Protective Role And Mechanism Of CD4⁺ T_RM In Mucosal Immunity Induced By Recombinant Staphylococcus Aureus Vaccine （rFSAV）

Background:Staphylococcus aureus（S.aureus）is a common opportunistic pathogen is a common pathogen that often causes severe infections such as acute pneumonia,osteomyelitis,endocarditis,arthritis,and sepsis.The incidence of S.aureus pneumonia accounts for about5%of community-acquired pneumonia and 10%-30%of hospital-acquired pneumonia,with high morbidity and mortality.The use of antibiotics has accelerated the emergence of resistant strains,posing great challenges to clinical treatment.Vaccination is an important means of effectively preventing bacterial infections,delaying bacterial resistance,and reducing the use of antibiotics.The team of the National Engineering Research Center of Immunological Products has developed the world’s first recombinant five-antigen Staphylococcus aureus vaccine（rFSAV）by preparing a“cocktail”of antigen components:（1）alpha-hemolysin（Hla）,（2）staphylococcal enterotoxin B（SEB）,（3）manganese transport protein C（Mnt C）,（4）staphylococcal protein A（Sp A）,and（5）iron surface determinant B N2 domain（Isd B-N2）.These core antigens target multiple key pathogenic points of the bacteria,and the immune response they induce plays a role in neutralizing toxins,preventing bacterial adhesion,slowing down bacterial metabolism,and inhibiting immune evasion.The vaccine has completed Phase II clinical trials,which showed good safety and immunogenicity,and is currently undergoing Phase III clinical trials（CTR20221329）.This vaccine is mainly used to prevent S.aureus infection after closed fracture surgery,and whether it has a protective effect on bacterial infections in mucosal tissues such as acute pneumonia is not clear,and the protective mechanism urgently needs to be clarified.Objectives:This study aims to evaluate the immune protective effect of rFSAV mucosal vaccine in preventing S.aureus pneumonia,and to elucidate the protective mechanism of IL-17A⁺CD4⁺tissue resident memory T cells(T_RM)induced by rFSAV mucosal immunization.Methods:1.rFSAV was immunized in mice via intranasal drip on days 0,7,and 14,and the survival rate after vaccination was evaluated by infecting mice with lethal doses of S.aureus Xen29,MRSA252,USA300,NEWMAN,and 8325-4;a living imaging instrument was used to monitor the infection process and proliferation of Xen29 in vivo.2.ELISA was used to detect antigen-specific Ig G and Ig A in serum,bronchoalveolar lavage fluid,and nasal lavage fluid;ELISPOT was used to detect IL-4,IL-17A,and IFN-γexpression levels in lung lymphocytes.3.B cells,CD4⁺T cells,CD8⁺T cells,and T_RMwere depleted separately in mice to evaluate the role of these cells in rFSAV-induced protection.4.Parabiosis experiments with na（?）ve WT CD45.1 and immunized CD45.2 mice were used to confirm the presence of CD4⁺T_RM,and lung IL-17A⁺CD4⁺T_RMinduced by the vaccine was detected by flow cytometry.After blocking peripheral circulating lymphocytes with FTY720,mice were infected with a lethal dose of Xen29 to evaluate the immunoprotective effect of lung IL-17A⁺CD4⁺T_RM.To observe the long-term protective effect of the vaccine,mice were infected with a sublethal dose of Xen29 on day 150.5.Anti-Ly6G antibody was used to deplete neutrophils.Using FTY720 to blocks lymphocyte egress from lymph nodes,the sublethal dose of Xen29 infected mice.Through bacterial colonization and in vivo imaging experiments,explore the effector cells that play a role in the downstream of CD4⁺T_RM;Study the dynamics of neutrophils and observe the recruitment of neutrophils by CD4⁺T_RM.6.To analyze the immune cell subsets of immunized mice infected with Xen29 by single cell sequencing,and to systematically evaluate the immune protective mechanism of vaccines.Results:1.Intranasal immunization with rFSAV could protect mice from lethal dose of S.aureus infection and inhibit the proliferation of S.aureus in lungs.The protective rates of rFSAV against Xen29,MRSA 252,USA300,NEWMAN,8325-4 were 100%（P=0.0002）,87.5%（P=0.0010）,77.8%（P=0.0002）,77.8%（P=0.0005）and 66.7%（P<0.0001）,respectively.rFSAV specific Ig G antibodies were detected in serum,alveolar lavage fluid and nasal lavage fluid,and high levels of mucosal Ig A antibodies were induced by mucosal immunization.rFSAV mucosal immunization enhanced the secretion of IL-17A（P<0.0001）and IFN-γ（P=0.0079）in lung lymphocytes,but had no effect on the expression of IL-4（P=0.7958）.2.After depletion of CD4⁺T cells by Anti-GK1.5 antibody,the vaccine protection in the lung was completely abrogated,and the bacterial colonization in the lung（P=0.0043）and spleen（P=0.0043）was significantly increased.After depletion of CD8⁺T cells in the whole body by Anti-CD8 antibody,the vaccine still had protective effect,and there was no significant difference in bacterial colonization in the lung（P=0.1592）and spleen（P=0.1342）between the two groups.There was no significant difference in the amount of bacterial colonization in the lungs between the immunized mice and the immunized mice（P=0.0608）,but the amount of bacterial colonization in the spleen was significantly higher than that in the immunized mice（P=0.0043）,after the B cells are eliminated.3.The existence of CD4⁺T_RMwas confirmed by parabiosis experiments.The paired mice shared circulating CD4⁺T cells,and the numbers of CD45.1（P=0.6857）and CD45.2（P=0.5306）cells in the blood did not differ significantly between the two types of mice.However,lung CD45.2 cells were mainly found in immunized CD45.2 mice,which were significantly higher than those in non-immunized CD45.1 mice（P<0.0001）.4.After rFSAV mucosal immunization,（CD44⁺CD69⁺CD62L^-）CD4⁺T_RMcells were induced in the lung tissues of mice,which was significantly higher than that in non-immunization mice（P=0.0079）,and these CD4⁺T_RMcells secreted more IL-17A（P=0.0079）.There was no significant difference in IFN-γ（P=0.0543）.FTY720 could block the entry of peripheral circulating lymphocytes into the tissues.There was no significant difference in the number of CD4⁺T_RMcells induced in the lungs of immunized mice after treatment with FTY720（P=0.9385）.After lethal dose Xen29 challenge,we found that the survival rate of FTY720-treated immunized mice was 66.7%,which was higher than that of non-immunized mice（P=0.0165）,but there was no significant difference in survival rate between immunized mice and FTY720-treated immunized mice（P=0.5514）.Mice were infected with Xen29 at a sublethal dose 150 days after immunization.CD4⁺T_RMcells in the lungs of the immunized mice proliferated rapidly after infection,and the amount of bacterial colonization in the lungs of the immunized mice was significant less than that of the non-immunized mice（P=0.0095）.5.After eliminating neutrophils,the immunized mice could not inhibit the proliferation of bacteria in the lungs,and the fluorescence intensity was significantly higher than that of the normal immunized mice（P=0.0286）.The amount of bacterial colonization in lung（P=0.0286）and spleen（P=0.0286）of the immunized mice with neutrophils depletion was much higher than that of the immunized mice without neutrophils depletion.The recruitment kinetics of neutrophils into BALF from non-immunized and immunized mice were studied,and it was found that the recruitment of neutrophils into lung of immunized mice was accelerated at 2h after infection（P=0.0286）.The results of immunohistochemistry also showed that more neutrophils were accumulated in the lung parenchyma of the immunized mice at 2h after infection（P=0.0022）.6.Single cell sequencing showed that CXCL1 and IL-23a were up-regulated in neutrophils of immunized group after S.aureus infection.S100A9 expression is significantly enhanced in non-immunized mice,and the accumulation of S100A9 impairs the killing ability of neutrophils.The analysis of neutrophils in the immunized mice showed that there were four heterogeneous subsets,which were"pro-inflammatory".Among them,Neutrophils1 subgroup accounted for the largest proportion.Enrichment analysis showed that Neutrophils1 was related to IL-17 signaling pathway and may be regulated by IL-17A⁺CD4⁺T_RMcells.There were also three heterogeneous subsets of macrophages,but all of them tended to be"M1 type"and played anti-inflammatory and bactericidal roles.The pseudo-time analysis revealed the transition from histone modification to pro-inflammatory immune function of macrophages after bacterial lung infection in mice.Conclusion:1.rFSAV mucosal vaccine can protect against pulmonary infection of different S.aureus strains and induce humoral and cellular immune responses.2.CD4⁺T cells are indispensable for vaccine efficacy and essential for the control of S.aureus infection.The induced lung IL-17A⁺CD4⁺T_RMcells could protect the body from bacterial infection independent of circulating memory T cells,and could persist in the lung tissue of mice for a long time.3.Pulmonary IL-17A⁺CD4⁺T_RMcells may rapidly recruit neutrophils to the lungs through IL-17 signaling pathway to enhance the mucosal immune protective effect of rFSAV,and cooperate with M1 macrophages to play an anti-infection role.