The Immunogenicity Investigation Of Recombinant Rabies Virus Expressing IL-21

Rabies is a zoonotic disease that is caused by rabies virus?RABV?.About 59,000 people died of rabies each year,and China is one of the countries with high rabies incidence.Once the clinical symptoms of rabies appear,the mortality is almost up to 100%,and vaccination is the most effective way to prevent rabies.The rabies vaccine can activate the B cell immune response through T cell-dependent and T cell-independent pathways after immunization,thereby producing virus-neutralizing antibody?VNA?and providing protection against lethal rabies challenge.Previous studies have shown that lacking of IL-21 signaling could affect the production of VNA after immunization with RABV,suggesting that IL-21 plays an important role in the immunogenicity of RABV.Therefore,in order to further explore the mechanism of IL-21 in the immunogenicity of RABV,recombinant RABV expressing IL-21 was constructed by reverse genetic manipulation,and was investigated through in vitro and in vivo experiments in this study:First,we constructed recombinant RABV expressing IL-21 through reverse genetic manipulation,named LBNSE-IL21.The expression of IL-21 was detected by ELISA.The results showed that LBNSE-IL21 infected BSR cells could express IL-21 in a dose dependent manner.In addition,the growth curves of LBNSE-IL21 in BSR cells and NA cells were similar with those of parent virus LBNSE,indicating that the insertion of exogenous gene IL-21 did not affect virus replication.In terms of viral pathogenicity study,the mice were intracranially infected with high dose of LBNSE-IL21;obvious clinical symptoms were observed in LBNSE-IL21 infected mice,and the mice body weight changes were similar with that of LBNSE,suggesting that expression of IL-21 had no significant effect on viral pathogenicity.To further study the influence mechanism for over-expression of IL-21 on RABV immunogenicity,Balb/c mice were immunized with LBNSE-IL21 or LBNSE,and flow cytometry were performed to detect various types of immune cells in humoral immune response.Theses results indicated that no significant differences on activation of dendritic cells?DCs?were observed in lymph nodes or peripheral blood between LBNSE-IL21 and LBNSE immunized mice.However,significantly more follicular helper T?Tfh?cells and germinal center B?GC B?cells generation were detected in the lymph nodes of mice immunized with LBNSE-IL21 than those immunized with LBNSE.These results indicated that LBNSE-IL21 could affect the development of germinal centers by directly promoting the generation of Tfh cells and GC B cells.Furthermore,it was also found that LBNSE-IL21 could promote significantly more B cells differentiate to long-life plasma cells in the bone marrow of immunized mice than those immunized with LBNSE.Finally,to investigate the efficacy of LBNSE-IL21 as a rabies vaccine,ICR mice were vaccinated with LBNSE-IL21 or LBNSE,and serum samples from immunized mice were collected weekly from the first week to the seventh week post-immunization to detect the VNA titres by FAVN.The results showed that the mice immunized with LBNSE-IL21 produced significantly higher levels of VNA as early as 1 week post-immunization compared to those immunized with LBNSE,and these levels could last for 6 weeks.All these results indicated that over-expression of IL-21 could enhance the induction of VNA in mice immunized with LBNSE-IL21 compared to mice immunized with LBNSE.In addition,at the third week after immunization,mice were challenged by intracranially injecting with 50 MICLD₅₀ of CVS-24.As a result,92% of the mice immunized with LBNSE-IL21 were protected against the pathogenic RABV challenge,which was significantly higher than that of the mice immunized with LBNSE?68%?.In summary,our results indicate that immunization with LBNSE-IL21 generated significantly more Tfh cells,GC B cells and PCs than LBNSE immunization,resulting in enhanced VNA production and improved protection.Our study suggests that LBNSE-IL21 could be a promising rabies vaccine candidate.