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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Virology. 2010 Jan 19;398(1):115–124. doi: 10.1016/j.virol.2009.11.032

Temporal analysis of HIV envelope sequence evolution and antibody escape in a subtype A-infected individual with a broad neutralizing antibody response

Katherine A Bosch 1,2, Stephanie Rainwater 1, Walter Jaoko 3, Julie Overbaugh 1,*
PMCID: PMC2823950  NIHMSID: NIHMS167868  PMID: 20034648

Abstract

The origin of broadly neutralizing HIV-specific antibodies and their relation to HIV evolution are not well defined. Here we examined virus evolution and neutralizing antibody escape in a subtype A infected individual with a broad, cross subtype, antibody response. The majority of envelope variants isolated over the first ~ 5 years post-infection were poorly neutralized by contemporaneous plasma that neutralized variants from earlier in infection, consistent with a dynamic process of escape. The majority of variants could be neutralized by later plasma, suggesting these evolving variants may have contributed to the elicitation of new antibody responses. However, some variants from later in infection were recognized by plasma from earlier in infection, including one notably neutralization-sensitive variant that was sensitive due to a proline at position 199 in V2. These studies suggest a complex pattern of virus evolution in this individual with a broad NAb response, including persistence of neutralization-sensitive viruses.

Introduction

There is a complex interplay between neutralizing antibody (NAb) responses and viral evolution over the course of HIV-1 infection. The early stages of infection are characterized by rapid viral escape, suggesting a role for NAb in driving HIV evolution soon after the resolution of acute infection (Albert et al., 1990; Richman et al., 2003; Wei et al., 2003). In turn, as the host responds to these accumulating, antigenically diverse variants, HIV diversity could contribute to the generation of a broader repertoire of NAbs. The molecular details of this so-called ‘clash of the titans’ (Burton, Stanfield, and Wilson, 2005) remain relatively poorly defined, particularly during chronic infection. Several studies have elucidated aspects of this dynamic in the first one to two years of infection; these studies suggest that early responses are rather focused and specific to the infecting virus (autologous virus), leading to relatively rapid escape (Gray et al., 2007; Li et al., 2006a; Li et al., 2006b; Moore et al., 2009; Richman et al., 2003; Rong et al., 2009; Wei et al., 2003). Less is known about viral changes in response to NAb in chronic infection. During this period, the NAb responses often broaden to recognize not only autologous, but also some heterologous viruses, supporting a role for the evolving viruses in driving new antibody responses (Albert et al., 1990; Deeks et al., 2006; Richman et al., 2003).

Most studies to date have focused either on the early responses and corresponding sequence variation, or on studies of sequence populations, rather than individual HIV variants. Thus the molecular details of envelope escape in relation to autologous antibodies over the course of a typical HIV infection are not well-defined, although cross-sectional studies of HIV-infected mothers support the notion that there is often a mixture of neutralization-sensitive and resistant variants in most chronically infected individuals (Dickover et al., 2006; Wu et al., 2006). In one recent study, Mahalanabis et al (Mahalanabis et al., 2009) examined the relationship of autologous neutralizing antibodies and virus evolution in subtype B-infected individuals with broad antibody responses who had sustained low-level virus replication without antiviral treatment. In this study, there was an evolving mixture of both neutralization-sensitive and neutralization-resistant variants that was generally associated with the level of viral control. Given that the levels of virus replication are likely to be determined to a large extent by the properties of the infecting viral strain (Kimata et al., 1999) these studies provide important insights into the potential of HIV variants of low replication fitness to elicit robust NAb responses. To date, there has not been a detailed analysis of HIV evolution in relation to NAb responses in individuals with a robust viral infection and a correspondingly broad NAb response. Such a situation may provide insights into the role that continued virus evolution in response to NAb escape can play in shaping the breadth of the antibody response.

Here, we studied envelope evolution over time in an individual who was identified as having a notably broadly neutralizing antibody response in comparison to a group of 70 women at the same stage of infection (approximately five years post-infection), (Piantadosi et al., 2009). This subytpe A-infected individual had antibodies capable of neutralizing the majority of a panel of subtype A, B, C and D variants (Blish et al., 2009; Blish et al., 2007; Li et al., 2005; Li et al., 2006b)} at levels higher than the median of the 70 women tested. In this study, we examined temporal neutralizing antibody responses and evolution of viral envelope sequences in this individual with broadly neutralizing antibodies. We also identified one highly neutralization-sensitive variant, and defined a single amino acid that contributed to this neutralization sensitive phenotype in a context-dependent manner.

Materials and Methods

Study subjects and plasma samples

Plasmas and peripheral blood mononuclear cells (PBMCs) were obtained from an individual in an established prospective cohort of female sex workers in Mombasa, Kenya (Martin et al., 1994). This individual, QA255, was enrolled in the study in 1993 when she was HIV-seronegative, became infected in January of 1998, and provided regular samples of plasma and PBMCs until being lost to follow-up in 2004. This individual was identified as having a broad neutralizing antibody response compared to 70 women in the Mombasa cohort at the same stage of infection (~ 5 years, (Piantadosi et al., 2009) and unpublished data).

A pooled plasma sample was made by combining plasma collected between 1998 and 2000 from 30 HIV-positive individuals in Kenya, most of whom were also expected to be infected with subtype A viruses (Blish et al., 2007). All plasmas were heat-inactivated at 56° C for 45 minutes before use in neutralization assays.

Isolation of viral envelopes

DNA was extracted from ~5×106 banked uncultured PBMCs from QA255 using the QIAamp Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The proviral copy number in the extracted DNA was measured by real-time PCR amplification of a 300bp region of pol as previously described (Rousseau et al., 2004). This copy number was used to dilute DNA to a single estimated proviral copy per reaction, in order to avoid recombination between distinct proviruses during PCR and to avoid selective amplification of particular proviral sequences (Rudensey, Papenhausen, and Overbaugh, 1993). Full-length viral envelope was then amplified by nested PCR using the TaqPlus Precision PCR system (Stratagene, La Jolla, CA) as previously described (Wu et al., 2006). The first-round product was amplified using the 5′ primer vpr1 (5′-GATAGATGGAACAAGCCCCAG-3′) and an equimolar mix of the 3′ primers nef24 (5′-TACTTGTGATTGCTCCATGT-3′) and nef34 (5′-TACTTGTGACTGCTCCA TGT-3′). The second-round product was amplified using 2 μL of the 50 μL first-round reaction product as template, using the 5′ primer vpr11 (5′-ATACTAAG ACGCGTGAAGCACCCGGGAAGTCAGCCT-3′), which encodes the MluI restriction site (in bold), and the 3′ primer nef30 (5′-ATATTCTTGCGGCCGCGTCTCGAGATACTGCTCC-3′), which encodes the NotI restriction site (in bold). The final products were then digested with MluI and NotI, purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and cloned into the mammalian expression vector pCI-neo (Invitrogen, Carlsbad, CA), which had been digested with MluI and NotI and purified using the QIAquick Gel Extraction Kit, at an approximate 1:1 vector-to-insert molar ratio. Each cloned envelope variant was named with a number indicating its time point in days post-infection followed by a letter identifying the PCR reaction.

Sequence analysis

Envelope variants were sequenced throughout the open reading frame. Sequence data were analyzed in VectorNTI 10.3.0 (Invitrogen, Carlsbad, CA) and aligned using ClustalX 2.0.8 (Thompson et al., 1997) with reference sequences from subtypes A1, A2, B, C, and D obtained from the Los Alamos database (http://www.hiv.lanl.gov/content/hiv-db/mainpage.html). Sequences were codon-aligned, manually adjusted, and gap-stripped. A neighbor-joining phylogenetic tree (Saitou and Nei, 1987) was constructed in ClustalX. Genetic distances were calculated using the Kimura two-parameter model (Kimura, 1980).

Cells and viruses

To generate pseudovirus with the desired envelope, the cloned viral envelope of interest was co-transfected into pre-plated 293T cells along with a full-length subtype A proviral clone with a partial deletion in envelope (Q23Δenv) using the Fugene-6 transfection reagent (Roche, Indianapolis, IN) as previously described (Long et al., 2002). In most experiments, 1.3 μg of plasmid encoding envelope (approximately 8kb) was co-transfected in a T75 tissue culture flask pre-plated with 2×106 293T cells with 2.7 μg of plasmid encoding Q23Δenv (approximately 16kb), for an approximately equimolar envelope-to-backbone ratio; in some experiments, the ratio of envelope to proviral backbone was titrated as noted. After 48 hours, supernatants were harvested and filtered through a 0.22 μm Steriflip Filter Unit (Millipore, Billerica, MA) to remove cellular debris. Pseudoviruses were initially screened for infectivity on TZM-bl cells, a HeLa-derived indicator cell line that expresses high levels of CD4, CCR5, and CXCR4 and also expresses β-galactosidase under the transcriptional control of the HIV-LTR (Wei et al., 2002). Approximately one-third of proviral envelopes cloned were not functional as judged by their inability to infect TZM-bl cells, consistent with results observed in previous studies (Blish et al., 2009; Long et al., 2002). For functional pseudoviruses, infectious titer was measured by serially diluting viruses ten-fold, adding 50,000 TZM-bl cells in suspension in growth medium containing a final concentration of 20 μg/mL DEAE-dextran, incubating at 37° C for 48 hours, staining fixed cells for β-galactosidase activity, and directly counting stained cells.

Neutralization assays

Neutralization assays were performed using pseudoviruses to infect TZM-bl cells, as described previously (Rainwater et al., 2007; Wu et al., 2006). Five hundred infectious particles of virus were incubated with growth medium alone or with serial two-fold dilutions of heat-inactivated plasma in duplicate or triplicate in a total volume of 50 μL for one hour at 37°C. A suspension of 10,000 TZM-bl cells in 100 μL of growth medium containing a final concentration of 20 μg/mL of DEAE-dextran was then added to each well. Neutralization assays were incubated at 37° C for 48 hours and β-galactosidase activity measured using Galacto-Light Plus reagents (Applied Biosystems, Foster City, CA) on a Fluoroskan FL luminometer (Thermo Scientific, Waltham, MA). Percent neutralization was calculated as the percent reduction in β-galactosidase activity of a virus incubated with a given dilution of plasma compared to the same virus incubated with growth medium alone. Percent neutralization was then plotted against the logarithm of the plasma concentration and a dose-response curve was fit using Microsoft Excel in order to calculate the IC50, the reciprocal dilution of plasma required to inhibit infection by 50%.

Mutagenesis

Site-directed mutagenesis was carried out according to the manufacturer’s instructions for the Quik-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Ten nanograms of plasmid template were amplified using Pfu Turbo (Stratagene, La Jolla, CA) and primers containing the desired base change (sequences available upon request), with thermocycler parameters of 1 cycle of 30 seconds at 95° C followed by 16 cycles of 30 seconds at 95° C, 1 minute at 60° C, and 16 minutes at 68° C,. The PCR product was then digested with DpnI to eliminate dam-methylated template DNA and electroporated into DH5α electrocompetent cells. Colonies were screened for the presence of envelope by digestion with MluI and NotI to produce a 3.2kb band corresponding to the full-length envelope. Introduction of the desired mutation was confirmed by sequencing the region of envelope containing it; error-free amplification of the entire envelope was verified for clones containing the desired mutation by sequencing the entire open reading frame.

Western blot

To measure envelope expression on pseudoviruses, cloned envelopes were co-transfected with the Q23Δenv backbone in 293T cells as described above. Aliquots of transfection supernatant were taken for measurement of infectious titer and neutralization assays; the remaining transfection supernatant (approximately 7 mL) was concentrated to 1 mL using an Amicon Ultra 100K Filter Device (Millipore, Billerica, MA). This concentrated pseudoviral stock was then pelleted by microcentrifugation at 16,000 × g for 90 minutes at 4° C, and the pellet was resuspended in sodium dodecyl sulfate loading buffer with reducing agent (Invitrogen, Carlsbad, CA) and boiled for 5 minutes at 95° C. Samples were then resolved on a 4–12% gradient Bis-Tris polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were probed with rabbit polyclonal sera raised against an SF162 gp140 immunogen (Doria-Rose et al., 2005) as the primary antibody to measure cleaved and uncleaved envelope, and simultaneously with an anti-p24 monoclonal antibody to measure gag proteins. Secondary antibodies were a 700DX-conjugated goat-anti-rabbit IgG and an 800DX-conjugated goat anti-mouse IgG (Rockland Immunochemicals, Gilbertsville, PA). Imaging and protein quantification were done with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Results

Cloning and characterization of viral envelopes from individual QA255

Plasma from QA255 was identified as being among the top 5% in neutralization breadth in a screen of 70 women at a similar stage of infection that included an initial screen against 6 viruses(Piantadosi et al., 2009), followed by a more in-depth screen of the 20 women with the most breadth against a panel of 17 early variants representing subtypes A, B, C and D (Blish et al., 2009; Blish et al., 2007; Li et al., 2005; Li et al., 2006b). In this analysis, QA255 plasma at 1876 days (5.1 years) post-infection neutralized the majority of viruses tested at IC50 values above the median for all women tested ((Piantadosi et al., 2009) and unpublished). This individual had set-point viral load of 4.81 log10 RNA copies/ml, which is slightly higher than the median set point viral load for this cohort (4.46 log10 RNA copies/ml; (Lavreys et al., 2004).

To determine the timing of development of broad NAb in QA255, plasmas from various times post-infection from this individual were screened against a test virus panel that included four subtype A viruses that had a range of neutralization sensitivities (Q461d1 > Q842d16 > Q259d2.26 and Q769b9; (Blish et al., 2007)) and one easily neutralized subtype B virus (SF162; Table 1). Four of five viruses tested, including the easy-to-neutralize viruses, SF162 and Q461d1, were neutralized at all time points tested, starting at 189 days post-infection. The potency of neutralization stayed roughly constant over time in the case of SF162 (IC50s ranging from 473 to 834). By contrast, in the case of subtype A virus Q461d1 the potency increased from an IC50 of 313 at 189 days post-infection to a peak IC50 of 1079 at 1174 days post-infection. A similar increase in potency was observed in the neutralization of a moderately sensitive variant, Q842d16 (from an initial IC50 of 118 to consistently greater than IC50 of 500 starting at 462 days post-infection) and a harder to neutralize virus, Q259d2.26 (where the IC50 values ranged from an initial 62 to a peak of 319). The more neutralization resistant virus in this panel, Q769b9, was not neutralized at detectable levels at 189 days post-infection, but was neutralized at low levels (IC50 38–118) at each of the later times. The negative control virus, SIVMne CL8 (Overbaugh et al., 1991), was not neutralized by any of the plasma tested.

Table 1.

Neutralizing antibody responses (IC50 values) over time in subject QA255 against a panel of 6 viruses.

Virus Days Post Infection Kenya plasma pool
189 days 322 days 462 days 791 days 1174 days 1512 days 1876 days
SF162 473 575 809 673 834 702 650 1347
Q461d1 313 762 810 658 1079 807 995 610
Q842d16 118 198 505 504 510 545 606 182
Q259d2.26 62 127 264 188 249 319 257 35
Q769b9 25 47 38 50 118 88 42 25
SIVMneCl8 25 25 25 25 25 25 25 25
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Envelope clones were isolated from four times post-infection of QA255. The earliest and latest time points chosen for cloning, 189 days post-infection and 1729 days post-infection respectively, were selected in order to span extensive evolution of envelope and of NAb responses. Envelope was also cloned from two intermediate time points, 560 and 662 days post-infection, that were around the interval where the antibody responses increased in potency. In total, 25 distinct, functional, full-length envelope variants were cloned—six from 189 days post-infection, four from 560 days post-infection, four from 662 days post-infection, and eleven from 1729 days post-infection.

A phylogenetic analysis of the 25 functional envelope clones from individual QA255 showed that this individual was infected with a subtype A1 virus (Figure 1), the most common subtype in Kenya (Neilson et al., 1999; Rainwater et al., 2005). While all variants from QA255 grouped together in the phylogenetic analysis, the variants from different time points were intermingled to various extents in the phylogenetic grouping. Most variants from the earliest time point, 189 days post-infection, were closely related, with three (variants I, J, and K) almost identical (average genetic distance 0.08%) and three (C, F, and G) more distinct from each other (average genetic distance 0.56%) and from the other 189-day variants (average genetic distance 1.32%). Variants from 560 days post-infection were more diverse (average genetic distance from each other 1.38%); however, two of them (variants D and F) showed little divergence from variants present at 189 days post-infection (average genetic difference 0.17% from variants 189I, 189J, and 189K). Variants from 662 days post-infection had an average genetic distance from each other of 0.99%, with none grouping closely with earlier variants. Envelopes cloned from 1729 days post-infection, as expected, showed the greatest diversity (average genetic distance from each other 1.81%), but some were very similar in sequence to much earlier variants. For example, variant 1729E grouped closely with variants from 662 days post-infection, and variants 1729A, 1729G, and 1729M grouped closely with variants from 189 days post-infection, with an average genetic distance from 189-day variants of 0.97%, compared to an average genetic distance from 189-day variants of 1.89% for all 1729-day variants. Additionally, variants 1729A, 1729G, and 1729M showed much less divergence from the consensus sequence of the early variants (1.32% compared to 2.15% for all 1729-day variants.)

Figure 1. Neighbor-joining phylogenetic tree of all functional QA255 envelope clones.

Figure 1

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A phylogenetic tree was constructed using several reference strains of subtypes (those with names beginning with A1, A2, B, C, and D) as outgroups. All sequences with the designation QA255 are from the individual examined in this study. The subject ID is followed by the days post-infection, when the clone was isolated and a letter to designate the PCR. The following symbols also denote the days post infection:◆ = 189dpi, ⊘ = 560dpi, ■ = 662dpi, △ = 1729dpi.

Characterization of QA255 sensitivity to autologous neutralization

To elucidate patterns of escape from neutralization in individual QA255, all envelope variants were screened against autologous plasma, from the contemporaneous time point and from the next available time point (Figure 2). A number of viruses were not neutralized by contemporaneous plasma (represented by gray bars in Figure 2) but were by later plasma (represented by white bars in Figure 2), which is consistent with what has been seen in prior studies (Wei, 2003 #8090; Richman, 2003 #8122). However, nearly half of the variants, mostly those present late in infection, were not neutralized by later plasma. Conversely, 7 of 25 variants (560D, 560F, 662J, 1729A, 1729G, 1729K, 1729M) were potently neutralized not only by later plasma but also by contemporaneous plasma.

Figure 2. IC50s of all QA255 envelope variants against autologous plasma.

Figure 2

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The IC50s are plotted in relation to the phylogenetic tree of (Fig. 1). The gray bars show the IC50 value for each functional envelope variant when tested against contemporaneous plasma (189, 560, 662, and 1729 days post-infection); white bars are IC50 values against plasma from the next available time point (322, 662, 791, and 2016 days, respectively); black bars are IC50 values for each envelope variant against a plasma pool composed of equal parts plasma from all seven plasmas that were tested individually. Values represent average results from two independent experiments.

Next, in order to compare variants from all time points against the same plasma, all variants were screened against autologous plasma pooled from all time points used in the experiment with individual plasma (189, 322, 560, 662, 791, 1729 and 2016 days post-infection) (Figure 2, black bars). Most early variants were sensitive to neutralization by this pooled plasma, including two (189J and 189K) that were not sensitive to neutralization by contemporaneous plasma and only weakly so by plasma from 322 days post-infection, suggesting that an antibody response effective against these variants was eventually elicited. The majority of late variants were resistant to neutralization by the pooled plasma, which may be expected given that the pool contained only one plasma that was later in infection than the envelope variants. However, several late variants were sensitive to neutralization by the pooled plasma, with IC50 values ranging from ~200 to >1600. These included several of the late variants that clustered with earlier variants on the phylogenetic tree, 1729A, 1729G, and 1729M, all of which were particularly neutralization-sensitive to individual plasma samples. The two early variants within this cluster 189C and 189F, were also sensitive to neutralization by the pooled plasma, although less efficiently than the later variants 1729A and 1729M.

To more directly address whether antibodies capable of neutralizing some of the later variants were present at earlier times, we examined 4 variants from 1729 days post-infection as well as representative variants from earlier time points against 6 plasma samples from 322 days to 1512 days post-infection. As predicted from the pooled autologous plasma, some of the late stage variants, e.g. 1729G and 1729M, were neutralized by antibodies present at much earlier time points (Table 2.)

Table 2.

Autologous neutralizing antibody responses (IC50 values) over time against sequential envelope variants from QA255.

322 days 462 days 560 days 662 days 791 days 1512 days
189C 124 234 268 803 615 240
189K <100 <100 163 275 255 175
560A <100 <100 <100 <100 <100 <100
662C <100 <100 <100 <100 <100 <100
662J 2133 ND >3200 >3200 >3200 >3200
1729C <100 <100 <100 <100 <100 <100
1729G <100 <100 130 226 225 199
1729M 400 420 1189 1030 1430 1105
1729O <100 <100 <100 <100 <100 80
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Characterization of QA255 envelope sensitivity to heterologous neutralization

To evaluate neutralization sensitivity to heterologous antibodies, pseudoviruses expressing the panel of cloned QA255 envelopes were screened against pooled plasma obtained from 30 HIV-positive Kenyan individuals between 1998 and 2000. For 24 of the 25 envelope variants screened, the pooled plasma either failed to neutralize virus entry by 50% (N=18) or did so only at the highest concentration tested, a 1:100 dilution (N=6). However, one variant, 662J, was neutralized by more than 50% at the lowest concentration tested, a 1:3200 dilution, and was neutralized by 90% at a 1:800 dilution (Figure 3a). This same variant was also highly sensitive to autologous antibodies, as described above. To confirm that neutralization of this envelope variant was due to HIV-specific antibodies, clone 662J was screened against plasma from an HIV-negative donor and against autologous pre-seroconversion serum from individual QA255; both the heterologous negative plasma and the autologous negative serum failed to neutralize this variant by 50% at a 1:100 dilution, the highest concentration tested (Figure 3b). These results show that most envelope variants from individual QA255, including those that are sensitive to autologous neutralization, are not sensitive to neutralization by antibodies from other individuals.

Figure 3. Identification of an envelope variant that is highly sensitive to heterologous neutralization.

Figure 3

Figure 3

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A. Screen of QA255 envelope variants against plasma pooled from thirty HIV-positive individuals from Kenya. The percent neutralization is plotted against 2-fold plasma dilutions, and the results for each envelope variant are shown as a line. The bold line represents the extremely sensitive variant 662J; the other 24 similarly-resistant variants are not individually denoted as their results are similar. The dashed line represents the 50% neutralization threshold. Values represent average results from two independent experiments. B. Specificity of neutralizing antibodies to 662J. Triangles represent the same heterologous pooled plasma used in panel A; squares represent plasma from an HIV-negative donor; diamonds represent autologous serum from subject QA255 from before HIV seroconversion. The dashed line represents the 50% neutralization threshold. Values are representative of results of two independent experiments.

The variant 662J is extremely sensitive to both autologous and heterologous neutralization, making it very distinct from the other variants phenotypically, although it is unremarkable phylogenetically (Figure 1). Of note, this variant was neutralized effectively (with an IC50 of approximately 1:2000) by plasma from as early as 322 days post-infection, or approximately one year prior to the time of isolation of the envelope, and at an IC50 of greater than 1:3200 for every later time point tested (Table 2).

Molecular characterization of the neutralization sensitive QA255662J variant

To begin to assess the basis for the extreme neutralization sensitivity phenotype in clone 662J, its full envelope sequence was compared to the sequences of all 24 other variants obtained from this individual. There were three positions at which a unique residue was present in clone 662J—a glycine to tryptophan mutation in the signal peptide, a serine to proline mutation at position 199 at the base of the V1–V2 loop on the C-terminal side, and an isoleucine to valine mutation at position 535 in the transmembrane domain of gp41 (Supplemental Figure 1). The latter two mutations were examined for their effects on neutralization sensitivity. One mutant (662J P199S) was constructed in which the proline at position 199 was mutated to the subtype A consensus serine; a similar mutant (662J V535I) was constructed in which the valine at position 535 was mutated to the consensus isoleucine. 662J V535I had neutralization sensitivity equivalent to that of the parental 662J, with greater than 90% neutralization up to a 1:1600 plasma dilution. However, 662J P199S was completely resistant to the heterologous pooled plasma, similar to the other envelope variants from this individual, with less than 50% neutralization even at the highest plasma concentration tested (Figure 4), implicating the proline at position 199 in defining neutralization sensitivity.

Figure 4. Effect of P199S and V535I mutations on 662J neutralization sensitivity.

Figure 4

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The percent neutralization is plotted against 2-fold plasma dilutions of the heterologous pooled plasma. Squares represent the parental 662J variant, diamonds represent the corresponding P199S mutant, and triangles represent the corresponding V535I mutant. The dashed line represents the 50% neutralization threshold. Values are representative of two independent experiments.

To determine whether a proline at position 199 could confer a neutralization sensitive phenotype in other, neutralization-resistant variants from the same individual, we introduced this change into 8 other variants, sampled from 4 different times post infection. All of the envelope variants encoding the S199P mutation had reduced titers compared to the parental form; four (560A, 662F, 1729C, and 1729O) showed differences of >100-fold; one of these mutants (1729O) was rendered completely non-infectious by the mutation (Figure 5). Conversely, introduction of the reverse P199S mutation into the neutralization-sensitive variant 662J increased its infectious titer by approximately six-fold.

Figure 5. Effect of the S199P mutation on envelope expression, infectivity and neutralization sensitivity in different backgrounds.

Figure 5

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The top panel shows a comparison of infectious titer (first two bars) and envelope expression levels (last 2 bars) of the wild type and corresponding mutant at position 199. The designation of the wild type envelope variant is shown below each 4 bar grouping. For 662J, the wild-type variant is proline at 199 while the mutant variant is a change to serine, P199S. For all other variants, the wild-type variant is serine at 199 and the mutant variant is a change to proline, S199P. In the table below, titers and envelope expression levels are shown as fold-change from wild-type to mutant (P199 to S199 for 662J, S199 to P199 for all others). The IC50 values are shown below; ND means not done because titers were too low; <100 means that 50% neutralization was not detected at the lowest dilution tested, 1:100. Values are representative of the results of two independent experiments

The four mutants (189C, 662C, 1729G and 1729M, Figure 5) that yielded pseudoviruses of high enough titer to be evaluated for neutralization sensitivity showed a range of effects of the S199P mutation on neutralization sensitivity. For example, variant 189C and the mutant 189C S199P were similarly resistant to neutralization by heterologous plasma, with neither being neutralized by 50% even at the highest plasma concentration tested. By contrast, variants 662C, 1729G, and 1729M were all fully resistant to heterologous neutralization in their parental forms but significantly more sensitive in their S199P mutant forms, with 50% neutralization being reached at dilutions of 1:353 for 662C S199P, 1:692 for 1729G S199P, and 1:138 for 1729M S199P (Figure 5). However, no S199P mutant recapitulated the extreme neutralization sensitivity of variant 662J, which showed greater than 50% neutralization even by the lowest concentration of plasma tested. This suggests that other sequence characteristics of variant 662J may contribute to the phenotype of extreme neutralization sensitivity.

Contribution of envelope expression levels to neutralization sensitivity of the 662J variant

Western blot analysis of the parental and S199P mutants showed that the effect of the mutation on envelope expression generally paralleled that on infectious titer: envelope expression was consistently reduced in most S199P mutants compared to wild type, with the exception of 189C. Interestingly, the P199S showed the least disruption if titer in this virus background. Conversely, the introduction of the reverse P199S mutation in 662J restored envelope expression from its very low baseline (Figure 5).

The effect of the S199P mutation on envelope expression raised the possibility that the extreme neutralization sensitivity of the 662J variant encoding this S199P change could be due to its defect in envelope expression. In order to address this question, we generated parental 662J pseudoviruses with higher levels of envelope expression and mutant 662J P199S pseudoviruses with reduced levels of envelope expression and tested their neutralization sensitivity. To do this, ratios of envelope to backbone were varied in co-transfections, and envelope expression was measured by Western blot and normalized to p24 expression. While envelope expression relative to p24 expression did not increase or decrease in direct proportion to the amount of envelope transfected, we did generate 662J and 662J P199S preparations expressing similar levels of envelope (e.g. 662J at 2:1 and 662J P199S at 1:1). However, even with their envelope incorporation increased to approximately the level seen in the 662J P199S mutant, 662J preparations remained sensitive to neutralization even at a plasma dilution of 1:3200, while 662J P199S preparations remained completely neutralization-resistant even with reduced envelope expression (Figure 6). These results indicate that it is unlikely that impairment of envelope expression alone explains the extreme neutralization sensitivity of variant 662J.

Figure 6. Effect of titrating envelope expression level on 662J and 662J P199S sensitivity to heterologous neutralization.

Figure 6

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The top panel shows a western blot, with the ratio of plasmids encoding the envelope and proviral backbone used for the transfection shown above the blot. To the left, the locations of the proteins of interest are shown. In the lower panel, a neutralization graph is shown comparing pseudovirus derived from the 662J and 662J P199S (designated 662J PS here) at different envelope to backbone ratios, where closed symbols represent 662J parental variants, with darker shades of gray indicating a greater amount of envelope transfected, and open symbols represent 662J P199S mutant variants, with darker shades of gray indicating a smaller amount of envelope transfected, as shown in the key at the bottom. The dashed line represents the 50% neutralization threshold. Plasma used was the heterologous pooled plasma. Values are representative of the results of two independent experiments.

Role of glycosylation at position 199 in neutralization sensitivity phenotype

The S199P mutation, besides introducing a proline residue in place of the highly-conserved serine, also disrupts the highly-conserved potential N-linked glycosylation site at N197. To determine whether the effect of this mutation on neutralization phenotype was simply due to the loss of the glycosylation site or whether the proline was itself necessary to confer the phenotype, a 662J P199A mutant envelope was constructed. This variant, like the parent 662J variant with the proline at position 199, lacks the conserved potential N-linked glycosylation site at position 197. However, unlike the parent virus, the P199A mutant was completely resistant to neutralization (Figure 7), suggesting that loss of the predicted glycosylation site alone is not sufficient to confer the sensitivity phenotype; rather, the specific presence of the proline residue at 199 is important.

Figure 7. Effect of eliminating the position 197 glycosylation site with a P199A mutation.

Figure 7

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Plasma used is the heterologous pooled plasma. Closed triangles represent the parental 662J variant and open triangles represent the 662J P199A mutant. The dashed line represents the 50% neutralization threshold.

Discussion

In this study, envelope variants from a single individual with naturally-occurring broad NAb were characterized in detail, with the goal of defining the process of neutralization escape in both early and chronic infection in a person with a broad NAb response. While some envelope variants followed the expected pattern of NAb elicitation followed by rapid escape, others were recognized very poorly or not at all by autologous NAb. Most surprisingly, some sensitive variants seemed to persist throughout the course of infection despite the constant presence of antibodies capable of neutralizing them. Experiments to characterize the overall neutralization sensitivity of envelope variants in this individual revealed the presence of one variant, 662J, that was extremely sensitive to neutralization, and this phenotype was mapped to a single serine-to-proline mutation at the base of the V1/V2 loop.

Several studies have provided evidence that NAb typically rapidly select for escape variants in the first years of HIV infection [(Albert et al., 1990; Moore et al., 2009; Richman et al., 2003; Rong et al., 2009; Wei et al., 2003). Thus, it was surprising to find several variants (including the ultra-sensitive variant, 662J) that were sensitive to neutralization not only by contemporaneous antibodies but also by antibodies present several years earlier in infection. The present study differs from the prior studies in that it included ~5 years of follow-up in an individual specifically selected for the presence of broadly neutralizing antibodies, which was not a selection criterion in the prior studies. Our finding of persistence of neutralization-sensitive envelope variants is consistent with results of a recent study (Mahalanabis et al., 2009) that included individuals selected for both broad neutralizing antibody and strong control of viremia, studied at time points late in chronic infection.

Collectively, these studies raise several new questions. First, are the persistent sensitive variants representative of actively replicating viruses, and how prevalent are they in the viral population overall? Because these envelopes were cloned from integrated proviral DNA it is theoretically possible that they represent archived rather than currently-replicating virus variants. However, the fact that three of eleven variants isolated from 1729 days, or nearly five years, post-infection were similar in neutralization sensitivity to variants from four years earlier argues against this hypothesis, since HIV-infected activated T cells have a short half-life (Markowitz et al., 2003; Perelson et al., 1997; Perelson et al., 1996) and latently-infected, resting T cells are rare (Chun et al., 1997). Additionally, other studies have reported results consistent with these using envelope cloned directly from viral RNA (Mahalanabis et al., 2009; Skrabal et al., 2005).

Second, why are these neutralization-sensitive variants able to persist until late in infection in the face of antibodies capable of neutralizing them and in the presence of contemporaneous virus that is not sensitive to neutralization by those antibodies? Because changes in envelope can have major effects on replicative fitness (Rangel et al., 2003), it is possible that escape carries a cost in replicative fitness, leading to a balance between more-fit but neutralization-sensitive variants and less-fit but neutralization-resistant variants. Another, possibility is that neutralization assays such as the one used here, which measures neutralization of cell-free virus infection, do not accurately reflect the processes of virus infection and neutralization in an infected individual, which could potentially involve both cell-free and direct cell-to cell virus spread. In this model, variants identified as neutralization-sensitive could persist if they are not neutralized during cell-to-cell spread by antibodies capable of blocking cell-free virus infection.

QA255 was identified because she had a broad neutralizing antibody response at 5 years post infection relative to other women in the cohort (Piantadosi et al., 2009). This broad response was evident as early as 189 days after infection - a time at which 4 of the 5 test viruses examined here were neutralized, as were other variants of subtypes B, C and D (data not shown). The development of breadth in chronically infected individuals may be due to either continued antigenic stimulation with multiple diverse variants that evolve over time, as suggested by one recent study (Scheid et al., 2009) or to the elicitation of antibodies of broad specificity as recently demonstrated in one individual with exceptionally broad antibody responses (Walker et al., 2009), or both. In the individual studied here, there was a very narrow window of HIV infection prior to the development of broad neutralizing antibodies, suggesting the possibility that breadth in this case may be due to the elicitation of an antibody with broad specificity. Such individuals are of high interest because they may hold the clues to identifying antibodies capable of recognizing highly diverse, circulating HIV variants.

QA255 mounted a broad heterologous response despite not having an unusually potent autologous response. Neutralization potency was similar or, in some cases, even greater against the heterologous virus than the autologous early virus. The relatively weak autologous response in QA255 contrasts with findings in individuals infected with subtype C viruses, where neutralizing antibodies capable of potently neutralizing autologous virus, with a median IC50 of 2,363, were observed within the first 30 months of infection(Li et al., 2006a). HIV autologous NAb tend to target the variable loops of envelope and are therefore often type-specific (Rong et al., 2007; Sagar et al., 2006), whereas broad NAb capable of neutralizing circulating strains of HIV are more likely to target conserved, conformational epitopes such as the CD4 binding site.(Dhillon et al., 2007; Li et al., 2007). Thus, these data also suggest that the neutralizing antibodies elicited in this individual recognize a conserved target, leading to broad specificity.

One env variant in this individual, cloned from 662 days, was extremely sensitive both to autologous neutralization by plasma from a wide range of time points and to heterologous neutralization. A single amino acid change, from a highly conserved serine at position 199 to a proline, was found to be necessary and sufficient to confer extreme neutralization sensitivity to the variant 662J, though not sufficient to confer the same phenotype to other envelope variants. Functionally, this position lies within the bridging sheet that forms part of the co-receptor binding site (Groenink et al., 1993; Ogert et al., 2001; Sullivan et al., 1993) Importantly, the S199P mutation also disrupts a highly conserved predicted N-linked glycosylation site at N197 that was previously described as contributing to a glycan shield on the bases of sequence changes that contributed to NAb escape(Wei et al., 2003). However, here we demonstrated by site-directed mutagenesis that the phenotypic effect of the mutation is not due to disruption of the glycosylation site, but rather specifically requires the presence of the proline residue. These analyses highlight the potential limitations of predicting disruption of glycosylation sites based only on amino acid sequence motifs and underscore the importance of demonstrating that changes in glycosylation are actually driving the biological differences, as has been done with studies of SIV NAb escape (Chackerian, Rudensey, and Overbaugh, 1997).

While the S199P mutation altered envelope expression levels and infectivity, changes in the level of envelope expression did not result in dramatic changes in neutralization sensitivity, indicating that the neutralization sensitivity of variant 662J is more likely due to changes in envelope conformation leading to epitope exposure. Amino acid changes in this region have been shown in other studies to confer CD4 independence (Kolchinsky et al., 2001; Li et al., 2008), a phenotype associated not only with extreme neutralization sensitivity (Edwards et al., 2001; Kolchinsky, Kiprilov, and Sodroski, 2001) but also with the elicitation of broad neutralizing antibodies (Li et al., 2008; Zhang et al., 2002; Zhang et al., 2007). It is therefore tempting to speculate that the envelope conformation responsible for the 662J’s neutralization sensitivity may also present conserved epitopes and thus be related to the development of broadly neutralizing antibodies in this individual.

This detailed analyses of envelope evolution in a subtype A-infected individual with typical viral load and a broad NAb response suggest that neutralizing antibodies do not invariably drive immune escape in HIV infection. These findings reveal a complex dynamic between virus evolution and neutralizing antibody escape that was not apparent in studies focused on NAb escape at earlier stages of infection.

Supplementary Material

01

Acknowledgments

We would to thank like the Mombasa research team for their support and efforts, particularly Kishorchandra Mandaliya, J.O. Ndinya-Achola, R. Scott McClelland and the lab staff, and Anne Piantadosi for her comments on the manuscript. We particularly acknowledge the women who in the Mombasa cohort who contributed to this study. This research was supported by NIH grant R01 HD058304 to JO. KAB was supported in part by T32 AI07509.

Footnotes

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