Genetic and neutralization sensitivity of diverse HIV-1 env clones from chronically infected patients in China

Abstract

As HIV-1 continues to spread in China from traditional high risk populations to the general public, its genetic makeup has become increasingly complex. However, the impact of these genetic changes on the biological and neutralization sensitivity of the virus is unknown. The current study aims to characterize the genetic, biological, and neutralization sensitivity of HIV-1 identified in China between 2004 and 2007. Based on a total of 107 full-length envelope genes obtained directly from the infected patients, we found that those viruses fell into three major genetic groups: CRF01_AE, subtype B', and subtype C/CRF07_BC/CRF08_BC/B'C. Pseudotyped viruses built upon the viable env genes have demonstrated their substantial variability in mediating viral entry and in sensitivity to neutralization by subtype-specific plasma pools and broadly neutralizing monoclonal antibodies (bnmAb). Many viruses are resistant to one or more bnmAb, including those known to have high potency against diverse viruses from outside China. Sequence and structural analysis has revealed several mechanisms by which these resistant viruses escape recognition from bnmAb. We believe that these results will help us to better understand the impact of genetic diversity on the neutralizing sensitivity of the viruses and to facilitate the design of immunogens capable of eliciting antibodies with potency and breadth similar to those of bnmAb.

FIGURE 1.

Unrooted neighbor-joining tree depicting the genetic relationship among the 88 full-length envelope molecular clones. The branch length is drawn to scale so that the relatedness between different sequences can be readily assessed. Individual sequences clustered with CRF01_AE are colored in green, those clustered with subtype B′ are shown in red, and those clustered with subtype C/07/08/B′C are shown in blue. A number of commonly used reference sequences for classifying HIV-1 subtypes and CRFs were also included and highlighted in each group with a different color. Closed circles represent those clones capable of mediating viral entry, whereas those indicated by open circles failed to do so.

FIGURE 2.

Identification of putative novel recombinants by SimPlot analysis. Right, recombination patterns for envelope genes. Left, recombination pattern for the entire HIV-1 genome. The envelope genes analyzed include CNE15, CNE16, CNE18, CNE7, and CNE67 and were compared with prototype 07_BC.CN54 and 08_BC.GX6F as well as previously identified non-typical recombinants BC.YNRL9607 and BC.YNRL9613. CNE87, shown to be a possible novel recombinant by phylogenetic analysis (Fig. 1), is not included because it failed to mediate viral entry. Analysis of the full-length HIV-1 genome was conducted for two clones, CNE15-YN8 and CNE18-YN22. The y axis shows the percentage of identity within a sliding window of 400 bp, with a step size between plots of 40 bp. Comparison of these sequences was made against selected reference strains B.CN.RL42, C.95IN21068, and 01_AE.90.TH.CM240.

FIGURE 3.

Comparison of the number of amino acid residues in the variable loops (a) and number of potential N-linked glycosylation sites (b) among CRF01_AE (n = 12), C/07/08/B′C (n = 47), and subtype B′ (n = 12) sequences. Significant differences are indicated by horizontal brackets.

FIGURE 4.

Neutralization sensitivity of selected HIV-1 isolates to subtype-specific plasma pools. The top 31 envelope clones capable of mediating viral entry were evaluated for neutralization sensitivity using the three subtype-specific plasma pools from chronically infected individuals. The average reciprocal log₁₀ID₅₀ titer for each plasma pool is indicated by a representative symbol. The black filled circles indicate the average log₁₀ID₅₀ titer across all three plasma pools.

FIGURE 5.

Two-dimensional clustering heat map to group viruses with similar neutralization sensitivity to bnmAb (a) and to subtype-specific plasma pools (b) and to compare between the two. The top 31 envelope clones capable of mediating viral entry were assessed for neutralization sensitivities using either seven bnmAb and sCD4 or the three subtype-specific plasma pools from chronically infected patients. Individual bnmAb, sCD4, and subtype-specific plasma pools are indicated at the bottom of the heat map. The magnitude of neutralization (ID₅₀ titer) is indicated by color, where lower neutralization values are represented by lighter colors and higher values by darker colors. Viruses are grouped based on their overall neutralization sensitivity patterns and exemplified by the dendrogram beside the heat map. Straight lines connecting the same viruses between the two heat maps were used to assess whether viruses grouped by bnmAb were also grouped by the subtype-specific plasma pools. Clustering of bnmAb, sCD4, and subtype-specific plasma pools was also conducted simultaneously based on their similar neutralization profiles, shown by the dendrogram above the heat map.

FIGURE 6.

Top, close-up structural view of the interactions between gp120 of VRC01-resistant viruses and antibody VRC01. The interactions are shown wheat-colored, with loop D in blue and V5 in orange. The heavy chain and light chain of antibody VRC01 are shown in surface with green and cyan colors, respectively. Residue Arg-61 on the heavy chain of VRC01 that plays significant roles in binding is colored in yellow. Residues in the loop D and V5 regions of gp120 with bulky side chains predicted to interfere with the binding with VRC01 are colored red. Sequence alignment of VRC01-resistant isolates at the loop D and V5 regions is shown at the bottom. The bulky residues that may interfere with binding of VRC01 are highlighted in red. Dots represent identical residues, whereas dashes represent gaps introduced to preserve alignment with the reference 93TH057 sequence.