Impact On Viral Antigenicity Of Mutation In RABV G Protein And HIV-1 Env Protein Based On Pseudovirus System

Vaccination is the fundamental way to prevent infectious diseases,and the neutralizing antibodies induced by vaccine play an important role in anti-virus infection.However,for RNA viruses with high mutation frequency and evolution rate,their sequences or antigenicity of envelope glycoprotein change continuously,which present major challenges for the development and clinical use of current vaccines and antiviral drugs.China is a country with a high incidence of rabies and AIDS.Rabies and AIDS were caused by RABV and HIV infection respectively,and both RABV and HIV are RNA viruses which have a high mutation rate.Rabies vaccine has been widely used and has shown good protection,while the HIV-1 vaccine is a research hotspot,and there is no effective HIV-1 vaccine at present.Rabies has a mortality rate close to 100% after clinical symptoms appear.Although the current RABV vaccines showed good protection against RABV infection,the effectness of the vaccine may be affected by the different antigenicity of street RABV from the vaccine strains.For HIV-1,the N-linked glycosylation on Env protein changed through virus mutation and evolution.Affecting antigenicity by shielding conserved neutralizing epitopes of glycosylation is one of the influencing factors that obstruct the vaccine development.In this study,we used a HIV-1 pseudovirus system to study the effect of single amino acid mutation on RABV glycoprotein(G)antigenicity and N-linked glycosylation site mutation on HIV-1 Env antigenicity,to provide imformation for the use and development of relevant vaccines.A total of 2890 street RABV G protein sequences containing complete background information were obtained from Genbank(524 AA).Globally,the street isolates from 1960 has a trend of decreasing similarity to all seven vaccine strains(p<0.001),which indicated that the sequence of street RABVs is divergent and the antigenicity of G protein may be changed.We next analyzd the composition and distribution of amino acid at each position in the extracellular region of G protein,and chosed 103 candidate sites which may affect the antigenicity.The candidate sites were selected by the naturally occuring variations in both antigenic sites and outside the antigenic sites,and also the PNGS elimination or introduction.Using site-directed mutagenesis,we constructed a total of 103 G protein mutants containing single amino acid mutation,then the RABV pseudovirus were prepared by HIV-1 pseudovirus system.11 mutations significantly reduced the viral infectivity and excluded from the neutralization assay,we evaluated the neutralization phenotype of remaining 92 mutants to a series of neutralizing monoclonal antibodies,vaccine-induced antibodies and human rabies immunoglobulin(HRIG)to identify the mutations that could influence the antigenicity of the G protein.Using pseudovirus based neutralization assay,we found mutations that significantly affected the antigenicity,and these mutations were mainly located in antigenic site ?.Mutations at R333 and I338 T mutation in antigenic site ? resulted in strongest resistance to the neutralizing m Abs,vaccine induced antibodies and HRIG.Other mutations in antigenic site ? also have a certain effect on antigenicity,such as mutations at K330 and N336.Mutations in other antigenic sites also had effect on the antigenicity.For example,the K245M/Q mutation in antigenic site ? prevented the virus from being neutralized by m Ab NM57 s,W251R and R264 Q in antigenic site IV&G5 reduced the suspceptibility to vaccine induced antibodies.Mutations outside the antigenic sites and at PNGS had littile effect on the antigenicity.We also provide a structural basis for the observed effects of these mutations by modeled RABV G protein structure.Among all the mutations,we found W251 R,R264Q,K330Q/R/N/E/T,R333L/P/Q/N/H,N336D/G/S/K,and I338 T demonstrated greatest resistance to vaccine induced antibodies,and different vaccine strains varied in their neutralizing activities against these RABV mutants(the ID50 decrease of PM and a GV were significantly higher than Flury-LEP,p<0.01).Antigenic variants containing these mutations were mainly isolated around 2010,and showed wide geographical distribution.These variants were distributed globally,mainly in North America,Africa,Asia and South America.The USA is the major source of North America,China in Asian,and Brazil in South America.In Africa,no country was found to be the main source of variants.Some variants showed regional epidemic characteristics and were found in certain geographic locations,specifically,R333 P were only isolated in China and I338 T only isolated in China and USA.The antigenic variants also showed a wide range of hosts include dogs,cats,bats,Homo sapiens and other mammals,and raccoon is the major host.Although the antigenic variants constitute less than 10% of all RABV strains at this time,there appears to be a trend that these mutants have been increasing in recent years.Close monitoring of emerging mutants should be conducted as they may potentially render the current rabies vaccine less effective.HIV-1,which is also an RNA virus,has high mutation rate and this is one of the important factors hindering the development of an effective HIV-1 vaccine.The N-linked glycosylation obtained through virus mutation and evolution,could mask the conserved epitopes on Env,which is also the barrier in the development of HIV-1 vaccines.In previous study,we evaluated the effect of single PNGS removal on a CRF07_BC virus FE,and found some PNGS could affect neutralization suspceptibility to m Abs.However,the effects of elimination of combined PNGS located on different regions have not been systematically studied.In this study,we eliminated the PNGS in combination and evaluated the influence on antigenicity using a HIV-1 pseudovirus system.Through different combinations,we obtained twelve combined PNGS mutants,and one mutant 197 M.1(N197D + N301Q)completely lost the viral infectivity,thus,exclued from the neutralization assay.For the remained 11 mutants,the neutralization phenotype to neutralizing m Abs and serum antibodies were evaluated.We found that there is a “synergy effect” between the PNGS,compared to the wildtype virus FE and the single PNGS removed mutants,combined PNGS removal can significantly enhance the neutralization suspceptibility to m Abs and serum antibodies.Specifically,the combined deletion of N197 and N463 significantly enhanced(300~1300-fold)the suspceptibility to m Ab VRC01 and VRC03 which directed against CD4 binding site(CD4bs).Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various CD4 bs m Abs elucidates a molecular rationale for this experimental observation.The removal of these two PNGS can expose the epitopes recognized by m Ab VRC01 and VRC03.Clade 07_BC viruse is one of the most predominantly circulated HIV-1 strains in China,and using Env of this clade virus as an immunogen to design HIV vaccine is more suitable in China.Our study should provide valuable information for a better understanding of the interplay between HIV-1 glycosylation and VRC01/03,and may be helpful for immunogen selection and optimization targeting the CD4bs epitopes.