Rational Design Of A Chimeric Derivative Of PcrV As A Subunit Vaccine Against Pseudomonas Aeruginosa

Background:World Health Organization（WHO）announced the《Global priority list of antibiotic-resistant bacteria》and emphasized that Pseudomonas aeruginosa（PA）was grade one（the most dangerous level）.It was drug resistance and prevention of PA that had been the big problem to be solved.While developing the new antibiotics,the vaccines and antibodies are the important strategies for preventing and curing PA.Currently,four vaccines of PA have been in the stage of clinical trials,however,three of them failed.There were no available vaccines in the market.The antigen is the core component of the vaccine.So,protective antigen with high quality plays a key role in developing the vaccine.Type III secretion system（T3SS）is one of the key virulence factors,by which ExoU was injected into host cells.The V antigen of PA（PcrV）,an important structural protein of T3SS,acts as a switch in secreting and translocating toxins.Therefore,PcrV is an important immune target.Studies have demonstrated that anti-PcrV antibodies can block the function of T3SS in vivo and in vitro to prevent and treat an infection.Besides,DNA vaccines encoding PcrV exert a better protective effects in a mouse model.Nevertheless,recombinant wild-type PcrV is instability,and easy to degrade and precipitate,which limits the use of PcrV as a vaccine candidate.The progress of structural biology provides new strategies for the optimization of vaccine antigens.So,identified the predominant domains of PcrV based on the structure of full-length PcrV and generated new vaccine molecules,and then evaluated the immunogenicity and protective effect,which laid the foundation for developing PA vaccine.Objective:In this study,to solve the problem of precipitation and degradation of recombinant wild type PcrV,the immune-predominant domains of PcrV were screened and a PcrV mutant was designed and produced,of which the immunogenicity and protective effect were evaluated,as well.Methods:1.Identification of predominant domains.PcrV was composed of four domains including N_ter（Met1-Lys127）,H₇（Arg128-Ala158）,C_ter（Lys159-Pro250）,H₁₂（Leu251-Ile294）based on the structure of PcrV predicted by I-TASSER Suite.And then,recombinant four domains were prepared,respectively.The intensity between four domains and sera from convalescent patients infected by PA were determined by ELISA;Then,four anti-domains antibodies were respectively prepared and used to evaluate the effect on toxin secretion.Taken together,predominant domains of PcrV were screened.2.Design,preparation,and analysis of biochemical characteristics of new vaccine molecule of PcrV_NH.Two predominant domains,N_ter and H₁₂,were designed to be a new fusion antigen PcrV_NH based on analysis of charge,space structure and energy dynamics;PcrV_NH were expressed in Escherichia coli by vector pGEX-6P-1 and purified by affinity chromatography（GST Bestarose 4B）and ion-exchange chromatography(Hi^TMTrap Q HP);The state of PcrV_NHH in aqueous solution was determined by size-exclusion chromatography and the dynamic light-scattering assay.3.The study of protective effect and mechanism of PcrV_NH.Purified PcrV_NHH was formulated with aluminum adjuvant and then intramuscularly injected into mice three times.Firstly,the survival rate of mice intratracheally challenged with a lethal dose of PA was analyzed to evaluate the protective effect.Secondly,the health,weight,bacterial burden,inflammation and histological changes of mice challenged by intratracheal injection with sub-lethal dose of PA XN-1 were observed to investigate the protection mechanism.Thirdly,to evaluate the immunogenicity of PcrV_NH,immune response type and level of anti-PcrV_NHH antibodies were investigated by ELISA.In mouse acute pneumonia model,protective effect and mechanisms of anti-PcrV_NH antibodies were evaluated by opsonophagocytic killing effect and secretion of toxin in vivo and in vitro.Results:1.The results of identification of predominant domains:bioinformatics analysis showed that PcrV was composed of four domains including N_ter（Met1-Lys127）,H₇（Arg128-Ala158）,C_ter（Lys159-Pro250）,H₁₂（Leu251-Ile294）.Then,MBP-PcrV_full-length,MBP-N_ter,MBP-H₇,MBP-C_ter,and MBP-H₁₂ were respectively prepared.Purification were⁹0%,95%,90%,93%,94%.Response intensity of single domain relative to MBP-PcrV_full-length was calculated based on the reaction between the domain and sera of convalescent patients infected by PA.The results indicated that the means of the relative strength of N_ter and H₁₂2 were 50%and 60%,which were significantly higher than those of H₇（25%）and C_ter（27%）.Besides,anti-N_ter,-H₇,-C_ter,-H₁₂ antibodies were respectively prepared.The study suggested that anti-N_ter and-H₁₂ antibodies significantly inhibited the secretion of ExoU under inducing and non-inducing conditions.Additionally,anti-N_ter and-H₁₂ antibodies significantly decreased cytotoxicity mediated by T3SS in cytotoxicity inhibition assay mediated by ExoU.However,no obvious effects on this process between anti-C_ter and-H₇ antibodies were observed.The above results indicate that N_ter and H₁₂ are the predominant domains of PcrV.2.The results of design,preparation,and analysis of biochemical characteristics of new vaccine molecule of PcrV_NH:predominant domains,N_ter and H₁₂,were optimally designed and constructed a new fusion antigen PcrV_NH composed of N_ter-GSGGSG-H₁₂.The recombinant protein fused GST tags was expressed in soluble form in Escherichia coli.After being purified by affinity chromatography（GST Bestarose 4B）and ion-exchange chromatography(Hi^TMTrap Q HP),the purification of PcrV_NH was approximately 95%analyzed by SDS-PAGE.The volume of elution peak of standard proteins including Blue Dextran 2000,Conalbumin,Ovalbumin,Carbonic Anhydrase,Ribonuclease A,Aprotinin and PcrV_NH were respectively 40.0ml、47.3ml、54.6ml、62.1ml、73.8ml、88.2ml,67.3ml by Size-exclusion chromatography(HiPrep^TM 26/10 Desalting).The molecular weight of PcrV_NH was²1.2 kDa calculated by linear regression of standard proteins.Dynamic lighting scattering analysis of PcrV_NH demonstrated that it formed a symmetrical peak with a diameter of 4.0 nm and estimated that the molecular weight was²4.9 kDa,indicating that PcrV_NHH is a homogeneous and stable monomer in aqueous solution.3.The results of mice challenged by PA:after being challenged by PA XN-1,the survival rate of mice in PBS control group,MBP control group,adjuvant control group,PcrV_NH experimental group,MBP-PcrV_full-lengthull-length positive control group were 0%,0%,0%,50%,50%,respectively.The survival of mice in PcrV_NH experimental group and MBP-PcrV_full-lengthull-length positive control group were significantly higher than those in three control groups（P<0.05）.However,there is no significant difference between PcrV_NHH experimental group and MBP-PcrV_full-lengthull-length positive control group（P>0.05）;The survival of mice challenged with lethal dose of ZJ-01（O6）,GZ-18（O11）,BJ-15（O10）,KM-9（O2）were respectively 50%,60%,60%,50%,which were significantly higher than that of mice in PBS control group（P<0.05）;With mice being challenged by sub-lethal dose of PA XN-1,the score of spirit,appetite,body weight,dorsal seta,shiver of mice in PcrV_NH experimental group were significantly better than that in control group（P<0.05）;In addition,the vaccinated group shows significantly lower bacterial loads than the unvaccinated group（P<0.05）.Histopathology,neutrophils,and inflammation all showed that immunization of PcrV_NH significantly reduced the inflammatory response in the lungs（P<0.05）.4.The results of immunogenicity of PcrV_NH and type of antibodies response:the direct interaction between anti-PcrV_NHH antibodies and single domain suggested that N_ter or H₁₂formed right conformation.Additionally,splenic cell stimulation assay showed that PcrV_NH-elicited immunity was a systemic immune response including Th1,Th2,Th17.Meanwhile,significantly increased levels of antigen-specific IgG₁,IgG_2a,and IgG_2b were detected.The level of IgG₁ was significantly higher than that of IgG_2a or IgG_2b（P<0.05）.No significant difference between IgG_2a and IgG_2b was observed（P>0.05）.Results indicated that PcrV_NHH vaccination induced a Th2-predominant immune response.5.The results of protective effect and mechanisms of anti-PcrV_NHH antibodies:with mice being challenged by PA XN-1,the mortality of mice in PBS control group,non-specific IgGs group and anti-PcrV_NHH antibodies group were 100.0%,100.0%,50.0%,respectively.The mortality of mice in anti-PcrV_NHH antibodies group was significantly lower than that in the control group（P<0.05）.However,there was no significant difference between the non-specific IgGs group and the PBS control group（P>0.05）.The opsonophagocytic killing assay finded that the bactericidal effect of antibodies was significantly stronger compared to that of non-specific IgG in different concentrations of antidodies（P<0.05）.Moreover,the bactericidal effect was positively correlated with the concentration of antibodies.Also,the concentration of ExoU in the culture supernatant was negatively correlated with the concentration of antibodies under the inducing or non-inducing condition.In the cellular model infected by PA,cell activity was significantly increased when antibodies were added.Taken together,anti-PcrV_NH antibodies played a protective role mediated by opsonophagocytic killing effect,inhibition of T3SS and secretion of ExoU.Conclusions:1.PcrV is composed of four domains.N_ter（Met1-Lys127）and H₁₂（Leu251-Ile294）are predominant domains.A new vaccine is consist of N_ter-GSGGSG-H₁₂ and expressed in soluble form in Escherichia coli.PcrV_NH with high purity monomer is got by ion-exchange chromatography.2.PcrV_NH conferred equal protection with full-length PcrV in an acute PA pneumonia model and elicited Th2-predominant,Th1-and Th17-participation multifactorial immune response,which suggests that PcrV_NH is a good candidate antigen and lays the foundation for developing further PA vaccine.Besides,anti-PcrV_NH antibodies plays a protective role,which may be mediated by inhibition of the T3SS and opsonophagocytic killing activities.These findings provide experimental evidence for antibody therapy of PA in the future.