| The envelope region of HIV-1is the most variable gene, and employs many mechanisms to make virus escape from the neutralizing antibodies. The variation of the envelope has become one of the great challenges to vaccine design. During the past four years, comprehensive application of advanced technologies has led to the isolation of many human broadly neutralization monoclonal antibodies. VRC01, a broadly neutralizing monoclonal antibody (bnmAbs), is capable of neutralizing a diverse array of HIV-1isolates through recognition of the loop D and the V5regions within the CD4binding site on envelope glycoprotein gp120. Nonetheless, resistant strains have been identified. Detailed analyses of VRC01escape mechanisms and study the genetic and phenotypic diversity of VRC01-resistance strains under the selection pressure within the infected individuals, can enhance our understanding of interactions between the virus and the immune system in vivo, and will undoubtedly optimize the development process and maximize the potential use of VRC01in clinical settings.We examined two genetically related envelope clones CNE47and CNE48, derived from a single time point samples of a CRF08_B’C infected patient, which showed distinct VRC01neutralization profiles. Chimeric envelope clones were then generated by interchanging the loop D and/or V5regions between the original envelopes or by single alanine substitutions within each region. The resultant effects on VRC01neutralization sensitivity were subsequently studied in the context of pseudotyped viruses. Our results showed that interchanging the V5region between two clones swapped their neutralization sensitivity profiles. Mutagenesis analysis revealed that the potential N-linked glycosylation site (PNGS) at position460enhanced the sensitivity to VRC01, while other amino acid changes made no discernible differences. Then we confirmed these results in the genetically unrelated clones CNE23and CNE30. Structural analysis showed that the presence of Asn-460would be expected to, make Arg-61less accessible to regions of gp120critical for VRC01binding and increase resistance to VRC01. Furthermore, changes in resistance were found to positively correlate with VRC01binding activity to the corresponding envelope glycoprotein. In terms of sCD4inhibition, almost all of the V5region mutants showed increased resistance to sCD4. None of the substitutions significantly altered binding and neutralization sensitivity to bnmAb b12or ibalizumab, except for alanine mutants in the PNGSs that showed significantly increased resistance to ibalizumab. CNE47and CNE48were isolated from the HIV-1CRF08_B’C infected patient50. To better understand the genetic and phenotypic diversity of VRC01-resistance strains under the selection pressure from the immune system within the infected individuals, we characterized HIV-1envelopes from two later series samples of patient50. A total of36functional diverse env clones were isolated from the two series samples.Cluster analysis showed that the env genes from a single time point were closest relative. Focusing analysis on the key VRC01contact regions, compared with CNE47, no variation were found in the loop D and CD4-binding loop, and only one or five residues differences were observed in the V5region of the isolated env sequences. Compared with CNE47, representative virus showed no significant difference in neutralization sensitivity to VRC01,b12and ibalizumab antibodies. In summary, our conclusions are as follows:1、The V5region plays a critical role in determining viral sensitivity to VRC01.2、The PNGSs at position460in the V5region can facilitate viral escape from the VRCO1.3、Neutralization sensitivity is determined by the binding affinity with VRC01.4、HIV-1variants with highly diverse sensitivity to VRC01may co-circulate at the same time in infected individual.VRC01-resistant virus can out-compete other virus under selection pressure and become the major population. |
PDF Full Text Request