Heptamerization Of PcrV-OprI Mediated By GroES And GroEL Enhances Its Protection Against Pseudomonas Aeruginosa

Background:Pseudomonas aeruginosa（PA）is a Gram-negative opportunistic pathogen that is widely present in nature,it is particularly susceptible to nosocomial infections and infections in patients with low immune function.Infection caused by PA can lead to many acute or chronic diseases,such as hand dermatitis,pneumonia,osteoarthritis and urinary system inflammation.Currently,antibiotics aremainly used in clinical treatment,but the effect is not good because PA has strong drug resistance,and the mortality rate of pneumonia patients infected with PA is as high as 40.2%.In addition,the global spread of multidrug-resistant and extensively drug-resistant PA strains has become a serious public health problem,new strategies areneeded to prevent and control infection of PA.A safe and effective vaccine for PA is an economical and practical means to solve this problem.Although morethan 60 candidate vaccines for PA have been tested in animal experiments or human clinical trials,thereis still no successful vaccine for PA.The reason for this phenomenon may be that the lack of efficient delivery vectors leads to the limited protection.Pseudomonas aeruginosa V antigen（PcrV）is located at the tip of the bacterial type III secretion system and is highly conserved,it is the corecomponent of PA to secrete and transport toxins.Candidate vaccines based on PcrV antigen effectively protect against lung infection and burn wounds of PA.OprI is an outer membrane lipoprotein of PA,which has an adjuvant-like effect.The fusion of OprI with a polypeptide or protein antigen can improve its immunogenicity and induce a strong humoral and cellular immune response.In the previous study of the research group,PcrV and OprI wereconnected by flexible Linker to form a fusion antigen PcrV-OprI（rePO）,which showed a good protective effect in animal experiments and obtained national invention patent authorization.Delivering antigens through nanoparticle carriers is one of the effective ways to enhance their immunogenicity and protective effect.The heat shock proteins GroES and GroEL of PA can spontaneously assemble into polymers to form nanostructures with uniform particle size and good biocompatibility.Therefore,we speculate that the delivery of rePO using GroES or GroEL as a carrier may induce a stronger immune response and protective effect.Objective:In order to further improve the immunogenicity and protective effect of rePO,this study intends to use GroES or GroEL as a carrier to construct nanoparticle vaccines rePO-GroES and rePO-GroEL that deliver rePO,evaluate their protective effects and clarify the immune protection mechanism,laying a foundation for the development of efficient and safe PA vaccines.Methods and results:1.Preparation,physicochemical properties and safety detection of fusion protein rePO-GroESThe GroES gene sequence of PA（PAO1）was obtained from NCBI,p ET-28a（+）-reGroES and p ET-28a（+）-rePO-GroES recombinant plasmids wereconstructed by In-Fusion gene fusion.After prokaryotic expression and affinity chromatography,the qualified reGroES and rePO-GroES proteins wereobtained.The physicochemical properties of reGroES and rePO-GroES weredetected by size-exclusion,dynamic light scattering detection and transmission electron microscopy,the results showed that the estimated molecular weights of reGroES and rePO-GroES proteins in solution wereabout 70.0 k Da and 315.5 k Da,respectively,forming well-distributed nanoparticles with particle sizes of about 10.2 nm and28.2 nm,respectively.The safety of rePO-GroES was evaluated in vitro and in vivo by hemolysis test,CCK-8 cytotoxicity test and tissue section observation of mice after immunization,the results showed that rePO-GroES had no hemolytic reaction in the range of 10～80μg/m L,and had no significant effect on the viability of DC 2.4 cells in the range of50～200μg/m L.Therewas no significant difference between the tissue sections of mice immunized with rePO-GroES and PBS.The above results showed that rePO-GroES successfully self-assembled into heptamer nanoparticles with good safety.2.The immune protection effect and mechanism induced by fusion protein rePO-GroESIn order to explorethe immunogenicity of rePO-GroES,after mice wereintramuscularly immunized with rePO-GroES,the titers of specific antibody and antibody subtypes in the sera weredetected by ELISA,the results showed that the titer of anti-rePO Ig G antibodies in sera of rePO-GroES-immunized mice was significantly higher than that of rePO-immunized mice,the main subtype induced by rePO-GroES was Ig G₁.The results of ELISPOT showed that the number of splenocytes secreting IFN-γ,IL17A and IL-4 in mice immunized with rePO-GroES was significantly higher than those in the rePO group and control group.These results suggested that rePO-GroES induced a stronger immune response than rePO.The protective effect of rePO-GroES was evaluated by the acute PA pneumonia model,the results showed that the survival rate of mice immunized with rePO-GroES was significantly higher than that of mice immunized with rePO and PBS.In addition,the loss of body weight,infection score,lung bacterial load,inflammatory response and pathological section scoreof rePO-GroES-immunized mice werelower than those of the rePO group and control group.The above results indicated that the immune protection of rePO-GroES was significantly better than that of rePO monomer.3.Preparation,physicochemical properties and immunogenicity detection of the fusion protein rePO-GroELThe GroEL gene sequence of PA（PAO1）was obtained from NCBI,p ET-28a（+）-reGroEL and p ET-28a（+）-rePO-GroEL recombinant plasmids wereconstructed by In-Fusion gene fusion.After prokaryotic expression and affinity chromatography,the qualified reGroEL and rePO-GroEL proteins wereobtained.Their physicochemical properties weredetected by size-exclusion,dynamic light scattering detection and transmission electron microscopy,the results showed that the estimated molecular weights of reGroEL and rePO-GroEL proteins in solution wereabout420.8 k Da and 665.7 k Da,respectively,forming well-distributed nanoparticles with particle sizes of about 22.7 nm and 28.2 nm,respectively.It is suggested that reGroEL and rePO-GroEL aresuccessfully self-assembled into heptamer nanoparticles.In order to explorethe immunogenicity of rePO-GroEL,the specific antibody titer and the number of spleen cells secreting IL-4,IL-17 and IFN-γweredetected by ELISA and ELISPOT after mice wereintramuscularly immunized with rePO-GroES.The results showed that the titer of anti-rePO Ig G antibodies and the number of spleen cells secreting IFN-γ,IL17 A and IL-4 in mice immunized with rePO-GroEL weresignificantly higher than those in mice immunized with rePO and PBS.Both rePO-GroEL and reGroEL could induce high titer anti-reGroEL Ig G antibodies,and therewas no significant difference between them.These results indicated that rePO-GroEL significantly enhanced Th1,Th2 and Th17 immune responses compared with rePO monomer.Conclusion:In this study,rePO-GroES and rePO-GroEL nanoparticle vaccines with uniform particle size and good biocompatibility weresuccessfully prepared.The immunization of rePO-GroES had a significant protective effect on PA infection and was superior to rePO monomer.Both rePO-GroES and rePO-GroEL enhanced Th1,Th2 and Th17 immune responses against PA infection.The nanoparticles rePO-GroES and rePO-GroEL aregood candidate vaccines for PA,providing new ideas for developing other nanoparticle vaccines.