Identification And Evaluation The Immune Protection Of Candidate Antigens PA0833 And POH For Pseudomonas Aeruginosa Vaccine Development

Pseudomonas aeruginosa?PA?belongs to the genus Pseudomonas in non-fermentative bacteria.It is widely found in hospital wards and medical devices and is one of the most common pathogens in clinical practice.At present,it has become one of the the highest pathogenic bacteria infection rates in the global respiratory departments,burns,ICU wards,and geriatrics.With the increasing of multi-drug resistant PA strains,many clinical isolates are resistant to carbapenem antibiotics or polymyxin antibiotics,and PA ranked as the first level?the most critical level?in the“Global Antibiotic-Resistant Bacteria Priority List”published by the WHO in 2017.So PA infections are in urgent need of new treatment methods,then vaccines and antibodies are currently the ideal choice.Since the 1960s,Four vaccines and three antibodies have been clinically tested,but they have not been successful so far.Currently,none has reached the market.The main reason is that the pathogenic factors of PA are many and complicated.In the past,the antigens used in PA vaccine were too single,and they were difficult to induce a more comprehensive and effective protective immune response.Therefore,it should be closely combined with clinical practice to establish corresponding animal models.And new technologies for vaccine development should be applied to screen and identify vaccine conserved antigens efficiently and accurately,to construct multiple targets.The multi-component antigen combination enables the vaccine to effectively block multiple pathways of bacterial colonization and pathogenesis,then to remove bacteria and control infection.Based on the established PA strain library and clinical serum and lymphocyte sample pools,started from the whole genome level of PA strains,42 conservative,specific,and immunoprotection superiority antigen were screened through reverse vaccination technology.In this study,I established a pneumonia model of PA?the most serious clinical infection?,and successfully selected 11 protective antigens among these42 antigens.These 11 protective antigens included 5 reported protective antigens?such as Flic,type III secretory system key protein PcrV,outer membrane protein OprI,OprF,and exotoxin ToxA?and 6 unreported protective antigens.And the 6 unreported protective antigens included 3 functionally known proteins?such as Hcp1 of the type VI secretion system,outer membrane proteins PopB,OprL?and three hypothetical proteins?such as PA0833,PA0807,PA5505?.Among the protective antigen of unknown function,PA0833 was selected because of the highest protection rate.And its protective effects were further determined in three animal models of PA?pneumonia model,skin burn combined infection model and sepsis model?.The function of PA0833 was analyzed by bioinformatics,and verified by biophysical experiment and gene knockout experiment.Finally,the 11 protective antigens were screened for multiple compatibility and fusion.And fusion protein with high-expression,easy-purification,high-protection rates?such as PcrV-OprI-Hcp1,POH?were screened.The immunoprotective effects of POH were evaluated in three animal models of PA?pneumonia model,skin burn combined infection model and sepsis model?.And the humoral and cellular immune responses induced by POH were evaluated in vivo and in vitro experiments.A preliminary exploration for the immune protection mechanism of POH was carried out.The main results were as follows:?1?Establishment of animal models of Pseudomonas aeruginosa and screening of protective antigens.In order to screen and evaluate the protective antigen of PA,according to the epidemiological investigation results of PA,three animal models close to clinical infection were established:murine pneumonia model,murine skin burn and infection model and murine sepsis model.In the murine pneumonia model of PA,PAO1 can effectively infect mice with a dose of LD₅₀?5.0×10⁶ CFUs/mouse?.The bacteria mainly colonized in the lungs and can cause significant pathological changes of lungs;A dose of 2×LD₅₀?1.0×10⁷ CFUs/mouse?were able to kill approximately 90%mice by acute pneumonia.In the murine skin burn and infection model of PA,burns at 88°C for 8 s can cause skin III°burns in mice.PAO1 can effectively infect mice with III°burn at a dose of 300 CFUs/mouse.The bacteria mainly colonized in the liver,spleen and skin,and the mortality rate of the mice reached 80%within 7 days.PAO1 was able to effectively infect mice at a dose of LD₅₀?3.5×10⁷ CFUs/mouse?in murine sepsis model of PA.The bacteria mainly colonized in the liver and spleen;A dose of 2×LD₅₀?7.0×10⁷ CFUs/mouse?infected mice,the mortality rate can reach 90%in sepsis model.Then,the candidate 42 recombinant proteins?immune dominant antigens?were screened by acute pneumonia model,and 11 protective antigens were successfully screened out.?2?PA0833 is an OmpA C-Like protein that confers protection against Pseudomonas aeruginosa infection.PA0833 is a newly identified protective antigen of PA that was identified in a screen using a reverse vaccine strategy in our laboratory.In this study,we further confirmed its protective efficacy in murine pneumonia,burn and sepsis models.Immunization with PA0833 induced strong immune responses and resulted in reduced bacterial loads;decreased pathology,inflammatory cytokine expression and inflammatory cell infiltration;and improved survival.Furthermore,PA0833 was identified as an OmpA C-like protein by bioinformatics analysis and biochemical characterization and shown to contribute to bacterial environmental stress resistance and virulence.These results demonstrate that PA0833 is an OmpA C-like protein that induces a protective immune response in mice,indicating that PA0833 is a promising antigen for vaccine development.?3?Protective efficacy of the trivalent Pseudomonas aeruginosa vaccine candidate PcrV-OprI-HcpWe designed a novel trivalent vaccine,PcrV_28-294-OprI_25-83-Hcp_11-162?POH?,and evaluated its protective efficacy in murine pneumonia,burn and sepsis models.POH existed as a dimer in solution,it induced better protection efficacy in PA lethal pneumonia,burn and sepsis models than single components alone when formulated with Al?OH?₃ adjuvant,and it showed broad immune protection against several clinical isolates of PA.Immunization with POH induced strong immune responses and resulted in reduced bacterial loads,decreased pathology,inflammatory cytokine expression and inflammatory cell infiltration.Furthermore,in vitro opsonophagocytic killing assay and passive immunization studies indicated that the protective efficacy mediated by POH vaccination was largely attributed to POH-specific antibodies.Taken together,these data provided evidence that POH is a potentially promising vaccine candidate for combating PA infection in pneumonia,burn and sepsis infections.The main conclusions were as follows:?1?The hypothetical protein PA0833 in PA was identified as OmpA C-like protein,and its C-terminal domain was able to form a dimer and bind to peptidoglycan;it was confirmed that PA0833 is a new virulence factor,which contribute to bacterial environmental stress resistance and virulence and participate in the natural immune response process against PA.So It provides a basis for elucidating the pathogenic mechanism of PA and provides a potential target for anti-PA infection..?2?The designed trivalent fusion protein POH has the advantages of high expression,easy purification,stable solubility and uniformity,and high protection rates.Immunization with POH induced strong and comprehensive cellular and humoral immune responses and resulted in reduced bacterial loads and improve survival rate.It provided experimental basis for the exploration of antigenic combinations and synergistic immune enhancement strategies for PA vaccines.And,these results provided important experimental evidence and technical support for novel multivalent new PA Vaccine.