针对水痘带状疱疹病毒高度保守表位疫苗候选物在小鼠中诱导中和抗体.ppt

针对水痘带状疱疹病毒高度保守表位疫苗候选物在小鼠中诱导中和抗体AbstractIntroductionCurrently,two live-attenuated VZV Oka vaccines are licensed,one against varicella,another against HZ,which have remarkable and beneficial effects in preventing varicella and HZ 4,5.With the development of biochemistry,molecular vaccinology and genetic engineering technology and increased demand for vaccine safety,the need for more safe and stable vaccines in various forms continues to grow.Plasmids containing the gene encoding the full-length or truncated form of gE(prototype DNA vaccines),which could stimulate immunity in mice,were constructed by Hasan et al.Mullane et al.reported that the heat-treated zoster vaccine was generally safe and immunogenic in immunocompromised adults.Significantly,an adjuvanted HZ subunit vaccine based on VZV glycoprotein E(gE)was well tolerated and immunogenic,and more importantly,this vaccine induced a robust immune response and had a clinically acceptable safety.In view of this,more safe,stable and efficient vaccines including epitope-based vaccines now more prevalent.Epitope peptides are considered to be promising candidates for new generation vaccines,especially those with conserved and neutralizing epitopes.The potential advantages of epitope-based vaccines include increased safety;economical technology as they are easily produced and have simplistic compositions;the reduction of allergic reactions;and the ability of focus immune responses on selected epitopes.VZV is an enveloped virus that expresses at least nine membrane proteins,of which gE is the most essential and abundant.Only a few neutralizing VZV epitopes have been identified to date.Yasushi et al.reported that the gH neutralizing epitopes were conformational and comprised a cluster of the seven independent protein portions of gH.Marius et al.16 generated a recombinant monoclonal anti-VZVantibody that recognized by a conformational epitope within the gH/L protein complex,which could effectively neutralize VZV.Residues 1-134 and 101-161 of gE were identified as immunodominant regions by Western blotting and ELISA analyses,using serum from both varicella and zoster patients.Additionally,immunizations with the recombinant hybrid Ty-virus-like particles presenting the gE(aa1-134)and gE(aa101-161)regions in small animals could induce neutralizing antibody responses.Furthermore,monoclonal antibody(mAb)3B3 recognized an 11-amino acid(residues 151-161;QRQYGDVFKGD)epitope in the ectodomain of VZV gE by using the recombination PCR technique.Therefore,pinpoint neutralizing epitopes and epitope-based vaccines against VZV are still lacking in-depth research.Therefore,we are interested in developing a recombinant epitope-based vaccine with neutralizing abilities against VZV.Our group previously generated aVZV-gE-specific complementdependent mAb(named 4A2)by immunization with a recombinant gE antigen expressed by insect cells and the 4A2 epitope was identified as the amino acid sequence(SAQEDLGDDTGIHIV).In this study,we validated the gE-epitope that we identified and found that it was highly conserved.Then,we generated a fusion protein with 149 aa of the hepatitis B virus core(HBc)protein and the epitope,and confirmed that this protein was able to self-assemble into virus-like particles(VLPs)by electron microscopy.We further found that the VZV gE-specific IgG antibody in mice immunized with chimeric VLPs HBc-gE(aa121-135)were not lower than purified rgE protein and heated-inactivated vOka groups,in high-dose groups.More importantly,we proved that these chimeric particles were assessed for an ability to induce neutralizing antibodies to gE-specific epitope in mice.Our results suggest that chimeric HBc particles carrying the neutralizing epitope of gE can induce protective immunity against VZV.These findings have important implications for the development of epitope-based protective VZV vaccines.技术路线：制备VZV毒株vOka，制备热灭活的vOka抗gE的mAb（4A2）的制备，同源性分析与表位的鉴定HBC-gE(aa121-135)重组蛋白和HBC蛋白的制备SDS-PAGE+western（HBC-gE,HBC,V-OKa理解物）电子显微镜+3D模型分析免疫小鼠（HBC-gE,4A2,HBC）与血清学检验，纯化gE蛋白ELISA检验免疫荧光染色检验gE抗血清肽体外中和VZV检验Results：mAb-4A2识别表位的保守性分析gE（121-135）蛋白抗原表位精细定位纯化重组HBC-gE(aa 121-135)和VLPs的分析电子显微镜与3D模型分析HBC特异性抗体反应动态gE（121-135）小鼠抗血清的功能特性gE（121-135）抗血清体外中和VZV检验DiscussionDiscussion：Varicella is a ubiquitous highly contagious infectious disease of seasonally epidemic propensities that often follows primary infection in children.Herpes zoster is a sporadic endemic disease,which most frequently occurs in elderly people.Vaccination has proven to be the most effective way to prevent these two diseases,and the two current licensed live attenuated viral vaccines have significant efficacy.However,there are still some safety concerns regarding these vaccines and the vaccine formulations contains about 69 VZV proteins,whereas some proteins are unnecessary for immunity induction.Moreover,the varicellazoster virus(VZV)encodes nine glycoproteins gE,gI,gC,gH,gL,gB,gK,gM and gN 26,which are inserted into the viral lipid-rich envelop that acquired from cellular membranes of infected cells.Amongst the glycoproteins,gE is the major and predominant VZV membrane protein,which has sometimes been called the navigator glycoprotein due to its importance in VZV replication,viral assembly and infection.The viral glycoproteins are closely related to the pathogenicity and immunogenicity of the virus,and it can especially induce the production of neutralizing antibodies to neutralize the infectivity of the virus.In particular,gE,gH,gL and gB have been shown to induce neutralizing antibody responses.An extension of this logic would be thatthese proteins contain numerous necessary and positive epitopes.This has created an interest in epitope-basedvaccines as being promising candidates for VZV.Currently,few neutralizing linear epitopes from VZV have been identified.In our recent study,the complement-dependent neutralizing mAb,4A2,was identified and mapped further to a linear epitope spanning amino acids 121135 within gE of VZV.In the present study,a sequence alignment reveals that the epitope recognized by 4A2 of gE is highly conserved and the Ala scanning results suggest that six sequential amino acids are critical for the binding affinity.The glycoprotein E contains 623 amino acid residues and harbors both B and T cell epitopes,to date,several VZV gE mutant strains have been identified.Two VZV gE mutant strains were collected in North America,these mutants contain a nonsynonymous mutation(G448A,corresponding to amino acid exchange D150A).In 2006,a VZV gE mutant strain harboring a nonsynonymous mutation(D161G)was isolated from a child with varicella in Rome,Italy.Five novel gE mutations were discovered within 45 analyzed VZV wild-type strains of genotypes A and D.Although gE sequence variability has been reported,our identified gE-epitope(121SAQEDLGDDTGIHVI135)was found to be well conserved,suggesting that it is a candidate for inclusion in a epitope-based vaccine.Subsequently,we studied the antigenicity of this epitope carried on HBc-VLPs expressed in E.coli,comparing with 4A2 synthetic peptide,purified rgE protein and heated-inactivated virus in different dosage.In high-dose groups,mice showed high serum levels of anti-VZV antibodies following immunization with HBc-gE(aa121-135),which were not lower than the others.Furthermore,the anti-HBc-gE(aa121-135)antiserum were able to neutralize the virus,only in complement dependent neutralization in vitro,which is a phenomenon that is similar to previous results 18,36.The exact neutralization mechanisms for both antibodies and the antipeptide serum remain unclear.In addition,the corresponding antibody of this epitope can effectively capture VZV,suggesting that this epitope exposes more fully on the native virus antigen gE.These results indicate that the identified VZV neutralizing epitopes are immunodominant.In the further research,we will analyze the immunogenicity of the HBc-gE(aa121-135)particles in other different animal models,such as rat and guinea pig and evaluate whether the antibody and antisera are protective in the SCID-hu mouse infection model or the guinea pig model.Epitope peptides with low molecular weights exhibit rather weak immunogenicity,however,their immunogenicity can be enhanced via presenting target epitope sequences on the surfaceof phage,lipid molecules,recombinant viruses or VLPs.Chimeric VLPs,suchas HBV-VLPs,HIV-VLPs and HPV-VLPs,have beenconsidered as more ideal promising delivery vehicles of foreign epitopes suitable for designing highly immunogenic vaccines.In this study,we use the Hepatitis B virus core(HBc)protein as an immune-carrier to enhance the immunogenicity of epitope peptides.The HBc protein can induce strong B cell,T cell and CTL responses,meanwhile,act as a T-cell-dependent and T-cellindependent immunogen,which directly activates B cells.Our results demonstrated that the HBc protein could correctly fold and auto-assemble into VLPs in vitro and the inserting peptides could be exposed on the surface of VLPs,after inserting VZV gE(aa121-135)(foreign peptides)into the MIR,and this conclusion is in agreement with previously reported results.In further studies,we may attempt to display the selected epitopes in other display technology to find the better way.In addition,to increase the immunogenicity of the chimeric HBc-gE(aa121-135)particles,we may make changes in adjuvants,routes of administration and the number and types of epitopes available for components in the vaccine,such as other neutralizing epitopes of VZV.In conclusion,the identification,characterization and application of the gE(aa121-135)epitope is important for VZV vaccine design,and our finding about chimeric VLPs carrying the conserved neutralizing epitope of VZV gE will promote the development of avaccine candidate based on epitope.谢谢大家！结结 语语