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Identification and Characterization of HIV-1 CD8+ T Cell Escape Variants with Impaired Fitness

  1. Victor Sanchez-Merino1,
  2. Melissa A. Farrow2,a,
  3. Frank Brewster1,
  4. Mohan Somasundaran1 and
  5. Katherine Luzuriaga1
  1. 1Department of Pediatrics and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester
  2. 2Interdisciplinary Graduate Program, University of Massachusetts Medical School, Worcester
  1. Reprints or correspondence: Dr. Katherine Luzuriaga, Div. of Pediatric Immunology, Infectious Diseases, and Rheumatology, Depts. of Pediatrics and Molecular Medicine, University of Massachusetts Medical School—Biotech II, 373 Plantation St., Ste. 318, Worcester, MA 01605 (katherine.luzuriaga{at}umassmed.edu).

  1. Presented in part: Keystone Symposium, Keystone, Colorado, 27 March–2 April 2006.

 
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Abstract

In this study, amino acid sequence variation inhumanimmunodeficiency virus (HIV)—1 GagCD8+ Tcell epitopes was examined in untreated mother-infant pairs. Several HIV-1 CD8+ T cell escape variants were identified within maternal plasma viral p17 and p24 sequences that were either not detected or did not persist in the plasma of their non—HLA-matched HIV-1—infected infants. Viruses constructed with each of these mutations demonstrated reduced viral replication in vitro and reduced expression of p17 and p24 proteins compared with wild type. Reduced recognition of the variant sequences compared with wild-type sequence was also demonstrated by enzyme-linked immunospot assays. Nontransmission or reversion after transmission was thus associated with reduced viral fitness cost in vivo. Better understanding of the balance between CD8+ T cell selective pressures and viral fitness cost may facilitate the identification of optimal viral sequences for inclusion in HIV-1 vaccines.

CD8+ T cell responses play an important role in controlling the replication of HIV in humans and of simian immunodeficiency virus (SIV) in rhesus macaques [1–6]. HIV and SIV are characterized by the presence of multiple variants within individuals as a consequence of high viral turnover, high viral reverse-transcriptase (RT) error rate, recombination, and selective pressures exerted by the host's immune system (including CD8+ T cell responses [7–9]). The generation of 108–109 new viral particles per day in chronically infected individuals [10, 11] creates an environment in which, in the presence of immune selection pressure exerted by CD8+ T cells, a large number of CD8+ T cell escape variants should be produced every day. HIV-1 and SIV escape from CD8+ T cell recognition has been well documented in the acute and chronic phases of HIV-1 and SIV infections [12–18], and the transmission of viral escape variants to a new host has been documented in both horizontal and vertical HIV-1 infections [17, 19–23] The stability of CD8+ T cell escape variants after transmission may depend on the balance between CD8+ T cell—mediated selective pressures and cost to viral replicative capacity. Reversion to wild-type sequence will likely occur if the escape mutant is transmitted to a non—HLA-matched recipient (absence of the CD8+ T cell selective pressures) and the escape mutation is located in a region within the viral genome where an escape mutation has a low replication fitness cost for the virus [24]. During the last few years, we and others have begun to study the balance between CD8+ T cell selective pressures and viral evolution in the SIV macaque model [25–27] and in HIV-1—infected humans [20, 23, 28].

We undertook the present study to better understand the balance between CD8+ T cell selective pressure and structural constraints on CD8+ T cell escape in young infants during acute infection. In our study, maternal plasma sequences were analyzed at delivery along with early infant viral sequences to detect the presence of CD8+ T cell escape variants in maternal plasma and transmission or nontransmission of these escape variants to their infants. Serial plasma samples obtained from infants during the first year of life were then studied to determine the fate of HIV-1 CD8+ T cell escape variants after transmission to non—HLA-matched infants. Viruses representing these variants were generated by site-directed mutagenesis experiments, and competition assays were performed to characterize the fitness cost of those mutations in vitro.

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Materials and Methods

Study population. Three HIV-1—infected mother-infant pairs (M-1001/P-1024, M-1002/P-1031, and M-1005/P-1026) were selected from our previous study [23]. Diagnostic studies (DNA polymerase chain reaction [PCR] and viral isolation) were consistent with HIV-1 infection during the intrapartum period [29]. RNA was extracted from maternal plasma at delivery and from infant plasma obtained at 2–3 months of age and again between 4 and 15 months of age. This study examined plasma samples obtained in 1990–1992, before the routine use of antiretroviral therapy (ART) to interrupt mother-to-child HIV-1 transmission, and therefore none of the mothers were receiving ART during pregnancy or at delivery. Infants were studied at least twice before the initiation of ART. Characteristics of the mother-infant pairs studied are shown in table 1. The Human Studies Committee at the University of Massachusetts Medical School approved these studies, and informed consent was obtained for participation.

Amplification, cloning, and sequencing of maternal and infant viruses. Viral RNA was extracted from 200 μL of plasma by use of the High Pure Viral RNA Kit (Roche Diagnostics). Complete amplification of gag genes, cloning into PCR4 TOPO vector, and sequencing was performed as described elsewhere [23].

Peripheral blood mononuclear cell (PBMC) isolation, peptide synthesis, molecular HLA class I typing, and interferon (IFN)—γ enzyme-linked immunospot (ELISpot) assay. PBMC isolation, peptide synthesis, molecular HLA class I typing, and IFN-γ ELISpot assays were performed as described elsewhere [23].

Mutagenesis, recombinant viruses, DNA transfection, and infections. Mutations corresponding to those observed in the B*8-restricted epitope (K26R [p17]), A*2-restricted epitope (G116A and G116T [p24]), and mutation Q127K (p24) were introduced into pNL43, as described elsewhere [23] (primer sequences available on request). Mutated pNL43 plasmids were designated as pNL43p17E17K, pNL43p17K26R, pNL43p24G116A, pNL43p24G116T, and pNL43E17K/Q127K. The gag gene from M-1002, P-1024 at 4 months, and P-1024 at 15 months of age were introduced by creating 2 restriction sites (XbaI and ApaI). DNA transfections and infections were performed as described elsewhere [23]. Viral production and replication were monitored by p24 antigen (Ag) detection. Several independent experiments were performed to determine the best supernatant dilution for detecting accurate p24 values. Once dilutions were established for each sample, duplicate or triplicate wells were used.

Gag p17 and p24 expression from transfected HeLa cells by Western blot analysis. HeLa cells (5 × 106/flask) were transfected by the calcium phosphate precipitation technique with 20 μg of each proviral plasmid DNA (pNL43, pNL43p17E17K, pNL43p17K26R, pNL43p24G116A, and mock), as described above.

Viral growth competition assays. Viral growth competition assays were performed with MT-4 cells. First, 5 × 106 MT-4 cells were coinfected with wild-type virus (NL43) and viral mutants (NL43E17K, NL43K26R, NL43G116A, NL43G116T, recombinant NL43gagP1024-4mos, and recombinant NL43gagP1024-15mos) at 3 initial ratios (passage 0) of 1:1, 5:1, and 1:5 (ratio of wild type to mutant), according to p24 values obtained from a transfection experiment described elsewhere. Assays were performed for at least 3 passages (each passage was performed after 1 week in culture). For each competition passage, 5 × 106 fresh MT-4 cells were infected with 500 μL of supernatant from the previous passage. Supernatants from each passage were harvested. RNA extraction, gag amplification by RT-PCR, and cloning into TOPO vector was performed as described elsewhere [23]. The proportion of each mutant clone in the competition passages was determined by sequencing; 15–20 clones were then analyzed from each passage in the competition.

Nucleotide sequence accession numbers. The nucleotide sequences that we obtained from each pair have been submitted to GenBank (accession numbers AY786790—AY786869 for M-1001/P-1024 and M-1002/P-1031 and numbers AY786950—AY786979 for M-1005/P-1026).

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Results

Identification of maternal CD8+ T cell epitope variants that revert to wild-type sequences after transmission to non—HLA-matched infants in early infection. We have previously reported the common transmission of maternal CD8+ T cell escape variants to their infants. Most became fixed in the infant's viral population [23]. However, in mother M-1002 (B*4002 positive), we described a rare amino acid substitution, E17K, within an HLA-B*4002—restricted Gag p17 (aa 11–19; GELDRWEKI [GI9]) CD8+ T cell epitope. This amino acid substitution was present in all 10 maternal viral clones at delivery but was never detected in this mother's B*4002-negative child (P-1031) at 2 months of age [23] (figure 1A). This epitope was noted to lie in the α-helical region 1 of p17, which is known to be important for viral replication in culture [30]. We have found another 2 variants in Gag (another in p17 and 1 in p24) that either were never detected in the infant's plasma or reverted after transmission to the infant (table 1).

Figure 1
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Figure 1

Deduced amino acid alignment of epitopes GI9, GL8 (Gag p17), and TM9 (Gag p24). Consensus (Cons.) B amino acid sequence is also shown. Dots represent amino acid identity with each maternal sequences at delivery (clones 3M-1002, b5, and 9s). A, Alignment of the B*4002-restricted epitope GELDRWEKI (GI9) with maternal (M-1002) and infant (P-1031) plasma viral sequences at 2, 4, and 11 months of age. B, Alignment of the B*8-restricted epitope GGKKKYKL (GL8) with maternal (M-1001) and infant (P-1024) plasma viral sequences at 2, 4, and 15 months of age. C, Alignment of the A*2-restricted epitope TLQEQIGWM (TM9) with maternal (M-1005) and infant (P-1026) plasma viral sequences at 3 and 15 months of age.

In mother M-1001 (B*8 positive), amino acid substitution K26R was detected within a HLA-B*8—restricted Gag p17 (aa 24–31; GGKKKYKL [GL8]) CD8+ T cell epitope. This K26R amino acid substitution reverted to wild type after transmission to her HLA-B*8—negative infant, P-1024, during the first year of life (figure 1B). The amino acid substitution K26R has been shown to distort the α-helical region 1 of p17 [31].

Finally, in mother M-1005 (A*2 positive), amino acid substitution G116T was observed within an HLA-A*2—restricted Gag p24 (aa 110–118; TLQEQIGWM [TM9]) CD8+ T cell epitope. This amino acid substitution was not detected after transmission to her HLA-A*2—negative infant, P-1026, at 15 months of age (figure 1C). According to p24 structural analysis, the TM9 epitope lies on the side of helix 6 in proximity to the CypA-binding loop, and when CypA binds to the p24 protein, methionine-248 in the binding loop packs against G116A [28]; a mutation could thus disrupt the binding and consequently affect viral replication.

Altogether, the presence within the Gag protein of these 3 amino acid substitutions in conserved and important regions for viral replication (E17K and K26R in p17; G116T in p24) and either reversion or posttransmission selection to wild-type sequences after transmission to non—HLA-matched infants in early infection suggested that these mutations may confer a fitness cost for viral replicative capacity in vivo.

Reduced recognition of variant sequences. Using ELISpot assays, we next determined whether maternal CD8+ T cells recognized the transmitted CD8+ T cell epitope variants. PBMCs from the mothers' infants were used as controls to confirm lack of recognition by non—HLA-matched individuals. We have reported elsewhere that maternal PBMCs from M-1002 poorly recognized the variant sequence GELDRWKKI compared with the wild-type sequence, GELDRWEKI [23–]. PBMCs from her B*4002-negative infant, P-1031, did not recognize either wild type or variant, as expected [23].

Maternal PBMCs from M-1005 also showed reduced recognition of the variant sequence TLQEQITWM compared with wild type (figure 2A). Neither this variant nor the wild type were recognized by PBMCs from her A*2-negative infant, P-1026 (figure 2B). Variant TLQEQIAWM was less robustly recognized at a high peptide concentration than was the wild-type sequence by PBMCs from M-1005. A small difference in recognition was observed in comparison with the wild-type sequence (figure 2A). PBMCs from her A*2-negative infant, P-1026, did not recognize either wild type or variant, as expected (figure 2B). Finally, the third CD8+ T cell epitope variant detected in maternal plasma samples from M-1001 in p17 (GGRKKYKL) has been well described as a B*8 CD8+ T cell escape mutant [31].

Figure 2
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Figure 2

HIV-1 specific CD8+ T cell responses. Maternal (M-1005) (A) and infant (P-1026) (B) peripheral blood mononuclear cell (PBMC) responses to TLQEQIGWM (TM9 wild type [WT]), TLQEQITWM (TM9 mutant G116T), and TLQEQIAWM (TM9 mutant G116A), as demonstrated by peptide titration assays. Responses are recorded as spot-forming cells per 106 PBMCs. Two criteria were used to define positive responses. First, each well (from triplicate wells) must have had a minimum of 6 sfc (corresponding to 60 sfc/106 PBMCs). Second, the experimental spot-forming cell frequency must have exceeded the mean of all triplicate negative control wells (PBMCs with no peptide) by 2 SDs. Phytohemagglutinin stimulation of PBMC samples from all study time points provided positive controls for interferon-γ release.

Replicative capacity of viruses harboring mutations E17K, K26R, and G116T compared with wild-type sequence. Reversion or posttransmission selection to wild-type sequences of E17K, K26R, and G116T mutants in non—HLA-matched infants suggested reduced replicative capacity of these variants in vivo. We were next interested in determining whether these mutations had an impact on viral replicative capacity in vitro. Site-directed mutagenesis experiments were performed to introduce these mutations into plasmid pNL4—3 background. We also introduced mutant G116A to confirm that this amino acid substitution had a low impact on viral replicative capacity, as has been previously suggested [28]. HeLa cell cultures transfected with proviral plasmid DNA pNL43p17E17K had shown markedly lower levels of particle production compared with the wild-type virus [23]. After transfection, fewer viral particles were also produced from pNL43p17K26R (P < .001, t test), pNL43p24G116A (P < .004, t test), and pNL43p24G116T (P < .001, t test) mutants, as measured by p24 Ag detection when compared with wild type (figure 3A and 3C). To detect viable virions obtained from transfection, RT activity was also measured (figure 3B and 3D). All but 1 mutant (pNL43p17K26R, which showed similar levels of RT activity compared with that of wild type) (figure 3B) demonstrated lower levels of RT activity than did wild type (for comparison with pNL43p24G116A, P < .051, t test; for comparison with pNL43p24G116T; P < .002, t test). Viruses produced by transfection were collected and then used to infect MT-4 cells. After infection with equivalent amounts of p24 Ag (10 ng/mL), viral mutants NL43G116A and NL43G116T showed lower levels of replication than did the wild-type virus. Mutant NL43K26R replicated as well as wild-type pNL4—3 in MT-4 cells (figure 4A). NL43G116A and NL43G116T mutants replicated 1.7- and 2.3- fold less than did wild-type pNL4—3, respectively, at the peak of viral replication (day 6 after infection) (figure 4B).

Figure 3
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Figure 3

Viral production after transfection of wild-type (WT) pNL43 and mutant plasmids pNL43K26R, pNL43p17K26R, pNL43p24G116A, and pNL43p24G116T in HeLa cells. HeLa cells (3 × 106/flask) were seeded into 75-cm2 tissue culture flasks 24 h before transfection. HeLa cell cultures were then transfected by the calcium phosphate precipitation technique with 20 μg of each proviral plasmid DNA. Forty-eight hours after transfection, supernatants were collected and passed through 0.45-μm-pore filters, and viral production was quantified by p24 antigen detection assay and reverse-transcriptase (RT) production. P values (unpaired t test) are shown. A and B, p24 and RT values, respectively, obtained from WT and mutant pNL43K26R transfection. C and D, p24 and RT values, respectively, obtained from WT and mutant pNL43G116A and pNL43G116T transfection.

Figure 4
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Figure 4

Viral replicative capacity of wild type (WT) and mutants and recombinant virus. Equivalent p24 amounts (10 ng/mL) of NL43, NL43XbaI, NL43E17K, NL43K26R, NL43G116A, and NL43G116T and recombinant NL43gagM1002 were used to infect 4 × 106 fresh MT-4 cells. Supernatants from different intervals after infection were collected and passed through 0.45-μm-pore filters, and viral production was quantified by p24 antigen detection assay. A, NL43 (WT) and mutant NL43K26R replication kinetics. B, NL43 (WT) and mutant NL43G116A and NL43G116T replication kinetics. C, NL43(WT), NL43XbaI (control for introduction of XbaI into NL43), NL43E17K (control for lack of replication), recombinant NL43gagM1002, and double-mutant NL43E17K/Q127K replication kinetics. XbaI was introduced into pNL43 from M-1002 and P-1024 (4 months). ApaI was created only in M-1002 because that restriction site was already present in pNL43 (the introduction of the ApaI restriction site in gag genes from M-1002 did not change the amino acid present at that position).

To confirm whether mutants NL43E17K, NL43K26R, NL43G116A, and NL43G116T had impaired viral replicative capacity, viral growth competition assays were performed. Infections were initiated at 3 initial ratios (passage 0) of 1:1, 5:1, and 1:5 (ratio of wild type to mutant), according to p24 Ag values obtained from a transfection experiment described elsewhere. Viruses were allowed to compete for 3 passages. Analyses of ratio 1:1 at passage 1 (7 days after initial coinfection) showed a quick replacement of the viral population of mutant clones by wild-type clones (100% wild type), except for mutant NL43G116A, in which a mixture between mutant and wild type (57% wild type and 43% mutant) was observed. This proportion was maintained at passages 2 and 3 (data not shown). In the analysis of ratio 5:1, only wild-type clones were detected at passage 1 in all of them (including mutant NL43p24G116A). At an input ratio of 1:5 (16% wild type and 84% mutant), wild type outgrew mutants NL43E17K, NL43K26R, and NL43G116T. Mutants NL43E17K and NL43G116T were not detected from passage 1, and NL43K26R clones represented 20% of the population compared with input (64% reduction) at passage 1. At passages 2 and 3, only wild-type clones were detected (figure 5A, 5B, and 5D). However, the initial proportion of mutant NL43G116A clones was maintained (84%) at passages 1 and 2, and only a small reduction in the proportion (20%) of mutant clones was observed at passage 3 (64% mutant sequences were detected) (figure 5C).

Figure 5
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Figure 5

Viral growth competition assays. Analysis of ratio 1:5 (ratio of wild type to mutant) is shown. First, 5 × 106 fresh MT-4 cells were coinfected with wild type (NL43); mutants NL43E17K (A), NL43K26R (B), NL43G116A (C), and NL43G116T (D); and recombinant viruses NL43gagP1024—4mos and NL43gagP1024—15mos (E), with an initial ratio (passage 0) of 1:5. The cultures were allowed to compete for 3 passages. For each competition passage, 5 × 106 fresh MT-4 cells were infected with 500 μL of supernatant from the previous passage. Supernatants from each passage were harvested. RNA extraction, gag amplification by reverse-transcriptase polymerase chain reaction, and cloning into PCR4 TOPO vector were performed. The proportion of each mutant clone in the competition passages was determined by sequencing 20 clones.

Next, we were interested in determining whether viral replicative capacity in the maternal virus harboring the E17K mutation was restored with secondary compensatory mutations in Gag protein. We compared plasma viral sequences from M-1002 at delivery with infant's sequences at 2 months of age. We observed 6 amino acid changes in the infant's sequences compared with maternal sequences in the whole Gag protein (data not shown). Only 1 amino acid change (Q127K) was not common in the HIV-1 database and was present at subsequent time points. The virus harboring this double mutant E17K/Q127K did not replicate in MT-4 cells (figure 4C), indicating that Q127K was not a compensatory mutation for E17K. We decided next to introduce M-1002 gag gene into the pNL43 backbone. The recombinant virus NL43gagM1002 did not infect MT-4 cells either (figure 4C), suggesting that secondary compensatory mutations are located outside the Gag protein.

To further estimate the true cost of fitness of escape mutations in the primary isolates, we performed a competition assay with recombinant viruses expressing gag genes from P-1024 at 4 months of age (NL43gagP1024-4mos) and P-1024 at 15 months of age (NL43gagP1024-15mos). At an input ratio of 1:5 (16% NL43gagP1024-15mos and 84% NL43gagP1024-4mos), NL43gagP1024-15mos outgrew NL43gagP1024-4mos. Clones from NL43gagP1024-4mos represented 26% of the population compared with input (58% reduction) at passage 1. At passage 2, only clones NL43gagP1024-15mos were detected (figure 5E).

Reduced expression of Gag p17 and Gag p24 proteins from mutants NL43E17K, NL43K26R, and NL43G116T compared with wild-type NL4-3. To determine whether the mutations with significant fitness cost for viral replicative capacity were also affecting Gag p17 and Gag p24 protein viral expression, we performed Western blot analyses with mutant proviruses obtained after transfection in HeLa cells. (We also analyzed Gag p17 and Gag p24 in mutant NL43G116A.) Proteins made in HeLa cells represent products of the original DNA transfected because these cells do not support HIV-1 replication. We detected less mutant proviral p17 and p24 protein expression in HeLa cells 48 h after transfection compared with wild-type pNL43 (figure 6A and 6B).

Figure 6
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Figure 6

Gag p17 and p24 expression by transfected HeLa cells, as shown by Western blot analysis. Forty-eight hours after transfection, cells were collected, washed twice with PBS, and lysed in 1% NP-40 for 1 h, and then the lysate was cleared by centrifugation at 16,000g. Aliquots from the supernatants obtained after centrifugation were resolved by SDS-PAGE analysis and electroblotted onto an Immobilon-P polyvinylidene difluoride membrane (Millipore). Samples were normalized based on the amounts of α-tubulin. Gag p17 and p24 were visualized with the ECL Western Blotting Analysis System (Amersham Biosciences) using anti-p17 and anti-p24 monoclonal antibodies. WT, wild type.

Table 1
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Table 1

Study population characteristics and maternal CD8+ T cell epitope variants: transmission and posttransmission stability.

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Discussion

The present study was undertaken to better understand the balance between CD8+ T cell selective pressures and viral fitness cost after transmission to non—HLA-matched individuals. Several HIV-1 CD8+ T cell escape variants were identified within maternal plasma viral p17 and p24 sequences that were either not detected or did not persist when transmitted to non—HLA-matched infants. Viruses constructed with mutations associated with a lack of recognition by maternal PBMCs demonstrated reduced viral replication in vitro and reduced expression of p17 and p24 proteins compared with wild type, suggesting a fitness cost to viral replicative capacity. Nontransmission or reversion of these mutations in the absence of CD8+ T cell selective pressure supported viral fitness cost in vivo.

CD8+ T cell escape has been associated with increased viral replication and disease progression [15]. Fernandez et al. [25] and Friedrich et al. [32] have proposed that CD8+ T cell escape variants targeting regions critical for viral replication may result in impaired viral fitness, thus facilitating control of viral replication. Several studies in the macaque model of SIV infection have provided data supportive of this hypothesis [25, 26, 32], but to our knowledge only one published study has successfully addressed this issue in humans [28], and all epitopes reported in that article were in the capsid protein.

In the present study, we have described 2 mutations in Gag p17 (E17K and K26R) and 1 in Gag p24 (G116T) with reduced fitness compared with wild type. Mutation E17K was detected in all plasma sequences from a B*40-positive mother (M-1002) at delivery but was not detected in any plasma samples obtained from her B*40-negative infant (P-1031) at 2, 4, and 11 months of age. Lack of detection of the p17E17K mutant in the infant's plasma sequences suggests either that a minor wild-type sequence from another compartment (for example, wild-type virions in vaginal secretions) was transmitted and later selected after transmission or that the mutation rapidly reverted to wild type in the absence of CD8+ T cell selective pressure because of fitness cost. Outgrowth of wild-type virus in the competition assays would support the latter hypothesis. Obviously, M-1002 had sufficient viral replication to transmit the virus to her child, and it is possible that compensatory mutations partially restored viral fitness in vivo. Infection assays showed that neither Q127K nor whole Gag protein from M-1002 restored viral replicative capacity, suggesting that secondary compensatory mutations are not within Gag protein.

The other mutation in Gag p17 (K26R) was also detected within a B*8-restricted CD8+ T cell epitope in maternal plasma samples at delivery from a B*8-positive mother (M-1001). This mutation reverted to wild type after transmission to the B*8-negative infant P-1024 during the first year of life, suggesting a fitness cost. Competition experiments with wild-type NL43 and recombinant viruses demonstrated that a virus harboring K26R showed impaired fitness. It is important to point out that E17K and K26R are located very closely together in a highly conserved region known to be important for viral replication [30], and several epitopes within this region have been identified as restricted by different HLA alleles. Inclusion of this region into HIV vaccines may therefore be highly desirable.

Mutation G116T also reverted after transmission to a non—HLA-matched recipient. We detected this mutation in Gag p24 within an A*2-restricted CD8+ T cell epitope from maternal plasma samples (M-1005) at delivery. It is of interest that amino acid substitution G116T changed to alanine (A) instead of reverting to glycine (G). In p24 protein, the most common amino acid at position 116 is glycine, but alanine is also well represented (44% and 33.6%, respectively) in clade B. By contrast, alanine at position 116 is the most common amino acid in clade C (http://www.hiv.lanl.gov). To revert from GAC (threonine) to GGC (alanine), only a single nucleotide change is needed (A→G). To revert to GGA (glycine), 2 nucleotide changes (A→G and C→A) are needed. This amino acid substitution, G116A (G248A according to Gag protein numeration), has been described elsewhere as a common mutation within an overlapping B*57/5801-restricted epitope TSTLQEQIGW (TW10) [20, 28]. The G116A amino acid variant arises commonly in clade B infections associated with amino acid variant T242N and persists in the population on transmission to B*57/5801-negative individuals. This G116A mutation alone causes a partial loss of recognition of the epitope; however, when it is combined with T242N, recognition by B*57/5801 is abrogated [20].

In our study, mutant G116A was recognized almost as well as wild type by maternal PBMCs, and we observed only a minimal reduction in viral replicative capacity when this mutation was introduced into an NL4-3 background. Furthermore, competition experiments with wild type demonstrated that a virus harboring this mutation showed little cost to viral replicative capacity. Our findings are compatible with persistence of G116A in individuals without B*57/5801-restricting HLA alleles. The small reduction in infectivity observed with mutant G116A when compared with wild-type NL4-3 did not correspond with the increase described by von Schwedler et al. [33]. This difference is possibly due to different methods used to infect or to cellular factors, because we used single infection of MT-4 cells and von Schwedler et al. [33] used MAGIC assays, in which P4.HeLa.CD4.LTR.β-gal indicator cells are infected.

These findings confirm our prior observation that CD8+ T cell selective pressures contribute to the evolution of the viral quasi-species in HIV-1—infected women and their infants [23]. Moreover, our studies are among the first to document in humans that CD8+ T cell escape mutations may be associated with impaired viral fitness. The discordance between in vivo viral loads and the diminished fitness measured in vitro suggests that secondary compensatory mutations have been able to restore fitness and are playing an important role in CD8+ T cell escape. These compensatory mutations may be external to the region or gene with CD8+ T cell escape mutations. The identification and characterization of additional CD8+ T cell escape mutants with impaired fitness may allow the selection of viral sequences suitable for inclusion in HIV-1 vaccines.

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Acknowledgments

We thank the infants and their families for their participation in these studies. We also thank Margaret McManus for data management; Linda Lambrecht, Joyce Pepe, and John Latino for technical assistance; Mei Gong and Bhavana Priyadharshini for assistance with viral cloning; and Wanda DePasquale for preparation of the manuscript.

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Footnotes

  • ↵a Present affiliation: College of the Holy Cross, Worcester, Massachusetts.

  • Potential conflicts of interest: none reported.

  • Financial support: National Institutes of Health (grants AI 32391 and HD 01489 to K.L.); University of Massachusetts Center for AIDS Research (grant AI 42845).

  • Received March 9, 2007.
  • Accepted July 31, 2007.
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  1. J Infect Dis. (2008) 197 (2): 300-308. doi: 10.1086/524845
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