Attenuated Salmonella Carrying The Plasmid Expressed BPV1-L2 For Inducing Neutralization Anti-HPV Antibodies

Papillomavirus (PV) are small DNA viruses infecting humans as well as many domestic and wild animal species, including birds, causing benign hyperproliferative lesions of both mucosal and cutaneous epithelia.Cervical cancer is the second leading cause of cancer deaths in women worldwide, and virtually all of these tumors are attributable to infection with some subset of human papillomaviruses (HPVs), of which HPV16 and HPV18 are found most frequently.Bovine papillomaviruses (BPV) is a family member of PV. BPV induces diseases of considerable veterinary importance in farm animals, but has also an enormous value as an in vivo model for HPV. In fact, due to the high species-specificity of Papillomaviruses, virologists have used BPV as a model for the study of HPV infection, its interaction with the host and with environmental co-factors.However, the requirement for multiple injections for a vaccine whose anticipated target population will be older than the population that receives childhood vaccines and the costing of polyvalent vaccines also will be higher than that of the general vaccines may represent a substantial hurdle for widespread implementation. This is particularly true in the developing world, which accounts for more than three-quarters of the worldwide cases of cervical cancer.BPV1-L2 1-88a.a,nowadays,is considered as a substantial candidate which could induce effect of cross-neutralization of Papillomavirus types including HPV16 and HPV18. Recombinant attenuated Salmonella strains that are attenuated yet invasive have been widely used as mucosal vaccine vectors to deliver pathogen-specific protective epitopes into the mucosal-associated lymphoid tissues. Via this route, both mucosal and systemic immune responses against the carrier and the foreign antigens may be obtained.In the present study, the BPV1-L2 gene was amplified from bovine neoplasitc organ genome DNA by PCR and cloned into pMD-18T. PCR amplification, double restriction enzymes digestion and sequencing analysis confirm the successful construction of the recombinant pMD-18T-L2 plasmid. The L2 gene was cloned into the prokaryotic expression plasmid pET28a(+) as pET28-BPV1-L2,which was then transformed into the E.coli JM109/BL21. The positive clones were confirmed by PCR and double restriction enzymes digestion。The recombination protein was expressed upon induction with IPTG as identified by SDS-PAGE.The BPV1-L2 gene was amplified from pET28a-BPV1-L2 plasmid by PCR and cloned into pMD-18T. PCR amplification, double restriction enzymes digestion confirm the successful construction of the recombinant pMT-18T-L2(Z) plasmid. The L2 gene was cloned into the eukaryotic expression plasmid pcDNA3.1a(+) as pcDNA3.1-BPV1-L2,which was then transformed into the E.coli JM109. The recombination eukaryotic plasmid to express 1-88a.a antigens in vitro was investigated in Vero Cells. pcDNA3.1-BPV1-L2 liposome mediated transfection of Vero cells. SDS-PAGE revealed a band of 12 kDa from cell lysates suggesting a protein is expressed.The recombinant eukaryotic expression plasmid pcDNA3.1a(+)/pcDNA3.1- BPV1-L2 was transformed by electroporation into attenuated salmonella strains Ty21a and PhoP/PhoQ, resulting in Ty21a-pcDNA3.1,Ty21a-pcDNA3.1-BPV1-L2,PhoP/PhoQ-pcDNA3.1 and PhoP/PhoQ- pcDNA3.1-BPV1-L2. The efficacy of the recombination attenuated salmonella(described above) were compared. Six to eight-week-old female BalB/c mice were divided randomly into six groups(six mice each).Mice in group 1 were administered intranasally with 1×PBS under anesthesia, Mice in group 2 were administered intranasally with 107 to 108 cfu of Ty21a-pcDNA3.1 under anesthesia, Mice in group 3 were administered intranasally with 107 to 108 cfu of Ty21a-pcDNA3.1-BPV1-L2 under anesthesia, Mice in group 4 were administered intranasally with 1×PBS under anesthesia, Mice in group 5 were administered intranasally with 107 to 108 cfu of PhoP/PhoQ-pcDNA3.1 under anesthesia, Mice in group 6 were administered intranasally with 107 to 108 cfu of PhoP/PhoQ-pcDNA3.1-BPV1-L2 under anesthesia. The mice were boosted three with the frequency of 7d with the same at the same dosage levels. Blood and vaginal fluid samples were collected at 28d point before challenge for antibody titration.The results of Western bloting of recombinant protein showed the target protein was reactive to BPV1-L2 antibody. Also the data of cell immunofluorescence and Western bloting of HPV16 showed that the vaccine can induce effect of anti-sera of HPV16. ELISA results showed that the neutralizing antibody against HPV16/HPV18 were induced in groups 3 and 6. compared to the other groups, the difference had statistical significance(p<0.05) after immunization. There were no statistical significance between blank groups of 1×PBS and control groups of Ty21a-pcDNA3.1/PhoP/PhoQ-pcDNA3.1. ELISA data showed that Cytokines including IL-2 and INF-γwere increased in groups 2,3,5 and 6.compared to the blank group, difference had statistical significance(p<0.05) after immunization. There were no statistical significance between 2,4 groups and 3,5 groups.In conclusion, the present study clearly indicates that the BPV1-L2 gene was expressed both in vitro and in vivo.the DNA vaccines via intranasally containing BPV1-L2 gene could be delivered by attenuated salmonella for induction of immune response.