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P01.08

Fusion of CTA and CTB Gene to Immunogen Significantly Enhances the Immunogenicity of DNA Vaccine


INTRODUCTION:Cholera toxin and its two subunits (CTA,CTB) have been intensively investigated as mucosal adjuvants for protein based vaccine. In this study we evaluated the adjuvanticity of CTA and CTB in modality of either mixing their encoding plasmids with DNA vaccine or fusing their encoding genes to immunogen encoding gene.

METHODS:DNA and recombinant vaccinia vaccines expressing HIV-1 AE strain tat, rev, intergrase(C-half), vif, nef fusion gene(designated as TRIVN) have been constructed. Overlapping PCR was employed to link CTA/CTB and TRIVN gene, tthen the fused genes(TRIVN-CTA and TRIVN-CTB)were cloned into eukaryotic expression plasmid vector(pSV1.0). Six groups of female BALB/c mice were immunized with mock control, pSV-TRIVN, pSV-TRIVN-CTA, pSV-TRIVN-CTB, pSV-TRIVN mixed with CTA or with CTB respectively in a DNA priming-recombinant vaccinia boosting regimen. Two weeks after the final injection, mice splenocytes were collected and IFN-γ ELISPOT assay were used as readout for specific T cell response. Statistical analysis was performed by using Prism5.0 software.

RESULTS:Figure 1. Recombinant plasmid AE-TRIVN-CTA, AE-TRIVN-CTB in vitro expression verification. For  improving the immugenity of the DNA vaccine, we construct plasmid AE-TRIVN-CTA, AE-TRIVN-CTB with overlap PCR. Our data showed that all constructed plasmids are capable of efficiently expressing their inserted genes.

Table 1. Mice were immunized with 100 μg of DNA  vaccine.Six groups of female BALB/c mice were immunized with mock control, pSV-TRIVN, pSV-TRIVN-CTA, pSV-TRIVN-CTB,pSV-TRIVN mixed with CTA or with CTB respectively in a DNA priming-recombinant vaccinia boosting regimen.

Figure 2.  Total Specific T cell responses against AE peptides elicited by pSV1.0, AE-TRIVN, AE-TRIVN-CTA, AE-TRIVN-CTB, AE-TRIVN+CTA, AE-TRIVN+CTB.All groups immunized with vaccines raised significant more T-cell response than mock control. ELISPOT based on IFN-γ secreting showed that the specific T cell responses elicited by pSV1.0-TRIVN (520±150 SFCs/106 splenocytes), which was lower than the regimen together with CTB (734±240 SFCs/106 splenocytes), CTA regimen(692 ±220 SFCs/106 splenocytes). T-cell responses elicited by pSV-TRIVN-CTB (1548±330SFCs/106splenocytes) and pSV-TRIVN-CTA (1642±514SFCs/106splenocytes) were significantly higher than that by pSV1.0-TRIVN (520±150SFCs/106splenocytes), pSV1.0-TRIVN mixed with CTA (692±220SFCs/106splenocytes) and pSV1.0-TRIVN mixed with CTB (734±240SFCs/106splenocytes). Data showed  that  AE-TRIVN-CTA, AE-TRIVN-CTB could  induce significantly higer T cell response than AE-TRIVN, AE-TRIVN+CTA,TRIVN+CTB. No significant differences were observed among groups inoculated with TRIVN alone or adjuvanted by CTA/CTB subunit proteins.

Figure 3. Specific T cell responses against AE peptides(Tat, Rev, Intergrase(C-half), Vif, Nef) elicited  by AE-TRIVN, AE-TRIVN-CTA, AE-TRIVN-CTB.Though TRIVN-CTA and TRIVN-CTB fusion vaccines mounted comparable level of total IFN-γ+ T-cell responses, only TRIVN-CTB elicits significantly T-cell responses against Tat, which is a subdominant component in the fusion immunogen.

CONCLUSION:CTA and CTB could serve as potent adjuvants for DNA vaccine in immunogen-CTA/CTB fusion modality. Compared with CTA,CTB may enhance T-cell responses against subdominant epitopes in the immunogen and broaden the T-cell immune responses.

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