgG (1 : 5,000) (Western).
Mentions: Total IgG antibody elicited in mice by fused-proteins was analyzed by ELISA (Figure 3(a)) and Western blotting (Figure 3(b)). The DnaK and the leptospiral proteins were used to individually coat ELISA microplates. The immune sera from mice injected with the fusion proteins, DnaK-Lsa21, DnaK-rLIC10494, DnaK-Lp95 C-terminal region, and DnaK-rLIC12730 were employed to probe the respective coated protein. The ELISA data show that mice responded with IgG antibodies against both proteins (Figure 3(a)). These results were strengthened by Western blotting data (Figure 3(b)), which show that both blotted proteins are individually recognized by their corresponding mice antisera injected with the fusion proteins, except for the proteins Lsa21 (DnaK-Lsa21) and Lp95 C-terminal region (DnaK-Lp95), where the place of the expected bands are indicated by symbols (*).

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