Establishment And Application Of Detecting Methods For HIV-1 Humoral Immune Response

Since Human Immunodeficiency Virus(HIV)was identified as the pathogen of Acquired Immunodeficiency Syndrome(AIDS)in 1983,the epidemic of AIDS has brought great influence on society,economy and public health in the world,which has attracted the attention of the WHO and the governments.It has become a great public health and social problem.The most effective way to prevent and control AIDS is to develop a successful HIV vaccine.Because of the increasing genetic diversity,regional differences in epidemic strains,unknown protection mechanism,no case of natural clearance of HIV,and ominous protection related immunological indicators,the development of HIV vaccine is very difficult.It is widely believed that HIV-1 neutralizing antibody plays an important role in preventing HIV-1 infection.The envelope(Env)is the target of HIV-1 neutralizing antibody.HIV-1 CRF07/08 BC,AE,B and B' subtypes are the three main epidemic clades in China.To evaluate the neutralizing antibody-based HIV-1 vaccine in China,panels of HIV-1 Env pseudoviruses involving the 2 major subtypes CRF07/08_BC?CRF01_AE were constructed.Recently.28 strains of subtype B,B' Env pseudoviruses including 15 strains B'(named as B'_CN)and 13 strains B(named as B_CN)were isolated from China and constructed in our laboratory.When comparing B'_CN and B_CN with 18 strains subtypes B isolated from foreign countries(named as B_EU),great differences were presented on the env gene,and the neutralization phenotypic characteristics against neutralizing plasma samples.In order to further analyze the neutralization characteristics of the virus against monoclonal antibody,three groups of 46 pseudoviruses(including 15 strains B'_CN,13 strains B_CN and 18 strains B_EU)were tested against the recombinant human soluble CD4 protein(sCD4)and 16 broad neutralizing monoclonal antibody(bnmAbs).Significant differences were observed when sCD4 was tested against the B_CN and B_EU strains.It suggested that the CD4 binding sites exposed on B_CN were different from B_EU strains.Among the antibodies targeting the trimeric apex.PGT145 and CHO1-04 showed similar neutralizing activities,showing greatest potency against B_EU strains and lowest against B'_CN strains.The susceptibility of B'_CN and B_CN against 2F5 targeting MPER differed significantly.By analyzing the epitope of the bnmAbs.the changes in the amino acids of the epitopes might lead to the difference in phenotypic propertiees of the subtype B and B' strains from China and those from abroad.To evaluate the proficiency detection level of HIV-1 neutralizing antibody in domestic laboratories,we have conducted collaborative studies on the pseudovirus neutralization assay based on TZM-bl.Proficiency test kits containing pseudoviruses and plasma reagents were assembled and distributed to 9 laboratories by National Institutes for Food and Drug Control(NIFDC).19 pseudovirus strains including 9 HIV-1 B and B'and 10 HIV-1 CRF01_AE were chosen for their high,medial and low neutralization phenotype,and 10 plasma samples including 5 HIV-1 B and B,plasmas and 5 HIV-1 CRF01_AE plasmas were selected for their high,medial and low neutralization potency.A laboratory qualifies if more than 80%of their ID50 values lie within the acceptance ranges.The scores of the six laboratory achieved the cutoff limit of 80%.The results showed that 6 laboratories passed the collaborative study and possessed the qualified ability to detect neutralizing antibodies.The results show the proportions of ID50 neutralization titers are within 3-fold of each other among the 6 laboratories.The ID50 values of each plasma-pseudovirus combination were independently compared between two laboratories.Two laboratories were considered equivalent when the pairwise-agreement achieved above 80%.The equivalent rate of the 6 laboratories was 46.7%.The equivalent rate between laboratories of this round was higher than that of the first round(46.7%vs 37%).It indicated that the laboratories' ability to detect HIV-1 neutralizing antibody had been improved through the collaborative study,and the comparability of results had been ensured.Clinical trial of the RV144 showed that the level of V1V2 antibodies had a negative correlation with the risk of infection,suggesting that the role of non-neutralizing antibody with protective function should not be ignored in the evaluation of HIV-1 vaccine.To assess the level of V1V2 antibodies induced by HIV-1 vaccine,the indirect ELISA method was established by using the gp70(MLV)-VIV2(HIV-1/CN54)and gp70(MLV)-VIV2(HIV-1 CRP01_AE)as the coating antigen.Plasma from patients who has infected different subtypes of HIV-1 from HIV-infected MSM cohorts were detected by this method.The results showed that the V1V2 antibodies were widely detectable and was cross-clade-reactive in the HIV-infected patients.And they were durable once the seroconvesion occurred.The results showed that there was no correlation between the level of V1V2 antibody and the number of CD4 T cells of the HIV-infected individuals.The difference in the length of V1V2 sequence,the numbers of glycans may lead to the difference in antigenicity of V1V2.Studying the neutralization characteristics of HIV-1 B and B' subtype against sCD4 and the bnmAbs could provide information for the immunogen design and immunogenicity evaluation of HIV-1 subtype B and B' vaccine.Collaborative study had evaluated the detection level of HIV-1 neutralization antibody in domestic laboratories.It enhanced the comparability across different laboratories.The detecting methods of V1V2 antibodies could provide a method and technical support for the evaluation of the levels of the V1V2 antibodies induced by HIV-1 vaccine in future.