Study On The Mechanism Of HMGB1 And CXCL13 Promoting Humoral Immune Response Induced By Rabies Virus

Rabies is a zoonotic disease induced by rabies virus?RABV?.When rabies occurs in human or animal,the mortality rate is almost 100%.In most parts of the world,rabies is still an important public security issue and causes more than 59000 human deaths every year.Rabies poses a serious threat to human health and safety,and brings the people a heavy psychological burden.Mass vaccination has largely controlled the spread of human rabies in developed countries,but the incidence of human rabies in developing countries continues to be widespread.Virtually all?99%?of human rabies deaths are caused by dog bites,so it is an urgent need to acquire an affordable and efficacious vaccine to control or eliminate dog-mediated rabies in developing countries.At present,there are many kinds of rabies vaccines,but the development of RABV live attenuated vaccine and recombinant live vector vaccine is the most potential way to control or treat rabies.High mobility group box 1?HMGB1?is a highly conserved and non-histone chromosomal protein,promotes the maturation of DCs and mediates immune responses in the noninfectious inflammatory response.The chemokine CXCL13 recruits follicular helper T cells and B lymphocytes to the secondary lymphoid organs,which contributes to the formation and development of B lymphoid follicles in secondary lymphoid organs.Previous studies have shown that overexpression of cytokines or chemokines such as macrophage colony stimulating factor?GM-CSF?or macrophage inflammatory protein-1??MIP-1??can enhance the immunogenicity of RABV live attenuated vaccine.Continuation of this idea,HMGB1 and CXCL13 were selected for the development of RABV vaccines,and their functionary mechanisms were studied.The following two studies were carried out in this paper:In the first study,all serine residues in both nuclear localization signals of HMGB1 were ^mutant into alanine residues and a signal peptide sequence was ligased in front of the HMGB1 gene,so the ^mutant HMGB1 could not enter the nucleus and was conducive to secrete in the extracellular,so as to construct the ^mutant HMGB1(HMGB1^mut).The HMGB1 ^mut gene was inserted into the genome of RABV strain LBNSE,and then the recombinant RABV expressing HMGB1 ^mut was rescued.The effect of HMGB1 ^mut on the immunogenicity of RABV and its mechanism was studied.Indirect immunofluorescence and Western blotting results showed that HMGB1 ^mut was expressed in the BSR cells infected with LBNSE-HMGB1 ^mut and secreted into the culture supernatant.Interestingly,the replications of RABV in BSR cells and NA cells were inhibited by the wild-type HMGB1?HMGB1wt?or HMGB1 ^mut,and the apoptosis of BSR cells induced by RABV was inhibited by the expression of HMGB1 wt or HMGB1 ^mut,but the cell activity was not changed.It was showed that HMGB1 ^mut significantly promoted the maturation of bone marrow derived dendritic cells in vitro and significantly recruited and/or matured dendritic cells?DCs?,thereby increasing the number of follicular helper T?Tfh?cells,germinal center B?GC B?cells and plasma cells in vivo.Further studies showed that mice immunized with LBNSE-HMGB1 ^mut produced more RABV virus-neutralizing antibody?VNA?than mice immunized with parent virus LBNSE or LBNSE-HMGB1 wt and provided better protection without harmful effects on mice.Together,these findings allow us to better understand the role of HMGB1 ^mut in RABV-induced humoral immune response and provide some clues for the development of more efficient RABV vaccines.It was the first time approved that HMGB1 was a promising adjuvant in a live attenuated virus vaccine without significantly affecting the viral replication.In the second study,the mouse chemokine CXCL13 was inserted into the genome of the RABV strain LBNSE,and the recombinant RABV expressing CXCL13 was rescued to study the effect of CXCL13 on the immunogenicity of RABV and its mechanism.The expression of CXCL13 was identified by ELISA.In vitro and in vivo experiments showed that the growth characteristic of RABV was not affected by the expression of CXCL13.The CXCL13 expressed by LBNSE-CXCL13 had biological activity which recruited the cells expressing its receptor CXCR5 in vitro and in vivo.Further studies showed that the growth and development of the draining lymph nodes and the formation of GCs were promoted by overexpression of CXCL13.Recombinant RABV expressing CXCL13 recruited more GC B cells than parent virus LBNSE or LBNSE-GM-CSF,and then activated more plasma cells,increased the production of VNA and provided better protection.In addition,there were highly significantly positive correlations among plasma concentration of CXCL13,RABV VNA titers and GC activity,so CXCL13 was a plasma marker for the detection of RABV-induced humoral immune response.In conclusion,CXCL13 is a promising RABV adjuvant molecule,and RABV-induced humoral immune response can be indirectly evaluated by the concentration of plasma CXCL13.Combined with our previous results,it was found that CXCL13 had a different mechanism to promote humoral immune response induced by RABV compared with previous studies.CXCL13 recruits follicular helper T cells and B cells other than promotes the maturation of DCs,activates more GC B cells,and activates more plasma cells,thus promots humoral immune response in mice.This study lays the theoretical foundation and provides new ideas for the development of novel RABV vaccines.