Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity

Abstract

Over the course of infection, human immunodeficiency virus type 1 (HIV-1) continuously adapts to evade the evolving host neutralizing antibody responses. Changes in the envelope variable loop sequences, particularly the extent of glycosylation, have been implicated in antibody escape. To document modifications that potentially influence antibody susceptibility, we compared envelope variable loops 1 and 2 (V1-V2) from multiple sequences isolated at the primary phase of infection to those isolated around 2 to 3 years into the chronic phase of infection in nine women with HIV-1 subtype A. HIV-1 sequences isolated during chronic infection had significantly longer V1-V2 loops, with a significantly higher number of potential N-linked glycosylation sites, than the sequences isolated early in infection. To assess the effects of these V1-V2 changes on antibody neutralization and infectivity, we created chimeric envelope sequences, which incorporated a subject's V1-V2 sequences into a common subtype A envelope backbone and then used them to generate pseudotyped viruses. Compared to the parent virus, the introduction of a subject's early-infection V1-V2 envelope variable loops rendered the chimeric envelope more sensitive to that subject's plasma samples but only to plasma samples collected >6 months after the sequences were isolated. Neutralization was not detected with the same plasma when the early-infection V1-V2 sequences were replaced with chronic-infection V1-V2 sequences, suggesting that changes in V1-V2 contribute to antibody escape. Pseudotyped viruses with V1-V2 segments from different times in infection, however, showed no significant difference in neutralization sensitivity to heterologous pooled plasma, suggesting that viruses with V1-V2 loops from early in infection were not inherently more neutralization sensitive. Pseudotyped viruses bearing chimeric envelopes with early-infection V1-V2 sequences showed a trend in infecting cells with low CD4 concentrations more efficiently, while engineered viruses with V1-V2 sequences isolated during chronic infection were moderately better at infecting cells with low CCR5 concentrations. These studies suggest that changes within the V1-V2 envelope domains over the course of an infection influence sensitivity to autologous neutralizing antibodies and may also impact host receptor/coreceptor interactions.

FIG. 1.

Phylogram of envelope sequences from the nine subjects described here, sequences from other subjects previously isolated in our laboratory, and reference subtype sequences from the Los Alamos HIV-1 database. The sequences analyzed include nucleotides 340 to 1107 relative to the coding sequence for the HXB2 reference strain gp160 envelope gene. Sequences were aligned using Clustal X and further manually codon aligned using MacClade (version 4.01). After gap stripping, a neighbor joining tree was constructed in the software package Phylogenetic Analysis Using Parsimony and Other Methods (PAUP 4.02b2a) (39) with a general-time-reversible model using a gamma distribution rate parameter of 0.5. For the sake of clarity, only one sequence from a set of identical sequences was included in this phylogram. Early-infection sequences are denoted by black boxes and chronic-infection sequences are denoted by gray boxes. Numbers at the nodes represent the bootstrap values for those nodes from 100 bootstrap resamplings. H.BE.93.VI was used as an outgroup because it was likely to have the least homology with the other sequences. The clusters formed by the subjects' sequences and the HIV-1 subtype grouping are labeled to the right of the trees. Q23, the backbone envelope used for the construction of all chimeric envelopes, is also labeled.

FIG. 2.

Distribution of the number of amino acids (A) and PNGS (B) within V1-V2 loops in sequences isolated during early (black bars) and chronic (white bars) infection. The numbers of amino acids (A) or PNGS (B) are shown along the x axes, and the fractions of the sequences are shown along the y axes. Each subject's graph shows the Z and P values for a Wilcoxon rank sum test comparing the distribution of the V1-V2 lengths (A) or PNGS (B) for sequences isolated early in infection versus that for chronic-infection sequences.

FIG. 3.

Changes in the positions of PNGS in the chronic-infection sequences relative to that in the paired predominant early-infection sequences within each subject. The PNGS positions were assigned according to the HXB2 envelope amino acid numbering. Changes in the numbers of PNGS occurred as a result of either insertions/deletions (ID) or mutations (M). The proportions of the chronic-infection sequences with PNGS changes are listed in parentheses. The numbers or ranges for the PNGS differences in the chronic-infection sequences relative to those in the early-infection sequences are shown outside the parentheses.

FIG. 4.

Subjects' predicted early- and chronic-infection V1-V2 amino acid sequences used to create chimeric envelopes in the Q23 backbone. Each sequence is aligned to the HXB2 reference sequence, shown in single-letter amino acid code. The parent Q23 V1-V2 sequence is shown below the HXB2 reference. The name of each sequence identifies the subject and the time, in number of months after estimated date of infection, when the V1-V2 sequences were isolated. Relative to the HXB2 sequence, a dash indicates no amino acid change and a dot indicates an insertion or deletion. All PNGS are identified in bold. The V1 and V2 domains and the HXB2 envelope amino acid number are indicated above the HXB2 sequence.

FIG. 5.

Neutralization of viral pseudotypes with plasma collected at various times after infection. Each panel represents neutralization of pseudotyped viruses with chimeric envelope proteins with plasma from the subject from whom the V1-V2 sequences were derived. The designation for the subject is listed in the top left corner of each panel. The x axis shows the time, in number of months after the estimated date of infection, when the plasma sample was collected. The y axis shows the IC₅₀ (the reciprocal of the plasma dilution required to inhibit infection by 50%). All IC₅₀s represent mean values from two or more independent experiments with viral stocks from two separate preparations. The error bars show the 95% confidence intervals. Any plasma that did not achieve at least 50% inhibition at the highest dilution of 1:25 was assigned an IC₅₀ value of 12.5. Pseudotyped viruses with early-infection V1-V2 sequences are denoted by white triangles and chronic-infection V1-V2 sequences by white squares. The parental Q23 clones, with their own native V1-V2 segments, are denoted with black circles. For two subjects (QA203 and QB424), another pseudotyped virus with a chimeric envelope incorporating a second chronic-infection V1-V2 sequence was tested and is denoted by white squares with dashed lines. For QC168, pseudotyped virus with the full-length early-infection envelope gp160 was also tested and is denoted by open triangles with dashed lines. The two arrows on each subject's graph indicate the time, in months after estimated date of infection, when the early-infection and chronic-infection sequences were isolated. The y axis scale ranges from 1 to 100 except for QA284, QB424, QC168, and QC890, for whom the scale was expanded because more-potent antibody responses were detected. In each panel, the approximate time when antibody responses against pseudotyped viruses with early-infection V1-V2 sequences were detected is denoted in months.

FIG. 6.

Neutralization of pseudotyped viruses with pooled plasma. Plasma pools were collected by combining plasma from 30 different HIV-1-infected Kenyan subjects. The virus names on the x axis identify the subjects and the times, in number of months after estimated date of infection, when the V1-V2 sequences were isolated. The y axis shows the IC₅₀s (the reciprocals of the plasma dilutions required to inhibit infection by 50%). All IC₅₀s represent mean values from two or more independent experiments with viral stocks from two separate preparations. The error bars show the 95% confidence intervals. QC168(gp160) denotes a pseudotyped virus with a full-length early-infection envelope. Q23 denotes the parent Q23 virus pseudotyped with an envelope carrying the native V1-V2 loops from the original clone.

FIG. 7.

Relative infection levels for the low-CD4, medium-CCR5 (RC-49) (A) and high-CD4, low-CCR5 (JC-10) (B) cell lines (25) with recombinant pseudotyped viruses with chimeric envelopes. The virus names on the x axes identify the subjects and the times, in number of months after estimated date of infection, when the V1-V2 sequences were isolated. The y axes show the infection levels for the respective cell lines. These values are normalized relative to the infection levels for the high-CD4, high-CCR5 (JC-53) cell line (25). All infection levels represent mean values from two or more independent experiments with viral stocks from two separate preparations. The error bars show the 95% confidence intervals.