Analysis Of Main Genes Of Mosquito-borne ZIKV And CHIKV In Yunnan Province And Experimental Immunization Study Of Recombinant Genetically Engineered Vaccines

Zika virus(ZIKV)is a mosquito-borne virus transmitted by Aedes mosquito and was first discovered in Uganda in 1947.It broke out in Brazil in 2015 which spread rapidly to multiple countries and regions in America and Asia.ZIKV causes symptoms such as nervous system diseases or microcephaly in newborns.In February2016,the World Health Organization declared ZIKV as“a Public Health Emergency of International Concern”.Chikungunya virus(CHIKV)was first discovered in Tanzania in 1952 and has burst on a global scale again since 2004.The virus is mainly transmitted by Aedes mosquito,causing symptoms of fever,joint pain and rash in patients.With the exchange of global economic trade and tourism,imported ZIKV or CHIKV carriers have increased in China.Cuttently,there is no effective vaccines against ZIKV or CHIKV infection.In this study,the prevalence of mosquitoes carrying ZIKV and CHIKV in parts of Yunnan Province was tested in 2018,and their genetic evolution was analyzed.By using a human type 5 adenovirus vector and a vaccinia Tiantan strain vectors,recombinant vector virus candicate vaccines co-expressing ZIKV and CHIKV structural proteins were constructed.Mice immunization experiment was used to verify the immunogenicity and protection of the recombinant vector vaccines.The main findings are as follows:(1)The detection of ZIKV and CHIKV in mosquitoes in Pu'er City and Dali Autonomous Prefecture of Yunnan Province in 2018 shows thar no ZIKV was detected in the collected mosquito samples and the positive infection rate of CHIKV was 12.5%.Among the samples,CHIKV was mainly carried by Aedes aegypti and Culex pipiens.Genetic evolution analysis shows that CHIKV detected in Yunnan belongsd to the ECSA genotype,and was highly homologous with Indian strain and Sri Lanka strain.It is speculated that the CHIKV carried by mosquitoes on Yunnan border was spread to China through Sri Lanka and India.Besides,CHIKV used in this study did not contain the mutaiton of E1 A226V,indicating that the detected CHIKV may not be transmitted by Aedes albopictus.(2)In this study,prME and E genes of ZIKV SY012016 strain and E2,E1 and E2-6K-E1 genes of CHIKV 0706a TW strain were used respectively to construct the candidate vaccines of recombinant human type 5 adenovirus and the vaccinia Tiantan strain,and mice immunization experiment was carried out.The results show that ZIKV or CHIKV candidate vaccines expressed by adenovirus or vaccinia virus could induce humoral and cellular immune responses in mice.When challenged with ZIKV or CHIKV,the vaccines could inhibit ZIKV or CHIKV replication in mouse organs.(3)ZIKV prME gene and CHIKV E2-6K-E1 gene were cloned into human type5 adenovirus and vaccinia Tiantan shuttle plasmids to construct the recombinant adenovirus candidate vaccine of rAd-ZIKV-CHIKV and the recombinant vaccinia candidate vaccine of rdVTT-ZIKV-CHIKV co-expressing ZIKV prME protein and CHIKV E2E1 protein.Mice were immunized with adenovirus Prime-vaccinia virus Boost strategy and a challenge text was carried out.The results show the Prime-Boost immunization strategy could induce stronger humoral and cellular immune responses in mice.The viral load of organs and blood of the immunized group was two orders of magnitude lower than that of the control when challenged with ZIKV or CHIKV,indicating that the heterologous Prime-Boost immunization strategy could enhance humoral and cellular immune responses of mice while reducing the immunity of viral vector,and inhibit viral replication and viremia in mouse organs.(4)Based on ZJ01 adjuvant and ZJ02 adjuvant,components A,B,C and D were added respectively to construct the second-generation composite vaccine adjuvants.The adjuvants were respectively mixed with the recombinant vaccinia virus of rdVTT-ZIKV-E and ZIKV E proteins,and then the tested mice were immunized to verify the immunopotentiating effect of different adjuvants on vecotr vaccines and subunit vaccines.The results showed that ZJ01 adjuvant and ZJ02 adjuvant supplemented with A and D could promote stronger humoral and cellular immune responses in vector vaccines and subunit vaccines.The levels of IgG,IG1,IgG2a,and IgG2b antibodies and specific antibodies in serum were 1.50 to 3.35 times the adjuvant-free group at 35 dpi.The neutralizing antibodies were also 1.75-2.5 times the adjuvant-free group at 35 dpi.The CD4~+and CD8~+T lymphocytes were 1.22-1.28 and 1.02-1.09 times the adjuvant-free group respectively at 35 dpi.It is shown that the ZJ01 adjuvant and ZJ02 adjuvant containing components A and D could better enhance the immune effect of the vector vaccines and the subunit vaccines.In summary,this study examined the status of ZIKV and CHIKV carried by mosquitoes in Yunnan province,and provided reference for the epidemiological investigation of ZIKV and CHIKV in Yunnan,China.The construction of candidate vaccines co-expressing ZIKV and CHIKV by using recombinant adenovirus and vaccinia vaccines and the immunizaiton of mice via heterologous Prime-Boost immunization strategy could induce high levels of humoral and cellular immune responses while reducing auto-immunity of vaccine vectors,and provide effective protection for challenged mice.The adjuvants used in vector vaccines and subunit vaccines were successfully screened through the study of compound adjuvants,which could significantly enhance the immune effect of vector vaccines and subunit vaccines.This study lays foundation for the study of ZIKV and CHIKV genetically engineered vaccines and complex adjuvants.