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[Construction of prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model].

AIM
To construct the prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluate its protective efficacy and immunogenicity in the Guinea pig Keratoconjunctivitis model.


METHODS
The HPV58 L1 gene was cloned into PUCMT, and the recombinant plasmid HPV58 L1-pCVD442 was constructed and introduced into attenuated Shigella (Sf301: deltavirG) with helper plasmid PRK2013 by filter mating. After homologous recombination, the positive colonies in Sf301: deltavirG strains contained HPV58L1 gene were selected and verified by PCR. The expressed HPV58L1 protein was harvested and analyzed by SDS-PAGE and Western blot. The bio-activity of the interested protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was proved with transmission electron microscope. Guinea pig Keratoconjunctivitis model was used to evaluate protective efficacy and immunogenicity of the vaccine. 20 days after immunization, serum HPV58L1-IgG, IgA level and serum sf301 LPS- IgG, IgA level were measured by ELISA, and HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes (SVCLN, MDLN, MSLN and pp) were measured by ELISPOT assay.


RESULTS
HPV58 L1 protein with MW 60 kDa was confirmed by Western blot. The ability of the interested protein to self-assemble into VLPs was identified by transmission electron microscope, and the result of murine erythrocyte hemagglutination assay indicated that the given proteins expressed by the recombinant bacillus had similar characteristics as the natural HPV58L1 protein.In the Guinea pig Keratoconjunctivitis Model, animal immune results showed that there were no Keratoconjunctivitis occurred in the immune group(HPV58-attenuated Shigella), and after Sf301 attacking, 80% eyes had no Keratoconjunctivitis occurrence. 20 days after immunization, serum HPV58L1-IgG, IgA level were obviously increased; but serum sf301 LPS-IgG position was just slightly increased; ELISPOT results showed that HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes were also obviously increased.


CONCLUSION
Recombinant HPV58L1-attenuated Shigella vector live vaccine could induce strong humoral immune responses in the immunized animals.