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Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs.

ABSTRACT: The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21-42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-? and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC6669209 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs.

Zhang Zhencang Z Luo Yan Y Zhang Yanming Y Guo Kangkang K

Journal of veterinary science 20190701 4

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of <i>Yersinia pseudotuberculosis</i> invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phospha ...[more]

PMID: 31364320

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Project description:Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.

Project description:(1) Background: There are currently limited treatments for castration-resistant prostate cancer. Immunotherapy involving Sipuleucel-T has increasingly drawn attention for prostate cancer management. BCG plays a vital role in treating bladder cancer, mainly by inducing immune activation, but is rarely used for prostate cancer. (2) Methods: The TCGA database, PCR, and Western blotting were used to analyze the expression of STEAP1 in mouse and human tissues. Then, we constructed a fusion protein vaccine with Mycobacterium tuberculosis Ag85B and three repeated octapeptide epitopes of a six-transmembrane epithelial antigen of the prostate 1 (STEAP1186-193), Ag85B-3×STEAP1186-193. The uptake of the fusion protein vaccine by DCs was evaluated by confocal microscopy, and DC markers were detected using flow cytometry after incubation with the fusion protein. The immune response against prostate cancer was evaluated by the LDH assay and xenografts in vitro and in vivo. Then, the tumor microenvironment was determined using IHC and ELISA. In addition, the epitope was mutated using CRISPR-Cas9 to illustrate that the fusion protein elicited immunization against STEAP1. (3) Results: The TCGA database analysis, PCR, and Western blotting showed that STEAP1 was highly expressed in human and murine prostate cancer. After the uptake of the purified fusion protein vaccine by DCs, CD11c, CD80, CD86, and MHC II were upregulated and triggered a cytotoxic T lymphocyte (CTL) response against TRAMP-C1 and RM1 cells in vitro. Furthermore, the fusion protein vaccine inhibited tumor growth and improved the tumor microenvironment in vivo, with more CD3+ cells and fewer FOXP3+ cells in the tumor. Serum IFN-γ and IL-2 were significantly higher than in the control group, while IL-4 expression was lower, indicating that the fusion protein vaccine activated Th1 immunity. The immune response against prostate cancer was greatly suppressed when the antigen targets were knocked out using CRISPR-Cas9. (4) Conclusion: In summary, our results provide the first evidence that a vaccine based on a fusion protein consisting of Ag85B and a prostate cancer octapeptide epitope with complete Freund's adjuvant (CFA), triggers a robust immune response and inhibits tumor growth in murine prostate cancer.

Dataset Information

Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs.

Publications

Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs.

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