| The data from many existing viral vaccines (such as influenza and measles) demonstrated that neutralizing antibody (Nab) plays a pivotal role in protective immunity. For Human Immunodeficiency Virus type 1 (HIV-1), passive transfer of broadly neutralizing antibodies completely blocked infection by a chimeric simian-human immunodeficiency virus in nonhuman primate studies and passive transfer of broadly neutralizing antibodies delayed HIV-1 rebound after cessation of antiretroviral therapy in clinical study. These previous studies in animal models and humans reaffirm that broadly humoral immunity responses may be required to provide immune protection functions. During the course of natural HTV-1 infection, NAbs against the infecting strain (autologous virus) appear months later but are not able to neutralize more divergent viruses isolated from other individuals (heterologous viruses). However, broad and potent neutralizing antibodies (Nabs) usually were produced in chronic HTV-1 infection. Thus, the present research efforts have focused on the following aspects.I:Profiles of neutralizing antibody response in chronically HIV-1 clade B’infected former plasma donors naive to ART in Central ChinaHere, we measured Nab responses using a panel of 25 Env-pseudoviruses composed of 8 clade B strains,4 A strains,4 C strains,4 CRF07BC strains,3 CRF01_AE strains and 2 tier 1 viruses (SF162.LS for clade B and MW965.26 for clade C) against plasma samples from 103 subjects in a former plasma donor cohort in central China, who were infected HTV-lclade B’for at least 10 years and naive to ART at the time of sampling. We found 64% of samples (n=66) neutralized at least half of the viruses tested,2%(n=2) neutralized all the viruses while 5%(n=5) neutralized none of the viruses tested. Strikingly,29% of plasma samples (n=30) neutralized more than 80% of the viral strains tested indicating the presence of broad reactive Nabs in these patients. When magnitude (GMTs) or breadth of neutralization was assessed for correlation with CD4 count or plasma viral load, the only positive association was observed between viral load and the neutralization magnitude (r=0.218, p=0.0268)/breadth (r=0.197, p=0.0464). A moderate difference between progressors and LTNPs was observed in both the breadth (p=0.0339) and the potency (p=0.0350). This observation concurs with data from other cohorts in multiple geographic areas including USA, South Africa, European, Kenyan, etc. These findings showed that, compared to patients with higher levels of viremia, LTNPs make weak Nab responses, perhaps due to reduced antigenic stimulation of B cell.A significant difference was found in the geometric mean ID50 titer (GMT) between intra-clade and inter-clade strains (p<0.001). Heatmap analysis based on Kmeans clustering of plasma identified three statistically robust clusters for both plasma and virus. These samples would provide physical biomaterials for further virological and serological studies from which useful insights into rational HIV-1 vaccine development and therapeutic design can be derived.II:Profiles of neutralizing antibody responses in chronically HIV-1 CRF07BC infected intravenous drug users in western ChinaIt is essential to characterize neutralizing antibody (Nab) responses in individuals infected with diverse HTV-1 strains to reveal the potential target for HIV-1 vaccine development. In this study, we assess the prevalence, breadth and potency of Nab responses in CRF07BC chronic infectors (n=100) with infected time around 3 years, and compare the neutralization pattern with that of clade B’chronically infectors (n=103) with infected time at least 10 years. 114 plasma samples were collected from intravenous drug users infected with CRF07BC virus in western China who were ART-naive. The Env pseudovirus-based TZM-bl assay was performed against a panel of 30 tier 2-3 pseudoviruses composed of CRF07_BC(10), clade C(5), B(7), A(4) and CRF01_AE(4) strains and 2 tierl viruses(SF162.LS and MW965.26). As negative control virus, SVA-MLV positive samples were excluded from further analysis. All 100 plasma samples (excluding 14 SVA-MLV positive samples) neutralized both tier 1 viruses. 53%of the samples (n=53) neutralized half of the viruses, and 17%of the samples (n=17) neutralized more than 80%strains tested.1%(n=l) neutralized all the viruses, and 2%(n=2) neutralized none of the viruses tested. Significant difference of geometric mean ID50 titer (GMT) between intraclade and interclade samples (p<0.001) was also observed in this cohort. Plasma samples from CRF07BC chronically infected subjects showed higher neutralizing titers (GMTs) against subtype-matched viruses than that of subtype B’infectors. In constrat, the plasma samples from FPD cohorts showed higher neutralizing titers against CRF01AE than that of IDU cohort. Plasma samples from both cohorts exhibited equavlentlly neutralizing titers. However, higher prevalence of broadly cross-reactive Nabs response were observed in clade B’chronically infection than that of CRF07_BC infection (29%vs.17%). Heatmap analysis based on Kmeans indentified three statistically robust cluster of plasma with higher, moderate and lower cross-reactive neutralizing reactivity. Further epitope specificity dissection of these broadly cross-reactive Nab samples will provide useful insights for rational HTV-1 vaccine design.Ⅲ:Preliminary identification of CD4bs monoclonal antibodies isolated from the best neutralizersHere, we obtained 17 broadly neutralizing sera from above two well-controlled HIV-1 chronic infection cohorts in China, and further investigated the antibody profiles in the same panel of 17 broadly neutralizing sera, using several validated epitope-specific probes in ELISA based assay. A total of 17 samples including 1-3 time points derived from these 9 top neutralizers were collected. The same sera were subjected to ELISA based analysis using validated pairing probes directed against CD4bs and V1V2 on HFV-1 Env spike for profiling the antibody repertoire in individual serum sample. Overall, eight sera collected at different time points from four patients showed various level of differential binding activity between RSC3 and ARSC3, indicating the presence of CD4 bs antibodies in the samples. Among them, five sera also exhibited various levels of differential binding activity between RSC3 (P369R) and ARSC3, implicating the neutralization potential of the CD4bs antibodies in the sera. Ten sera samples from seven patients did show differences in binding reactivity to JO8 compared to △JO8. Among them, five sera samples from three patients displayed differential binding activities to both sets of probes. Interestingly, DRVI-01-1/DRVT-01-2 sera showed detectable binding activity to RSC3 (B-LD/V5) probe, in which sequences in LD/V5 regions were replaced by corresponding sequences in VRC01-resistant clade B strain-BL01, although these sera are not particularly potent in neutralizing BL01 virus. However, both sera neutralized large numbers of VRC01-resistant viruses with respectful titers. Information obtained from these assays can provide essential details about the types of the neutralizing antibodies in the serum, and provide guidance for further isolation and identification of broad neutralizing monoclonal antibodies.When DRⅥ-03-1 was used as a sample for pilot study, ELISA data showed this serum could bind the RSC3 strongly, compared to ARSC3, indicating the presence of CD4bs antibodies in the samples. As such, we initially determined to sort the single B cells from the PBMCs of donor DRⅥ-03-1 We isolated 5 RSC3-specific memory B cells from this donor by FACS. By conducting nested RT-PCR utilizing customer-specific primers, we obtained 3 pairing(s) of positive heavy and light chains IgG genes. We got 3 IgG antibodies by co-transfection in 293F cells and purification by protein A. ELISA data indicated two (DRVIO3-1-5A and DRVI03-1-7A) of three IgG antibodies show strong binding activities. Further neutralization assay in TZM-bl demonstrated these 3 antibodies can only neutralize prototype strains-HXBII. Competition ELISA asaay showed the two antibodies could compete with CD4 and belong to CD4bs antibodies. Gene family analysis against the three mabs indicated that they had longer CDRH3 but possessed lower mutation compared with their corresponding putative germine genes which might explain their lack of neutralization against primary isolates. Although we do not get monoclonal antibodies with neutralization activity, the pilot study offers a valuable experience for further study.IV. Development of new 8b probes to isolate novel CD4bs monoclonal Abs other than VRC01DRVI01 showed broad and potent serum neutralization activities, and also neutralized VRC01-resistant strains. Neutralization competition assay data showed that the neutralizing antibodies in this sample may target the location different from VRCOl-like antibodies. Therefore, it is essential to design new probes to sort the singe B cell for this unique sample with CD4bs antibody activity. New 8b core is derived from a gp120 core of HXBII strain and can bind the J3 (a kind of immunization-induced llama antibody) and VRCOl-like antibodies. Based on structure knowledge of VRC01 complex with gp120 core, we replace the BV5, BLD, BLDV5, CV5, CLD, CLDV5 region with the corresponding gene sequences of two typical VRC01-resistant strains including one BLD.01 for clade B and TV29.11 for clade C. After mutants construct, protein expression and purification, the ELISA data indicated that the 8b-BLDV5 version can bind J3 but decrease the binding of the majority of VRCOl-like antibodies. We also found that this version can still bind the CD4. In order to better block the binding of VRCOl-like antibodies and CD4, we designed several mutants including 458RW, 458WR,458YY,458YY280W,280W,280R,282T, W427E and found the 8b-BLDV5-282T reachs the design goal. But when an extended mabs panel including bl2, bl3, F105 and 17b was tested, the 8b-BLDV5-282T can bind the b 12, b13,17b.To only bind the J3, two versions of mutants by adding a glycan at 419 and 421 were designed. We observed that the 8b-BLDV5-282T-419NT can finally reach the set-goal and work as potential positive control. Meanwhile, two different versions of potential negative control including 8b-BLDV5-282T-419NT-D368R and 8b-BLDV5-282T-419NT-Q363Nd371I were designed and evaluated. Finally, the potential pairing of probes-8b-BLDV5-282T-419NT and 8b-BLDV5-282T-419NT-D368R are ready for further testing more clinical samples to confirm if there are this type of antibodies produced during the course of HIV-1 infection. If so, this pairing of probes will be used to isolate the antigen specific single B cell for monoclonal antibodies production for further study including structure resolution to reveal potential novel subtle epitope directed the CD4 binding site. If this kind of mabs could be isolated, the further modified version of 8b-BLDV5-282T-419NT based on new structure knowledge will have the potential to serve as immunogen to test its efficacy in animal models except for the llamas. |
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