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Neutralization Site Variation And Immunogenicity Of Attenuated Poliovirus Type 2 Strain

Posted on:2021-08-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y N WuFull Text:PDF
GTID:1480306308981049Subject:Pathogen Biology
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Since 1988,the Global Polio Eradication Initiative by the World Health Organization(WHO)has dropped by 99.9%cases and the world have made incredible progress in eradicate polio.Among the three wild poliovirus(WPV)types,wild poliovirus type 2(WPV2)was declared eradicated globally by the Global Commission for the Certification of Poliomyelitis Eradication(GCC)in 2015,with the last virus detected in India in 1999.Type 3 wild poliovirus was declared eradicated in 2019.Vaccination with OPV2 replicates in the intestine,and then be excreted VDPVs with highly neurovirulent into the environment,causing circulating Type 2 vaccine-derived poliovirus(cVDPV2).Subsequently,for cVDPV2 outbreaks,a global withdrawal of Sabin oral poliovirus vaccine type 2(OPV2)switched from the trivalent oral polio vaccine(tOPV)to bivalent oral polio vaccine(bOPV).In addition,poliovirus is an enterovirus,which can survive in sewage systems,and these viruses in the environment occasionally infect people.Even,individuals in immunodeficiency patients can become exposed to OPV.iVDPV(immunodeficiency-related vaccine-derived polioviruse,iVDPV)excreters may shed iVDPV for months or years before they become paralyzed or initiate community.During this replication process,some of the vaccine-virus may genetically mutate from the original attenuated strain and become neurovirulent and transmission characteristics of wild poliovirus.For biosafety,the poliovirus type II(PV2)materials is contained and only allowed to be used in a few P3 laboratories approved by the WHO,which will unable to detect to monitor and evaluate of the human population beginning with PV2.In this study,bioinformatics was used to predict OPV2 neutralizing antigen variation,and prepare pseudovirus and immune animals,and conduct research on immunogenicity and cross-neutralization activity.These provide references for maintaining polio-free status and formulating vaccine immunization strategies.In summary,A novel PV neutralization test using pseudovirus to replace live poliovirus was established method and It would serve as a safe and simple procedure for serosurveillance of PV.This is of great significance to the final stage of polio eradication and the maintenance of polio-free status.In this paper,we developed type ? PV pseudovirus(referred to as the pseudo-S-?).Through Westren analysis,infectivity and electron microscopy analysis,its immunogenicity is similar to that of infectious virus particles,with a diameter of about 30 nm.The neutralization test was performed using type ? virus and pseudo-S-? to determine the specific neutralizing antibodies in 40 serum samples with good correlations in the neutralizing-antibody titers determined by cPNT and pPNT(R2=0.87).The average geometric titers(GMT)were 36.47 and 55.90,respectively.Eleven independent repeated experiments were performed,the 95%confidence interval was 2012.37-2828.36,and the CV value was 0.29.The American Disease Control and Prevention Center(CDC)selected a type? iVDPV(iVDPV2)isolated from immunodeficiency patients with OPV?Sabin IPV(sIPV)and Salk IPV(wIPV)vaccine human immune serum to carry out cross-neutralization tests and found that the neutralizing activity was significantly reduced.In order to explain the problem,the main neutralization sites of VP1,VP2 and VP3 of iVDPV were analyzed by bioinformatics technology.By comparing the primary amino acid structure of the neutralizing sites of and iVDPV2,There are 16 different amino acid sites,among which 13 amino acids were changed in their electric properties or polarity.We used Poliovirus type 2(strain Lansing)(PDB ID:1 eah)as the template in the homology-modelling server SWISS MODEL and made iVDPV2 and ? capsid protein modelling.Capsid protein modellings were displayed by Pymol 2.1 software.We found that conformation of two virus strains are different.there are changes including three amino acid residues of VP1,679?VP 1,680 and VP2,141 at the neutralization site.11 mutant pseudoviruses were packaged at these sites,which were respectively neutralized with rabbit polyclonal antibodies and human serum vaccinated against poliovaccines.It was found that all mutations would affect the protective ability of serum,and the protective ability of anti-serum against RA141679S-decreased most significantly(p<0.01).The affinity detecting of these pseudoviruses and CD155 receptors showed that mutation of VP1,679 amino acids(mutated to D or absent)could increase the affinity of CD 155.Therefore,it indicate that VP 1679 amino acid plays an important role in neutralizing sites and virus binding to receptors.Given that poliovirus vaccines had no cross protection on some VDPVs,and residues at VP 1679 and VP2141 had different influence on the neutralization site.R141S,A679D and A679-pseudoviruses were developed and purified to immunize Wistar rats.Compared with strain,R141S mutants with a single amino acid mutation in the VP2 protein,but these have cross protective effect on WPV2 and VDPV2 after immunizing rats.These GMT of R141S antiserum against WPV2(MEF-1),PV2 and 4 strains of VDPV were 16.64?10.28?14.56?16.32?30.50?30.50.The antibody positive conversion rate reached 100%,and there was no statistical difference(p>0.05).As a result,we developed a pseudovirus neutralization test(pPNT)using PV pseudoviruses in human serum.The pPNT would serve as a new test for serosurveillance of PV after global eradication of polio.Meanwhile,by site-directed mutation of OPV2?pseudovirus technology?the neutralization test and affinity test showed that the VP1,679 is an important site in both neutralization epitopes and binding to the receptor.This study has important value for the development of novel polio vaccine type ?.
Keywords/Search Tags:Poliovirus, type ?, pseudovirus, Neutralizing antibodies, Immunogenicity
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