The Mechanism Of TLR Signaling Pathway In Immune Responses To Adenoviral Vector-based Vaccines

Currently,adenoviral vectors?AdV?are the most commonly used viral vectors and are widely used in vaccines and gene therapies.AdVs have the advantages of high safety,easy amplification,and can induce antigen-specific humoral and cellular immunity at the same time.Secreting antibodies by B cells during humoral immune responses is an effective prevention mechanism against foreign pathogens.AdV-based vaccines have a great advantage in the rapid production and maintenance of antibodies.Therefore,the mechanism of AdV-based vaccines is largely studied,which is of great significance to the rational design of emergency vaccines.Toll-like receptors?TLRs?are a group of pathogen recognition receptors in innate immunity.The purpose of this study was to explore how AdVs activate innate immunity via TLR signaling pathways.According to the distribution and structural characteristics of TLR,TLR2?TLR4 and TLR7 signaling pathways were selected as the object of study,adenoviral vector serotype 5-based anthrax vaccine?Ad5-PA?was used as model immunogen.From the aspects of antigen presentation and germinal center responses,the expression of cytokines,co-stimulatory molecules and transcription factors was studied,systematically.In this study,the results showed that the expression of TLR2,TLR4 and TLR7were up-regulated after stimulation,suggesting that these TLRs are involved in the immune response induced by Ad5-PA.Flow cytometry and ELISA assays were used in this study,and the decline of PA-specific antibodies,cytokines?IL-6,TNF-?and IL-1??was seen after immunization.Besides,the decrease in the number of plasma cells,and the impaired activation and maturation of antigen-presenting cells were also occurred because of TLR deficiency.These results indicate that the innate immune responses generated by Ad5-PA immunization are affected by the TLR deficiency in different aspects.Flow cytometry was used to detect the expression of CD69 and CD86 on the surface of various APCs after Ad5-PA immunization.The results showed that Ad5-PA had significant effects on activation of B cells and NK cells in mice,and can significantly up-regulate the expression of CD86 on the surface of mDC and M?cells.Correspondingly,TLR deficiency significantly reduce the activation of NK cells,but only in TLR4-KO mice,the decreased activation of B cells and the down-regulation of CD86 expression and integrins CD11b on mDC was statistically significant.We also found that the number of neutrophils in TLR2-KO mice after immunization was significantly lower than that of WT mice.Among which,TLR4 has more significant effects on the antigen-presenting cells than TLR2 and TLR7.In addition,the expression of HMGB1 gene in RAW264.7 stimulated by Ad5-PA was also investigated by qPCR.The results showed that the expression of HMGB1 gene was significantly increased,suggesting that HMGB1 may be the potential ligand for the activation of TLR4signaling after immunization.The changes of germinal center?GC?after Ad5-PA immunization were observed by immunofluorescence.The results showed that the absence of TLR4 significantly reduced the GC area.The number of GC B cells in three TLR deficiency mice was significantly reduced,and the absence of TLR4 down-regulated the expression of Fas on the GC B cells,and the formation of Bcl6⁺T_FH,which may in turn affects the auxiliary effect of T_FH;Loss of TLR7 results in an increase in the number of T_FR,and a decrease in the proportion of proliferation in T_FH cells,which ultimately leads to a decrease in the number of T_FH cells and an effect on the differentiation of GC B cells.Depletion of TLR2 leads to a decrease in the expression of Bcl6 in GC B cells,which in turn affects GC B cell function.The above results indicate that the three TLR signaling pathways participate in different stages of GC B differentiation,and TLR4 signaling pathway plays an important role in the GC reaction after Ad5-PA immunization.Down-regulation of Bcl6 expression in Ramos cells after inhibited by STAT3 inhibitor indicated that Bcl6 expression in GC B after Ad5-PA immunization was regulated by STAT3.The mice were immunized with the Adenoviral structural proteins Fiber and Hexon,respectively.The results showed that Hexon could stimulate the expression of TLR4 on dendritic cells more effectively.It was also found that the up-regulation of TLR4stimulated by Ad5-PA could be neutralized by anti-HMGB1 antibody,again suggesting that HMGB1 may act as an endogenous ligand of TLR4 to induce a cascade of downstream pathways of TLR together with Hexon in Ad5-PA immunization.We then used the TLR4 ligand MPLA containing Addvax or Alum to co-immunize mice with the antigen PA,similar immunological effects as Ad5-PA were seen.Thus,simultaneous enhancement of the presentation of APCs and activation of TLR4 signaling pathways can more effectively enhance antigen-specific humoral immune responses.Taken together,this study found that TLR signaling plays an important role in the key steps of antibody production in AdV-based vaccines.This study also demonstrated that the significance of activating the intact immune responses,providing the basis for the rational design of emergency vaccines in the future.This study also found that the activation of TLR4 by Ad5-PA may depend on the Hexon in an HMGB1-dependent way.In addition,the particle adjuvant systems containing TLR4 ligand can enhance the immune effect of antigen and stimulate the rapid production of antibodies.The mechanism of immune responses to Ad5-PA can further provide insights to the design of novel vaccines.