The Enhancing Effect Of Manganese On The Efficacy Of Viral Vaccines And Its Possible Mechanisms

Manganese（Mn）is an essential metal involved in keeping growth,development and reproduction.Early researches showed that Mn²⁺could indue an increase in the expression of type I interferonα/β（interferonα/β,IFNα/β）in the serum of mice.Recently,Mn²⁺was found to activate the c GAS-STING signaling pathway to induce the transcription and production of interferonα/β.The induction of IFNα/βcould not only inhibit the spread of the virus between the infected cells and adjacent cells,but also markedly augmented the antibody responses against a poorly immunogenic soluble protein.Hence,Mn²⁺could be used as the adjuvant for virus vaccines to enhance the efficacy of viral vaccines.The underlying mechanisms by which Mn²⁺enhances the antibody responses to viral vaccines might involve in the signaling pathway of the induction of IFN-α/βand the interferon regulatory factors（IRF）.In addition to IRF3and IRF7 that were involved in regulating the transcription of IFN-α/βin the c GAS-STING signaling pathway,IRF5 was also a key transcriptional regulator of IFN-α/β.Furthermore,Mn²⁺could inhibit the expression of IL-10 in the cultured murine dendritic cells,and the activation of IRF5 could also inhibit the transcription of IL-10.The data suggested that Mn²⁺might enhance the antibody response for viral vaccines by inhibiting the expression of IL-10 and IRF5 signaling pathway might be involved in.IL-10,a cytokine secreted by a variety of immune cells,is immunosuppressive to inhibit the ability of antigen presenting cells（APCs）to present antigens to T cells and inhibit the phosphorylation activation of CD28 on CD4⁺T cells,leading to anergy or disability of antigen-specific T cells.Therefore,inhibition of the expression of IL-10 could have a synergistic effect on the adjuvanticity of Mn²⁺for enhancing the efficacy of viral vaccines.It has been reported that the anti-IL-10 antibodies or the anti-IL-10 receptor1 antibodies could be used as adjuvants to enhance the antibody response for vaccines,and IL-10 antisense oligos（ASOs）which could interfere the expression of IL-10 m RNA could also significantly increase the OVA antibody titer in the mice immunized with OVA antigens.These results indicated that IL-10 inhibitors could be used as viral vaccine adjuvant for enhancing the antibody response of viral vaccines and the adjuvanticity of Mn²⁺.Overall,in this study,we explore the possibility of using Mn²⁺as the adjuvant for viral vaccines,and unexpectedly,we found that the activation of IRF5 signaling pathway might involve in the adjuvanticity.We designed ASOs targeting the same sequences of both the human and mouse IL-10 m RNA and tested its inhibitory effects on IL-10 and its adjuvanticity for viral vaccines and Mn²⁺.The study might provide an experimental basis for the development of Mn as a new type of viral vaccine adjuvant.The results were as the following:1.Exploration of the possibility of Mn²⁺as the adjuvant of microbial vaccines.To explore whether Mn²⁺could be used as the viral vaccine adjuvant,we detected the effects of Mn²⁺on mouse macrophage-like cell line Raw264.7 cells,tested its effects on the immune cells in mouse splenocytes cultured in vitro and in peripheral blood and splenocytes in vivo,and detected its dose and time effect on immune cells in the spleen and draining lymph nodes isolated from the immunized mice.On the results of the above research,we tested the effect of Mn²⁺as a viral vaccine adjuvant on the antibody responses induced by recombinant protein vaccine and inactivated influenza virus vaccine and protective effect of Mn²⁺in the immunized mice challenged by influenza virus.The results showed that:1)Mn²⁺could activate macrophages,and might induce macrophages to produce IFN-α/βby activating the c GAS-STING signaling pathway.2)Mn²⁺alone was not limited to activating macrophages in vitro,but could also activate T cells,B cells and DCs in the splenocytes that were associated with the promotion of antibody responses;3)Mn²⁺used alone in vivo also had the same immunostimulatory activity as used in vitro;4)Mn Cl₂at 1000μg was the effective dose of Mn²⁺used as a viral vaccine adjuvant to activate immune cells,and could induce the activation B cell in mouse splenocytes at different time points or activate CD4⁺T/CD8⁺T cells,B Cells and DCs in draining lymph nodes;5)Mn²⁺significantly enhance the antibody responses for recombinant protein vaccines and inactivated influenza virus vaccine and protect the mice against the influenza virus infection.The data suggested that Mn²⁺could be used as the adjuvant for viral vaccines.2.Exploration the possible mechanisms of Mn exerting the adjuvanticity in viral vaccines.Since Mn²⁺could be used as the viral vaccine adjuvant,we tried to explore the possible mechanism of Mn exerting the adjuvanticity in viral vaccines.When Mn²⁺was cultured with murine splenocytes for different time,Mn²⁺could increase the m RNA expression of IFN-α/β,TNF-α,IL-6,IL-4 and BAFF,and when Mn²⁺was used as the recombinant protein adjuvant,Mn²⁺could also induce the m RNA expression of IFN-α/β、TNF-α、IL-4 and BAFF m RNA in the splenocytes or draining lymph nodes.To explore the reasons why Mn²⁺could induce the expression of these cytokines,we detected the expression and phosphorylation of STING,IRF3 and IRF7 in the splenocytes isolated from the mice immunized with Mn²⁺-formulated vaccines.In addition to IRF3 and IRF7,IRF5 is also a key transcriptional regulator of IFN-α/βand a transcription factor for TNF-αand IL-6 genes.Therefore,we also detected the expression and phosphorylation of IRF5 in the splenocytes isolated from the mice immunized with Mn²⁺-formulated vaccines.The data showed that Mn²⁺could obviously induce the expression and phosphorylation of IRF5 in the splenocytes isolated from the mice immunized with Mn²⁺-formulated recombinant Cp vaccine and could not significantly induce the expression and phosphorylation of STING,IRF3 or IRF7.And Mn²⁺could also induce the expression and phosphorylation of IRF5,not STING,IRF3 and IRF7,in the splenocytes isolated from the mice immunized with Mn²⁺-formulated recombinant hepatitis vaccine.Considering that TBK1 is an upstream kinase in IRF5 signaling pathway and could activate IRF5,we inferred that TBK1 might be involved in the activation of IRF5.The results showed that Mn²⁺could induce the expression and phosphorylation of TBK1 in the splenocytes isolated from Mn²⁺-formulated vaccines.The results indicated that Mn²⁺used as a viral vaccine adjuvant to enhance the efficacy of viral vaccines might be related to the activation of IRF5 via inducing the activation of TBK1.In order to detect whether Mn²⁺activated IRF5 could induce Blimp-1 expression,we used q PCR to detect the effect of Mn²⁺on the m RNA expression of Blimp-1 in the splenocytes.The results showed that Mn²⁺could increase the m RNA expression of Blimp-1 in the splenocytes isolated from na?ve mice or the mice immunized with Mn²⁺-formulated vaccines.To further detect whether Mn²⁺could induce the expression of IRF5 and Blimp-1 in the activated B cells,we tested the effective dose and time curve of Mn²⁺for activating B cells.We found that Mn Cl₂ at400μM could significantly activate B cells in the splenocytes cultured in vitro in 24h.And with the activation of B cells,Mn Cl₂ at 400μM could also increase the expression of IRF5 and Blimp-1 in 24h.Furthermore,the increased m RNA expression of Blimp-1in the splenocytes cultured with Mn Cl₂ for 24 h was abolished in the presence of an AAAG ODN,an oligodeoxynucleotide capable of interfering IRF5.Together,the results may reveal that the IRF5 signaling pathway involved in the adjuvanticity of Mn²⁺.3.Design of the anti-IL-10 antisense oligo as an adjuvant for enhancing the antibody response of viral vaccines and the adjuvanticity of Mn²⁺.To test whether the ASOs targeting the same sequences in human and mouse IL-10 m RNA could be used as a viral vaccine adjuvant and enhance the adjuvanticity of Mn²⁺,we designed two ASOs targeting human and mouse IL-10 m RNA（IO-1 and IO-2）and tested their activities.And then we selected the ASOs which could efficiently inhibit the expression IL-10 to be used as the viral vaccine adjuvant to test its effect on the antibody responses induced by the viral vaccine and the viral vaccine formulated with Mn²⁺.The data showed that IO-1could decreased the expression of IL-10 in Raw264.7 cells activated by LPS.And when IO-1 was used alone or combined with Mn²⁺,IO-1 could significantly enhance the antibody response induced by the viral vaccines.In conclusion,in this study,we found that Mn²⁺could be used as a novel type of adjuvants for viral vaccines,and the activation of IRF5 signaling pathway might involve in the adjuvanticity.In addition,we designed and screened anti-IL-10 antisense oligonucleotides（IO-1）which could be used as an adjuvant to enhance the antibody response induced by viral vaccine and the viral vaccine formulated with Mn²⁺.