Studies On Design And Generation Of A Dual-subunit Th17-stimulating Vaccine Against Pseudomonas Aeruginosa And Its Protective Mechanism

Background:Pseudomonas aeruginosa(PA)is one of the most common pathogens in wound infections and hospital-acquired infections.To deal with the high incidence and high antibiotic resistance of PA,an effective PA vaccine is also urgently needed while developing new antibiotics.Although clinical trials of many PA vaccines have been carried out previously,none were successful.PA vaccines at the current stage mainly rely on serum-specific antibodies and cannot take effect at the front line of invasion.Furthermore,the high genetic diversity of PA may also be the reason for the failure of the previously tested vaccines.Therefore,highly efficient induction of a broad-spectrum mucosal protective response will be the key to the successful development of PA vaccines.A vaccine-induced Th17 response plays a key role in the process of combating lung infections by multiple micro-organisms,because it does not rely on serum-specific antibodies and can provide broad-spectrum protection.Tissue-resident memory T cells(TRM)reside in non-lymphatic tissues and play the role of long-term immune monitoring.TRMs can rapidly respond to local infection in the skin,respiratory mucosa and similar barrier tissues.In the process of PA infection,the level of IL-17A increased significantly and the symptoms of infection increased significantly once IL-17A was neutralized.Attenuated PA vaccines can protect against multiple PA serotypes by inducing Th17 responses.Previous studies have identified 5 protective Th17-stimulating antigens using an in vitro translation system.However,due to their low production and limited protective effect,they were unsuitable for further translational studies.Therefore,an alternative strategy to identify novel Th17-stimulating antigens is needed to successfully develop PA vaccines.Objectives:1.To screen protective antigens that efficiently induce a Th17 response in the lungsfrom a genetically engineered PA antigen library.2.To identify epitopes in the protective Th17-stimulating antigens,design a subunit vaccine,and elucidate its protective mechanism.3.To clarify the protective effect and mechanism of TRM cells in the protection mediated by a mucosal delivered PA vaccine.Methods:1.Ten soluble recombinant PA antigens with high purity and stability were used to intranasally immunize mice.Flow cytometry was applied to detect the proportion and quantity of Th17 cells(CD3⁺CD4⁺IL-17A⁺)in the lungs.Mice were challenged with a lethal dose of PA(XN-1 1×10⁷ CFU/mouse)to evaluate the protective effect of candidate antigens.2.Experiments with antigen-specific CD4⁺T cells,IL-17A KO mice and IL-17A neutralization confirmed the contribution of Th17 to the immune protection elicited by the novel antigens re Pcr V and re Amp C.3.Mice were intranasally or intramuscularly immunized with re Pcr V,then challenged with PA,and the protective effects were compared in terms of survival rate,lung tissue pathology,bacterial colonization and concentrations of pro-inflammatory cytokines,the quantification of the anti-re Pcr V antibody titer and opsonophagocytic killing(OPK)activity,as well as the proportion and quantity of re Pcr V-specific Th17 in the lungs.4.The immunodominant epitopes in re Pcr V and re Amp C that induce a Th17 response were identified using an ELISpot assay,after which the dual-subunit antigen vaccine PVAC was constructed and produced.Mice were intranasally immunized with PVAC,after which their serum specific antibody titers and lung Th17 levels were determined.The mice were then challenged using four PA strains with different serotypes to evaluate the broad-spectrum protective effects.5.Mice were intranasally immunized with re Pcr V and the IL-17A⁺CD4⁺TRM(CD44^highCD4⁺CD69⁺CD62L^-IL-17A⁺)response in the lungs was assessed.FTY720 was used to deplete the peripheral lymphocytes,after which the protective effect of the above-mentioned cells was evaluated by comparing the survival rate,infection symptoms,weight loss,lung pathology and bacterial colonization in the mice.Results:Part I Screening of protective Th17-stimulating PA antigens1.The top three antigens that induced Th17 response were identified as re Opr L,re Amp C,and re Pcr V.The survival rates of mice after individual immunization with these three antigens were 50%,50%,and 100%,respectively.The survival rates of mice immunized with the other tested antigens were not significantly different from those of the control group.Thus,re Amp C and re Pcr V were identified as novel protective PA antigens that induce Th17responses in mice.2.The survival rate of mice in the re Pcr V intranasal immunization group(IN)was significantly higher than in the intramuscular immunization group(IM).Compared with the IM group,the inflammatory lung pathology of the IN group was less severe,while the amount of bacterial colonization and the levels of pro-inflammatory cytokines were significantly lower,indicating that the protective effect of intranasal immunization with re Pcr V was significantly better than that of intramuscular immunization.The serum anti-re Pcr V Ig G titer of the IN group was significantly lower than that of the IM group.The OPK test results showed that the sera elicited by the two immunization methods had a similar killing effect on PA.The proportion and number of Th17 cells in the IN group was significantly higher than in the IM group.Part II The protective effect of re Pcr V and re Amp C depends on the Th17 response1.After adoptive transfer of CD4⁺T cells from mice immunized with re Pcr V or re Amp C,the levels of bacterial colonization,inflammatory lung pathology and pro-inflammatory cytokines were significantly decreased.The survival rates of IL-17A KO mice immunized with re Pcr V or re Amp C after challenge were 30%and 10%,respectively,which was significantly lower than that of identically immunized wild-type mice.After intranasal immunization with re Pcr V or re Amp C,the survival rates of mice treated with a monoclonal IL-17A antibody were decreased to 30%and 0%,respectively,which was significantly lower than in the control group.2.Notably,the titer of anti-re Amp C antibodies in the sera of re Amp C immunized mice was not significantly different from that of unimmunized mice.No significant difference of OPK activity was observed between the immunized and na?ve sera.Part III Screening of Th17-stimulating epitopes and construction of epitope-based vaccines1.The Th17-stimulating epitopes in re Pcr V were identified as Pcr V_8-25,Pcr V_29-46,Pcr V_43-60,Pcr V_57-74,Pcr V_64-81,Pcr V_78-95,Pcr V_92-109,Pcr V_106-123,Pcr V_197-214,and Pcr V_218-235.Similarly,the Th17-stimulating epitopes in re Amp C were identified as Amp C_64-81,Amp C_78-95,Amp C_99-116,Amp C_155-172,Amp C_169-186,Amp C_183-200,Amp C_204-221,and Amp C_281-298.2.The multi-epitope fusion antigen PVAC(re Pcr V_27Glu-126Gly-GSGGSG-re Amp C_full-length)was constructed by connecting the N terminus of Pcr V to full-length re Amp C using a GSGGSG linker and successfully expressed in E.coli.The purity of PVAC reached as high as87.6%after purification.The titer of anti-PVAC antibodies and lung Th17 response was significantly higher in the immunized group.After challenged with a lethal dose of four clinical PA strains(464,XN-1,ZNJ004 and PA 451),the survival rates of PVAC-immunized mice were 70%,60%,100%and 50%,respectively which was significantly higher than in the unimmunized group.Part IV Study of the role of TRM cells in the protection elicited by intranasal immunization with re Pcr V1.After intranasal immunization with re Pcr V,the proportion of CD4⁺IL-17A⁺TRM in the lungs increased significantly compared with the non-immunized group.Nonetheless,the proportion of CD4⁺IFN-?⁺TRM did not change significantly.Immunofluorescence staining showed an increase of CD69⁺IL-17A⁺cells in the lungs of re Pcr V-immunized mice.Furthermore,the m RNA levels of Hobit,Blimp-1 and ROR?t in lung CD4⁺T cells from re Pcr V-immunized mice were significantly higher than in the non-immunized group.2.After depletion of peripheral lymphocytes using FTY720,the survival rate of re Pcr V-immunized mice after PA challenge fell to 50%,which was still significantly higher than in the non-immunized group.Consistently,after challenge with a sublethal dose of PA,the disease score,weight loss,and lung inflammation in the re Pcr V-immunized group were significantly lower than in the non-immunized group.The proportion of CD4⁺IL-17A⁺TRM cells in the lungs of the re Pcr V-immunized group significantly increased after PA infection.The survival rate of immunized mice decreased to 0 after treatment with anti-IL-17A antibody,which was significantly lower than that of the non-specific antibody treatment group.Conclusions:1.Re Pcr V and re Amp C were able to elicit Th17 responses and provided Th17-dependent protection in mice.2.Intranasal immunization with re Pcr V provided better protection than the intramuscular route,which probably relies on the induction of a Th17 response in the lungs.3.The Th17-stimulating epitopes of re Pcr V and re Amp C were identified and used to construct the fusion vaccine PVAC,which proved to be protective against infection with four different PA serotypes.4.Intranasal immunization with re Pcr V was able to induce the production of CD4⁺IL-17A⁺TRM in the lung,which conferred IL-17A-dependent protection.