Evaluation Of The Chikungunya Virus (CHIKV) Vaccine Based On Pseudovirus System

Chikungunya virus?CHIKV?is an arthropod-borne Alphavirus that is transmitted to humans primarily via the bite of an infected mosquito.CHIKV is becoming widely popular in Africa,South Asia and Southeast Asia,with high infection rate^[1].Infection of humans by CHIKV can cause Chikungunya fever?CHIKF?which is an acute febrile illness associated with severe,often debilitating polyarthralgias,fever,rash and even to death^[2].CHIKV has emerged in more than 100 countries,causing about one million people infected each year^[3].In 2017,WHO reported 10 potential infectious diseases for priority research,with CHIKV on the list.At present,there is no licensed vaccine and approved drugs against CHIKV.The process of drug discovery and vaccine research against these viruses is limited by the requirements of high level biosafety and the lack of easy-to-use and effective animal models.The aim of this project is to construct high-titer CHIKV pseudovirus,establish a live imaging mouse models and establish a set of safe,comprehensive and reasonable detection method of neutralizing antibody in vivo and in vitro,so that it can be used in screening and evaluation of vaccines and drugs in BSL-2laboratory environment.In this study,the expression plasmid pCMV3.1-CHIKV was used to construct pseudovirus,and the factors affecting the titer of pseudovirus were optimized.The packaging conditions were determined as follows:293T cells were co-transfected with membrane protein expression plasmid pCMV3.1-CHIKV and the modified HIV backbone plasmid pSG3?env.cmv.Fluc at ratio 1:2 using lipo3000.The culture supernatants containing the pseudovirus were harvested after 48h.With the optimization,the titer of CHIKV pseudovirus was up to 1.0×10⁷ TCID₅₀/mL.We also used the CHIKV pseudovirus to establish a series of safe and high-throughput neutralizing antibody assay.After determining the detection time,cell tropism,cell amount and virus dose,a high-throughput CHIKV pseudovirus neutralization antibody evaluation method was established:The samples were incubated with CHIKV pseudovirus of 400 TCID₅₀0 for 1 h,then added to 293T cells with 50000/well for 48h,and the neutralization antibody titer were calculated.This method is highly sensitive and it may provide a platform for screening and evaluation of antiviral products such as vaccine,monoclonal antibody and small molecular compounds.Meanwhile,a imaging mouse model of CHIKV pseudovirus was established based on the high titer pseudovirus.Four weeks BALB/c mice were infected by intravenous?IV?.The distribution and metabolism of the virus in mice were observed dynamically by using in vivo imaging technique,and the AID₅₀0 of BALB/c mice infected with CHIKV pseudovirus was 4.9×10³ TCID₅₀.The establishment of this imaging mouse model makes it possible to evaluate the CHIKV vaccine in BSL-2 environment.In the present study,the DNA vaccine against CHIKV was successfully constructed,and the serum of guinea pigs and mice immunized with DNA vaccine were detected by the established neutralizing antibody assay in vitro.It was found that high titer neutralization antibody in guinea pigs and mice could be induced by the DNA vaccine,and the ID₅₀0 was up to 1:8000.Using the established mouse model,the passive transfer of immune serum was evaluated in vivo.The results showed that the serum had protective effect on mice,and the protective effect was related to the titer of antibody?R²=0.66?.In addition,our results showed that by combination of DNA vaccine and GM-CSF,then followed by electroporation could enhance immune responses in mice?P<0.0005?,and the antibody titer was related to the protective effect?R²=0.73?.The protective effect of passive immunity was compared with active immunity.It was certain that there was no significant difference in the protective effect of the two immunity?P=0.46?.This result confirmed the main role of neutralizing antibodies in anti-CHIKV infection.In conclusion,we have successfully constructed a high titer CHIKV pseudovirus,and established a visualized pseudovirus mouse model for the first time.Based on this,a series of safe and high-throughput neutralizing antibody assay in vitro and in vivo were established.This detection platform can be operated in the BSL-2 laboratory and will be an ideal evaluation method for antiviral agents such as drugs,vaccines,and monoclonal antibodies.