Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular env Clones from Acute and Early Heterosexually Acquired Infections in Southern Africa^▿

Plasma samples, soluble CD4 (sCD4), and monoclonal Abs.

Plasma samples from chronically HIV-1-infected blood donors in South Africa who were presumed to be treatment naive were obtained from the South African National Blood Services. These individual plasma samples were designated BB8, BB12, BB28, BB55, BB70, and BB106 and were selected from a larger set of plasma samples based on their greater neutralizing activities in an initial screen against three subtype C Env-pseudotyped viruses (Du151.2, Du156.12, and Du172.17). All donors were infected with HIV-1 subtype C viruses as determined by gag and env sequences. Plasma pools for subtypes A, B, C, and D were described previously (45). These latter plasma pools were each comprised of plasma from 6 to 10 subjects who had pure subtype infections as verified by full HIV-1 genome sequencing of DNA from cryopreserved peripheral blood mononuclear cells (PBMC). The plasma samples and plasma pools were derived from chronically infected individuals and did not include the HIV-1-positive subjects who served as a source of env clones. A normal plasma pool was prepared from Leukopaks from four HIV-1-negative subjects (BRT Laboratories, Inc., Baltimore, MD). All plasma samples were heat inactivated at 56°C for 1 h prior to use. Informed consent was obtained from all study participants as approved by local institutional review boards and biosafety committees; the blood bank samples were obtained from anonymous donors, with all sample codes delinked from subject identifiers.

Recombinant sCD4 comprising the full-length extracellular domain of human CD4 and produced in Chinese hamster ovary cells was obtained from Progenics Pharmaceuticals, Inc. (Tarrytown, NY). Human monoclonal Ab immunoglobulin G1b12 (IgG1b12) was kindly provided by Dennis Burton (The Scripps Research Institute, La Jolla, CA). Human monoclonal Abs 2G12, 2F5, and 4E10 were obtained from the NIH AIDS Research and Reference Reagent Program (NIH ARRRP; Rockville, MD) as contributed by Hermann Katinger. Monoclonal Abs 17b, 23e, 31H, 21c, E51, 48d, 112d, 412d, and ED10 against CD4-induced (CD4i) epitopes on gp120 were isolated from Epstein-Barr virus-transformed B-cell lines established from subtype B HIV-1-infected individuals as described previously (66, 90). TriMab is a mixture of three monoclonal Abs (IgG1b12, 2G12, and 2F5) prepared as a 1-mg/ml stock solution containing 333 μg of each monoclonal Ab/ml in phosphate-buffered saline, pH 7.4.

Cells.

TZM-bl (JC53-bl) cells were obtained from the NIH ARRRP as contributed by John Kappes and Xiaoyun Wu. This is a genetically engineered HeLa cell clone that expresses CD4, CXCR4, and CCR5 and contains Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli β-galactosidase under regulatory control of an HIV-1 long terminal repeat (60, 83). 293T/17 cells were obtained from the American Type Culture Collection (catalog no. 11268). Both cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies) containing 10% heat-inactivated fetal bovine serum and 50 μg gentamicin/ml in vented T-75 culture flasks (Corning-Costar). Cultures were incubated at 37°C in a humidified 5% CO2-95% air environment. Cell monolayers were split 1:10 at confluence by treatment with 0.25% trypsin, 1 mM EDTA (Invitrogen).

Amplification and cloning of env/rev DNA cassettes.

Specimens for env/rev gene cloning were obtained from 18 HIV-1-infected individuals living in either South Africa or Zambia and who were judged to be in acute/early stages of infection as determined by either their last known seronegative clinic visit, time of onset of acute retroviral syndrome, or a combination of these two clinical parameters. All infections occurred through heterosexual contact. env-rev cassettes were cloned from either plasma viral RNA, uncultured PBMC DNA, or cultured PBMC DNA. In the latter case, fresh phytohemagglutinin-stimulated PBMC from healthy HIV-1-negative donors were inoculated with low-passage (<5 passages) primary isolates, and the infected PBMC were used as the source of DNA for PCR amplification of HIV-1 env-rev cassettes as described elsewhere (45). Virus-containing culture supernatants were clarified from cells by 0.45-μm filtration and stored at −80°C in 1-ml aliquots as archives of the immediate uncloned parent of the corresponding molecularly cloned Env-pseudotyped viruses. In some cases, uncultured PBMC direct from study subjects were used for DNA extraction and PCR amplification. In other cases, virion-associated plasma RNA was purified and subjected to cDNA synthesis prior to PCR amplification as described elsewhere (23, 44, 45). The inner antisense primer EnvNF (5′-GGCACCTGAGGTCTGACTGGAAAGCC-3′) replaced antisense primer EnvN in the second-round PCR and was used in conjunction with inner sense primer EnvA (23) to amplify full-length env from ZM197M. PCR products from cultured PBMC DNA were inserted directly into pcDNA 3.1D/V5-His-TOPO (Invitrogen Corp., Carlsbad, CA). PCR products from uncultured PBMC DNA and from plasma viral RNA were inserted into either pcDNA 3.1/V5-His-TOPO or pCR3.1 (Invitrogen Corp., Carlsbad, CA) by TA cloning.

Plasmid minipreps from multiple colonies of transformed JM109 cells were screened by restriction enzyme digestion for full-length inserts. Clones with inserts in the correct orientation were screened by cotransfection with an env-deficient HIV-1 (SG3Δenv) backbone in 293T cells to produce Env-pseudotyped viruses that were subsequently titrated for infectivity in TZM-bl cells as described previously (45). Env clones conferring the highest infectivity were selected for further characterization. Twelve of these molecularly cloned gp160 genes were chosen to comprise a new panel of standard reference reagents and have been donated to the NIH ARRRP (item no. 11326).

Other viruses.

A functional gp160 clone for the R5 subtype B virus SF162.LS (17, 73) was obtained from Leonidas Stamatatos. T-cell line-adapted HIV-1_MN (30) was obtained from Robert Gallo and was propagated in H9 cells as described elsewhere (53). Molecularly cloned gp160 genes from a standard panel of subtype B reference strains was described previously (45). The CD4-independent strain NL-ADArs (38) was obtained from Joseph Sodroski and was propagated in PBMC.

DNA sequence and phylogenetic analysis.

Sequence analysis was performed by cycle sequencing and BigDye terminator chemistry with automated DNA Sequenators (models 3100 and 3730; Applied Biosystems, Inc.) as recommended by the manufacturer. Individual sequence fragments for each env clone were assembled and edited using the Sequencher program 4.2 (Gene Codes Corp., Ann Arbor, MI). Nucleotide and deduced Env amino acid sequences were initially aligned using CLUSTAL W (35, 76) and manually adjusted for an optimal alignment using MASE (27). Pair-wise evolutionary distances were estimated using Kimura's two-parameter method (37) to correct for superimposed substitutions; sequence gaps and ambiguous areas within the alignment were excluded from all comparisons. Phylogenetic trees were constructed by the neighbor-joining method (67), and the reliability of branching orders was assessed by bootstrap analysis using 1,000 replicates (28). The sequences of all new gp160 genes are available from GenBank and the Los Alamos HIV Sequence Database (see Table 1 for accession numbers).

Analysis of coreceptor usage.

Coreceptor usage of 15 Env-pseudotyped viruses was determined in TZM-bl cells by measuring reductions in infectivity in the presence of the CXCR4 antagonist AMD 3100 and the CCR5 antagonist TAK-779 as described elsewhere (45). For the three CAP clones, coreceptor usage was determined by green fluorescence protein (GFP) reporter gene activation in GHOST cells that expressed either CCR5 or CXCR4 (19).

Neutralization assay.

Neutralization was measured as a reduction in Luc reporter gene expression after a single round of virus infection in TZM-bl cells as described previously (52). This assay is a modified version of the assay used by Wei et al. (83, 84). The 50% inhibitory dose (ID₅₀) was defined as either the plasma dilution or sample concentration (in the case of sCD4 and monoclonal Abs) at which relative luminescence units (RLU) were reduced 50% compared to virus control wells after subtraction of background RLU in cell control wells.

Statistical analyses.

Differences in the neutralizing potencies of subtype C plasma samples (six samples with a BB prefix) were determined by comparing their geometric mean titer (GMT) against each virus. Differences in neutralization sensitivity between subtype B and C viruses were compared using a two-sided Wilcoxon matched pairs test with a 95% confidence interval. Differences were considered significant if P was <0.05. Differences in envelope lengths and number of potential N-linked glycans (PNLG) between subtype B and C viruses were compared using the Wilcoxon two-sided rank test. The two-sided Fisher's exact test was used to determine the relative loss of specific PNLG in one HIV-1 subtype compared to another. Both of these latter tests were performed using the R Project for Statistical Computing (www.r-project.org).

Nucleotide sequence accession numbers.

GenBank accession numbers for the sequences identified in this study are DQ411850 to DQ411854, DQ388514 to DQ388517, DQ422948, DQ435682 to DQ435684, AY423971, AY423984, AY424079, AY424138, and AY424163.

Demographics and biologic properties of the molecularly cloned gp160 genes.

Candidate gp160 reference clones were obtained from studies of acute and early sexually acquired subtype C infections in South Africa and Zambia (Table 1). Five clones with a Du prefix were obtained in 1998 from commercial sex workers recruited from truck stops between Durban and Johannesburg (11); these women were participating in a multicenter clinical trial of a potential vaginal microbicide (81). Clones with a ZM prefix were obtained between June 1998 and July 2003 from a study of HIV-1-discordant couples in Lusaka, Zambia (1, 23). Clones with a CAP prefix were obtained in 2005 from a study of acute/early heterosexual HIV-1 transmission organized by the Center for the AIDS Program of Research in South Africa (CAPRISA). Twelve strains arose from male-female transmission and six strains arose from female-male transmission. All molecularly cloned Env-pseudotyped viruses used CCR5 but not CXCR4 for cell entry.

Sequence analysis.

Phylogenetic analysis of full-length gp160 nucleotide sequences confirmed that all 18 functional clones grouped within subtype C (Fig. 1). The clones comprised a wide spectrum of genetic diversity with only two clones (Du151.2 and Du422.1) that clustered with a high bootstrap value.

Deduced amino acid sequences showed that all 18 clones had an uninterrupted env open reading frame and 100% conservation of the cysteine residues that form the V1, V2, V3, and V4 loops of gp120 (Fig. 2). Considerable amino acid sequence variability was seen in V1, V2, V4, V5, and a region of approximately 34 amino acids immediately downstream of V3. V1, V2, V4, and V5 also exhibited substantial length variation (Table 2). V3 did not vary in length (35 amino acids in all clones) and exhibited only minor sequence variability that included position 320 (numbered as in Fig. 2) flanking the tip of the loop; this same position also shows greatest variability in the V3 loop of subtype B gp160 reference clones (45). Compared to a subtype C consensus sequence, amino acids in the V3 loop of these clones exhibited an average of 12% variability, whereas the 34 amino acids immediately downstream varied by an average of 39%.

Moderate variation was seen in the number and position of PNLG, where V1, V2, and V4 of gp120 were the most heavily glycosylated regions (Fig. 2; Table 3). All clones contained at least one PNLG in the relatively short stretch of amino acids comprising V5. Six PNLG in gp120 and three in gp41 were 100% conserved in all 18 clones (Fig. 2). Another three PNLG were conserved in all but one clone (Fig. 2). One highly conserved PNLG in the N terminus of V3 has been shown to mask neutralization epitopes in the V3 loop of subtype B viruses (2, 71).

Neutralization phenotype.

Neutralization phenotypes were characterized with six subtype C plasma samples, four subtype-specific plasma pools, sCD4, and the broadly neutralizing monoclonal Abs IgG1b12, 2G12, 2F5, and 4E10 (Table 4). They were also characterized with a series of monoclonal Abs against CD4i epitopes on gp120 (Table 5). Each subtype C Env-pseudotyped virus was broadly sensitive to neutralization by individual subtype C plasma samples and by subtype-specific plasma pools. Some subtype C viruses were clearly more sensitive than others, but the level of sensitivity in all cases was at least 10-fold lower than that of MN and SF162.LS (Fig. 3A). This diminished sensitivity relative to MN and SF162.LS distinguishes the subtype C viruses from easily neutralized “tier 1” viruses (47). Another interesting property of the subtype C Env-pseudotyped viruses was their greater sensitivity to neutralization by a subtype C plasma pool compared to plasma pools of subtypes A, B, and D (P = 0.0002). The overall potencies of these plasma pools were ranked in the following order: subtype C > subtype A > subtype D > subtype B pool. Significant differences also were seen for the subtype A pool compared to the subtype B (P = 0.0078) and subtype D (P = 0.0443) pools but not when the subtype B pool was compared to the subtype D pool (P = 0.107).

All subtype C Env-pseudotyped viruses were sensitive to inhibition by sCD4. ID₅₀ doses ranged from 0.2 μg/ml to 26 μg/ml, suggesting a broad spectrum of epitope exposure in and around the CD4 binding site of gp120. IgG1b12, which targets a complex epitope on gp120 that affects CD4 binding (13, 58), neutralized 10 of the 17 subtype C Env clones. No clear association was seen between the neutralizing activity of this monoclonal Ab and sensitivity to inhibition by sCD4.

All 18 gp160 clones were highly resistant to neutralization by the glycan-specific monoclonal Ab 2G12. Similar broad resistance to 2G12 has been reported for other subtype C viruses (7, 11, 33) and is associated with an absence of PNLG at critical positions that affect the 2G12 epitope (7, 14, 33, 69, 70). Each of our clones lacked a PNLG at one or more of these critical positions; most often this was position 295 at the N-terminal base of V3. Loss of a PNLG at position 295 has been associated with the general 2G12-resistant phenotype of subtype C viruses from chronically infected individuals (7) and pediatric infections (33). Our results indicate that the same is true for newly transmitted, heterosexually acquired subtype C viruses.

Monoclonal Abs 2F5 and 4E10, which recognize adjacent epitopes in the membrane-proximal external region of gp41 (3, 56, 57, 61, 74, 95), were quite different from one another in their ability to neutralize these subtype C viruses: 2F5 neutralized only 2 viruses, whereas 4E10 neutralized all 18 viruses. Neutralization by these two monoclonal Abs was highly predicted by amino acid sequence. Thus, both 2F5-sensitive viruses contained a DKW motif that has been reported to be a minimum requirement for 2F5 recognition (7, 45). Two additional clones (ZM214M.PL15 and CAP210.2.00.E8) contained this motif but had changes elsewhere in the 2F5 epitope. All clones except ZM215F.PB8 contained the WFXI motif that has been reported to be important for 4E10 recognition (7, 45). Clone ZM215F.PB8 contained a different motif (WFNM) yet it was highly susceptible to neutralization by 4E10, suggesting additional flexibility in the 4E10 core epitope. Six clones contained a potential PNLG in this core epitope that might be expected to mask the virus from 4E10, but biochemical evidence suggests this site is not glycosylated (41).

Consistent with the general resistance of these viruses to 2G12 and 2F5, their ability to be neutralized by TriMab (mixture of IgG1b12, 2G12, and 2F5) tracked with their sensitivity to IgG1b12. We saw no clear evidence of synergism with this triple combination of monoclonal Abs.

Neutralization phenotypes were further assessed by using CD4i monoclonal Abs to probe epitopes in the coreceptor binding domain of gp120. These are some of the most highly conserved epitopes on the gp120 molecule (22), but they are often concealed from Abs unless sCD4 is present to induce their exposure prior to virus-cell engagement (22, 68, 75). The CD4-independent NL-ADArs virus on which CD4i epitopes are spontaneously exposed (38) was highly sensitive to all of these monoclonal Abs, confirming that they possessed potential neutralizing activity (Table 5). Most of these CD4i monoclonal Abs also neutralized SF162.LS and, to a lesser degree, MN, although much higher concentrations were usually needed in these cases compared to NL-ADArs. In general, the subtype C Env-pseudotyped viruses were resistant to these CD4i monoclonal Abs regardless of whether subinhibitory doses of sCD4 were present (Table 5). An exception was E51, which neutralized 5 of 12 subtype C viruses but only at high concentrations. These latter results agree with a previous study in which E51 was superior to other CD4i antibodies (91). We observed two cases where sCD4 augmented the neutralizing activity of a CD4i monoclonal Ab: one case produced at least a fivefold increase in sensitivity (neutralization of Du172.17 by E51), whereas the other case was much less dramatic (neutralization of ZM135M.PL10a by 412d). Notably, this latter virus was the only new strain that was neutralized by four CD4i monoclonal Abs, including two that did not neutralize the other subtype C viruses. Although the potency of neutralization against ZM135M.PL10a was relatively weak, there appears to be greater exposure of CD4i epitopes on this virus compared to other subtype C viruses. Overall, the results suggest that epitopes in the coreceptor binding domain on newly transmitted subtype C viruses are mostly concealed from Abs.

Noting that our gp160 genes were cloned from either cocultured PBMC virus, uncultured PBMC, or directly from plasma, we performed an analysis to determine whether the source of gp160 influenced the general neutralization sensitivity of the corresponding Env-pseudotyped viruses. For this analysis, combined neutralization data from the subtype C plasma samples (BB samples) and subtype-specific plasma pools in Table 4 were compared between the three categories of clones. The results showed a trend in neutralization sensitivity such that cocultured PBMC clones (GMT, 344) > uncultured PBMC clones (GMT, 129) > plasma clones (GMT, 101), but this trend was not significant (P > 0.05). Also, greater resistance to IgG1b12 was seen in the uncultured PBMC group of clones, especially when compared to the cocultured PBMC clones. We caution that a larger number of clones will be needed to confirm this latter observation.

Selection of standard reference strains.

The demographic, biologic, genetic, and neutralization properties of many of the Env-pseudotyped viruses described in this report appear suitable for standardized assessments of vaccine-elicited NAb responses. For adequate statistical power, it has been recommended that each standard panel be comprised of 12 reference strains (47). Among the 18 candidate reference strains described here, 12 were selected to comprise a panel that represents the greatest genetic and antigenic diversity while avoiding strains that are unusually sensitive or resistant to neutralization. None of the 18 strains would be considered unusually sensitive to neutralization; however, 1 strain was omitted for being relatively insensitive to neutralization by HIV-1-positive plasma samples (CAP244.2.00.D3). Another strain (ZM215F.PB8) was omitted because careful analysis showed that all bulk-derived env genes from this patient (who had a heterogenous early infection) were in vitro recombinants. Also, the Du123.6 strain was excluded because it was easily neutralized by pool C plasma, with titers higher than those seen for MN and SF162.LS viruses. On the other hand, two strains (Du422.1 and Du151.2) were phylogenetically closer to each other than any other pair of strains, clustering together with a high bootstrap value (Fig. 1); to maximize the genetic diversity among members in the panel, only Du422.1 was selected while Du151.2 was excluded. Of the 12 selected strains, 7 originated in Zambia and 5 in South Africa (Table 1). Also, six arose by male-female transmission, while the remaining six arose by female-male transmission (Table 1).

Genetic comparisons between subtype B and C reference strains.

The variable loops and number and position of PNLG in gp120 have been shown to influence the neutralization phenotype of HIV-1 (8, 26, 48, 56, 62, 84). Recent results also suggest that acute/early subtype B and C strains differ from one another in their neutralization properties (23, 29). For these reasons, amino acid sequences of the 12 subtype C gp160 reference genes were compared to the sequences of 12 subtype B gp160 reference genes described previously (45). Comparisons also were made between subtype B and C sequences in the LANL database. These comparisons focused on the number of amino acids that comprise gp120 and its variable regions. They also focused on the number and position of PNLG in gp120.

LANL database sequence comparisons showed that subtype C gp120 was generally shorter and contained fewer PNLG compared to subtype B (Table 6). Differences in gp120 length were evident by shorter V1, V3, and V4 regions despite a slightly longer V2 region in subtype C. Fewer PNLG were present in V1, V3, V4, and V5 of subtype C gp120, whereas the number of PNLG in V2 remained constant in both subtypes. Many PNLG sites were common in both subtypes, but four were prevalent in only one subtype. Subtype-specific PNLG included positions 230 (between V2 and V3) and 442 (between V4 and V5) that were diminished in subtype B (HXB2 numbering). They also included positions 295 (proximal to N terminus of V3) and 362 (between V3 and V4) that were diminished in subtype C (HXB2 numbering).

No significant differences were seen in the lengths of V2 and V3 and the number of PNLG in V1 and V5 when sequences of the subtype B and C reference clones were compared. Thus, overall the subtype C panel of reference clones contained shorter gp120 regions (V1 and V4) and fewer PNLG in gp120 (V1, V3, and V4) than the subtype B panel of reference clones. Differences observed only in database (mostly chronic virus) sequences might be an indication that gp120 of certain subtypes evolved over time in infected individuals to give rise to viral variants that are rarely transmitted. In this regard, our intrasubtype comparisons suggest that transmission of subtype C favors variants with fewer PNLG in V4 compared to chronic subtype C variants (P = 0.01). The comparisons also suggest that transmission of subtype B favors variants with a greater number of PNLG in gp120 compared to chronic subtype B variants (P = 0.02).

Neutralization phenotype comparisons between subtype B and C reference strains.

Results obtained with the subtype C plasma samples from South Africa, subtype-specific plasma pools, sCD4, and monoclonal Abs were used to compare the neutralization phenotypes of subtype B and C reference strains. Overall, the subtype C reference strains were more sensitive to neutralization by subtype C plasma samples (P = 0.0005) (Fig. 3). Additional comparisons were made with published data on a panel of 12 subtype B reference strains (45). In general, the subtype C panel was less sensitive to neutralization by a subtype B plasma pool (P = 0.009), but no significant difference was found between the two panels of reference strains when compared in terms of their overall sensitivity to CD4, IgG1b12, 4E10, and the subtype A, C, and D plasma pools. The most dramatic difference between the two panels of reference strains was the uniform resistance of subtype C to neutralization by 2G12 and their infrequent neutralization by 2F5.