Genotypic and functional properties of early infant HIV-1 envelopes

Abstract

Background: Understanding the properties of HIV-1 variants that are transmitted from women to their infants is crucial to improving strategies to prevent transmission. In this study, 162 full-length envelope (env) clones were generated from plasma RNA obtained from 5 HIV-1 Clade B infected mother-infant pairs. Following extensive genotypic and phylogenetic analyses, 35 representative clones were selected for functional studies.

Results: Infant quasispecies were highly homogeneous and generally represented minor maternal variants, consistent with transmission across a selective bottleneck. Infant clones did not differ from the maternal in env length, or glycosylation. All infant variants utilized the CCR5 co-receptor, but were not macrophage tropic. Relatively high levels (IC₅₀ ≥ 100 μg/ml) of autologous maternal plasma IgG were required to neutralize maternal and infant viruses; however, all infant viruses were neutralized by pooled sera from HIV-1 infected individuals, implying that they were not inherently neutralization-resistant. All infant viruses were sensitive to the HIV-1 entry inhibitors Enfuvirtide and soluble CD4; none were resistant to Maraviroc. Sensitivity to human monoclonal antibodies 4E10, 2F5, b12 and 2G12 varied.

Conclusions: This study provides extensive characterization of the genotypic and functional properties of HIV-1 env shortly after transmission. We present the first detailed comparisons of the macrophage tropism of infant and maternal env variants and their sensitivity to Maraviroc, the only CCR5 antagonist approved for therapeutic use. These findings may have implications for improving approaches to prevent mother-to-child HIV-1 transmission.

Figure 1

Evolutionary relationships of HIV-1 env clones. Evolutionary history was inferred using the Neighbor-Joining method. (A) V1-V5 nucleotide sequences of cloned env and subtype reference sequences. Filled triangle = infant, empty circle = maternal sequence. (B) Full length gp160 nucleotide sequences. M = maternal, P = infant. The percentage of replicate trees in which the associated sequences clustered together >70% of the time in the bootstrap test (1000 replicates) are shown to the left of branches in (B). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated from the dataset. Horizontal scale bars represent (A) 5%, or (B) 1% genetic distance.

Figure 2

Highlighter analysis of infant P1024 V1-V5 sequences. The subject quasispecies consists of three variants. Sequences belonging to the same variant are indicated by colored arrows. Pink and blue variants arose from transmission of two very closely related maternal viruses, or by post-transmission diversification. The brown variant arose from transmission of a distinct maternal virus.

Figure 3

Infant quasispecies are more homogeneous than maternal. The percent of base substitutions per site over the V1-V5 region for each subject were computed using the Kimura 2-parameter method in the MEGA4 software program.

Figure 4

Receptor and co-receptor requirements of cloned env. Pseudoviruses expressing cloned env were titered on HeLa cell lines engineered to express various levels of CD4 and CCR5. To normalize between different pseudovirus preparations, titers are expressed as a ratio of the titer on the cell line divided by the titer on TZMbl cells. (A) TZMbl, (B) JC37, (C) JC10, (D) RC49, (E) RC23. Results are an average of 3 independent experiments performed in duplicate. Average number of receptor and co-receptor molecules per cell as reported by Platt et al [21] is inset in the charts. Filled triangle = infant, empty circle = maternal. Pairwise statistical analysis performed using the Mixed Model ANOVA with mother-infant pairings included as random effects indicated that mean maternal and infant titers did not vary significantly across pairs.

Figure 5

Macrophage infectivity. Pseudoviruses expressing cloned envelopes were titered on primary macrophage cultures. Macrophage infectivity is expressed as the percentage of the TZMbl titer achieved on macrophages. Data is representative of three independent assays performed in duplicate. Filled triangle = infant, empty circle = maternal. Clone M1002 G1 is highlighted red. Cont = Controls [42], see inset key: (macrophage tropic) NA20 B59, NA420 B33 and JRFL, (non-macrophage topic) JRCSF, NA420 LN40 and NA20 LN8.

Figure 6

Sensitivity of maternal and infant env to neutralization or inhibition. The sensitivity of infant and maternal clones to (A) autologous maternal IgG, (B) NAbs, (C) pooled seropositive plasma and (D) entry inhibitors was determined using pseudovirus infection of TZMbl cells. (A) Neutralization IC₅₀ of maternal and infant env clones. Lines indicate maximum concentration of IgG. (B-D) Values are an average of two different pseudovirus stocks run in the same experiment. Solid lines indicate infant and maternal means. Dotted lines in (B) indicate maximum concentration of NAb used. Filled triangle = infant, empty circle = maternal. Pairwise statistical analysis performed using the Mixed Model ANOVA with mother-infant pairings included as random effects indicated that mean maternal and infant IC₅₀ did not vary significantly across pairs.