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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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CD spectra of purified recombinant proteins depicted after refolding. (a) CD spectra (FAR-UV CD) of 10 μM of each recombinant protein in 10 mM Na-phosphate buffer (pH 7.4), except Lsa21 and Lp95 C-terminus region that were in 100 mM Tris (pH 12.0) 500 mM NaCl, performed at 20°C. Far-UV CD spectra are represented as an average of five scans recorded from 185 to 260 nm. Ellipticity (ϕ) is expressed in function of wavelength. (b) Percentage of secondary structure of the recombinant proteins according to the analysis of CD spectra data by the CAPITO software. ND: not determined.
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fig2: CD spectra of purified recombinant proteins depicted after refolding. (a) CD spectra (FAR-UV CD) of 10 μM of each recombinant protein in 10 mM Na-phosphate buffer (pH 7.4), except Lsa21 and Lp95 C-terminus region that were in 100 mM Tris (pH 12.0) 500 mM NaCl, performed at 20°C. Far-UV CD spectra are represented as an average of five scans recorded from 185 to 260 nm. Ellipticity (ϕ) is expressed in function of wavelength. (b) Percentage of secondary structure of the recombinant proteins according to the analysis of CD spectra data by the CAPITO software. ND: not determined.

Mentions: Genetically combined proteins were successfully obtained and high expression level of recombinant proteins was achieved using the pAE vector and E. coli BL21 (SI) (Figure 1(b)). DnaK and rLIC12730 proteins were expressed in a soluble form and purified from the supernatant. The other proteins were expressed in insoluble form, as inclusion bodies, and were purified after solubilization in 8 M urea and on-column chromatography refolding on Ni2+-charged beads. The expected recombinant protein bands either individually or fused with DnaK are visualized by Coomassie blue stained SDS-12% PAGE and shown in Figure 1(b): DnaK (69 kDa), rLIC10494 (25 kDa), DnaK-rLIC10494 (93 kDa), rLIC12730 (76 kDa), DnaK-rLIC12730 (143 kDa), Lsa21 (20 kDa), DnaK-Lsa21 (87 kDa), C-terminal portion of Lp95 (44 kDa), and DnaK-Lp95 C-terminal (111 kDa). Structural integrity of the purified proteins was assessed by circular dichroism (CD) spectroscopy. The method evaluates the secondary structure content of the protein and it is an important data to obtain after protein refolding. The CD spectrum of individually cloned and expressed protein and the spectrum of the corresponding fusion with DnaK are shown in Figure 2(a). Analysis of the spectrum data by CAPITO software is depicted in Figure 2(b), except for rLsa21 and Lp95 proteins that had their spectra badly resolved with very low absorption values. The data shows that DnaK, rLIC10494, and rLIC12730 secondary structure contents present a predominance of alfa-helix, the minima at 208 and 222 nm, and the maxima at 192 nm. The DnaK fusion proteins have had their secondary structure contents shifted to alfa-helix, similar to the DnaK spectrum alone. CD spectrum of DnaK presented higher absorption values than the ones of the proteins alone and has, probably, superimposed the CD spectra of the leptospiral proteins.


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)

CD spectra of purified recombinant proteins depicted after refolding. (a) CD spectra (FAR-UV CD) of 10 μM of each recombinant protein in 10 mM Na-phosphate buffer (pH 7.4), except Lsa21 and Lp95 C-terminus region that were in 100 mM Tris (pH 12.0) 500 mM NaCl, performed at 20°C. Far-UV CD spectra are represented as an average of five scans recorded from 185 to 260 nm. Ellipticity (ϕ) is expressed in function of wavelength. (b) Percentage of secondary structure of the recombinant proteins according to the analysis of CD spectra data by the CAPITO software. ND: not determined.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: CD spectra of purified recombinant proteins depicted after refolding. (a) CD spectra (FAR-UV CD) of 10 μM of each recombinant protein in 10 mM Na-phosphate buffer (pH 7.4), except Lsa21 and Lp95 C-terminus region that were in 100 mM Tris (pH 12.0) 500 mM NaCl, performed at 20°C. Far-UV CD spectra are represented as an average of five scans recorded from 185 to 260 nm. Ellipticity (ϕ) is expressed in function of wavelength. (b) Percentage of secondary structure of the recombinant proteins according to the analysis of CD spectra data by the CAPITO software. ND: not determined.
Mentions: Genetically combined proteins were successfully obtained and high expression level of recombinant proteins was achieved using the pAE vector and E. coli BL21 (SI) (Figure 1(b)). DnaK and rLIC12730 proteins were expressed in a soluble form and purified from the supernatant. The other proteins were expressed in insoluble form, as inclusion bodies, and were purified after solubilization in 8 M urea and on-column chromatography refolding on Ni2+-charged beads. The expected recombinant protein bands either individually or fused with DnaK are visualized by Coomassie blue stained SDS-12% PAGE and shown in Figure 1(b): DnaK (69 kDa), rLIC10494 (25 kDa), DnaK-rLIC10494 (93 kDa), rLIC12730 (76 kDa), DnaK-rLIC12730 (143 kDa), Lsa21 (20 kDa), DnaK-Lsa21 (87 kDa), C-terminal portion of Lp95 (44 kDa), and DnaK-Lp95 C-terminal (111 kDa). Structural integrity of the purified proteins was assessed by circular dichroism (CD) spectroscopy. The method evaluates the secondary structure content of the protein and it is an important data to obtain after protein refolding. The CD spectrum of individually cloned and expressed protein and the spectrum of the corresponding fusion with DnaK are shown in Figure 2(a). Analysis of the spectrum data by CAPITO software is depicted in Figure 2(b), except for rLsa21 and Lp95 proteins that had their spectra badly resolved with very low absorption values. The data shows that DnaK, rLIC10494, and rLIC12730 secondary structure contents present a predominance of alfa-helix, the minima at 208 and 222 nm, and the maxima at 192 nm. The DnaK fusion proteins have had their secondary structure contents shifted to alfa-helix, similar to the DnaK spectrum alone. CD spectrum of DnaK presented higher absorption values than the ones of the proteins alone and has, probably, superimposed the CD spectra of the leptospiral proteins.

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