![]() In this study, we used 454 pyrosequencing to probe the “antibodyome” of this elite neutralizer and utilized identity/divergence analysis to examine whether the CD4bs-directed NAbs in this donor originated from IGHV1-2*02, the germline gene precursor of VRC01. One individual in particular appeared to have developed CD4bs-directed NAbs, as indicated by epitope mapping analysis. These individuals showed a broad coverage of serum neutralization, including over 95 % of the circulating HIV-1 isolates. In a previous study, we identified treatment-naïve Chinese elite neutralizers using a panel of 25 Env-pseudotyped viruses, including clade B, C, A, CRF07_BC, and CRF01_AE, from 103 subjects who had been infected with clade B’ virus for at least 10 years. To date, almost all reported CD4bs-directed NAbs have been isolated from Africans and African-Americans, and it is unclear whether they can be found in HIV-1-infected individuals of a different genetic background (e.g., Chinese) and which germline genes are responsible for encoding such NAbs. Of these, the CD4bs-directed NAbs provide promising templates for vaccine development, as they recognize the CD4bs of the HIV-1 envelope glycoprotein in the same manner as the co-receptor molecule CD4. These newly identified NAbs target the CD4bs, MPER, the glycan shield, and the gp120-gp41 interface, respectively. The first-generation NAbs, including b12, 2G12, 2F5, and 4E10, were identified over a decade ago, while the second-generation of NAbs, such as VRC01-class antibodies, PG9/PG16, PGT121-145, 10E8, and 35O22 were reported more recently. Great efforts have been made to isolate such NAbs from elite neutralizers. Ībout 10-30 % of HIV-1-infected individuals can develop cross-clade NAbs during natural infection, but only 1-3 % of individuals are capable of developing high titers of potent NAbs after years of chronic infection. Reverse engineering offers a promising method for the rational design of immunogen candidates with desirable properties but relies heavily on the identification and molecular characterization of new NAbs from HIV-1-infected individuals. However, the genetic diversity and heavy glycosylation of the HIV-1 envelope protein have hindered vaccine immunogen design. An effective vaccine is urgently needed to curb the spread of HIV-1 infection. The number of HIV-1 infected individuals in China has been estimated to be ~800,000. The 454 sequencing data and NAb obtained from this study will provide useful insights into the CD4bs-directed B-cell response during HIV-1 infection as well as the diversity of neutralizing antibodies. Our study thus demonstrates that CD4bs-directed NAbs can be produced by rearrangement from other VH genes, such as IGHV5-51 in this donor, rather than IGHV1-2*02. Interestingly, the VH gene of this weak NAb belongs to the IGHV5-51 lineage, with a somatic mutation rate of 7.99 %. We next used a pair of CD4bs-specific probes (RSC3/ΔRSC3) to sort the B cells from this Chinese donor and identified a CD4bs-directed Ab that showed limited neutralization capability. When an identity/divergence plot was used to interrogate the 454 sequencing data, no VRC01-like sequences were found within the dataset. ![]() IGHV1-2*02, the heavy chain germline V gene (V H) of the CD4bs-directed bNAb VRC01, was found to have a relatively low somatic mutation rate. ![]() We first performed 454 pyrosequencing to capture the IGHV1, IGKV, and IGLV germline gene families. In this study, we combined deep sequencing and single memory B cell sorting to isolate CD4bs-directed NAbs from a Chinese HIV-1-infected elite neutralizer. Broadly neutralizing antibodies (NAbs) against the CD4 binding site of HIV gp120 (CD4bs) have provided important information for vaccine design.
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