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Original Article |
kelleher{at}worf.molbiol.ox.ac.uk
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Key Words: human immunodeficiency virus • immune escape • selection • CD8+ T lymphocyte • phylogenetic analysis
In patients who carry HLA-B2705, the HIV-specific CTL response, during the chronic phase of infection, is usually characterized by an immunodominant response to an epitope in the core protein, p24 (amino acids 263–272, KRWIILGLNK; references 7 and 9). The interaction of the arginine (R) residue at position 2 of this and other HLA-B2705–restricted epitopes with the B pocket of the HLA-B2705 plays a crucial role in stabilizing the MHC–peptide complexes 910111213. Substitution of either lysine (K) or glycine (G) for arginine (R) at gag residue 264 (R264K and R264G) results in an epitope that binds poorly to HLA-B2705, thus forming unstable complexes 714.
In two previously described patients, the mutation K (AAA) for R (AGA) at residue 264 (R264K) occurred late in the infection and coincided with disease progression 7. This mutation could have enabled viral escape from CTLs. Another nonsynonymous mutation, AGA to GGA (R264G), has not been detected in patients, but was engineered into the LAI strain of HIV. This synthetic mutant virus was replication competent in vitro 14. As viral turnover in untreated HIV-infected patients is high 1516 and HIV reverse transcriptase has an error rate of 10–4 base incorporations 171819, Nietfield et al. argued that R264G should be preferentially selected if CTLs exerted a significant selection pressure on the virus 14. However, this sequence is absent from the database 20, arguing against CTLs being important in control of virus replication 14. The epitope is within a structurally important region of p24 which is involved in the conformational multimerization of p24 during capsid formation 21. As this protein structure must be maintained, few amino acid substitutions within or near the HLA-B2705–restricted epitope are likely to be tolerated.
Here we describe five HIV-infected patients with HLA-B2705 in whom the appearance of mutations at R264 was observed. This selection occurred both after primary infection and during late HIV disease. When CTL selection pressure was lost, partial reversion to wild-type (w/t) virus sequence occurred.
Isolation of PBMCs.
Sequencing of Cell-associated Viral DNA.
Viral subtype was determined using "HIV subtyping using BLAST" software available on the Los Alamos HIV sequence data base web site (http://hiv-web.lanl.gov/). p24 sequence data from each patient were compared with several reference sequences of each subtype, scored for similarity to each, and a subtype allocated.
Phylogenetic Analysis.
Population Genetic Analysis.
Viral Loads and CD4+ Cell Counts.
HLA Class I Tetramers.
Bulk Culture and Cell Lines.
Online Supplemental Material.
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The ability of HIV to adapt to changes in its environment through mutation of its genome is well described. Evidence from the simian immunodeficiency virus (SIV)-macaque model of immunodeficiency virus pathogenesis indicates that CD8+ T cells can control retrovirus replication 12 and that SIV evolves to escape the CD8+ CTL response 34. These data add substantial support to earlier studies in humans which demonstrated that HIV-1 mutants can escape CTL responses 5678. Viral escape from CTLs may occur rapidly during primary infection, or late in the disease as features of AIDS appear, but the constraints on the evolution of escape mutants are unclear.
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Patients.
HIV-1–infected patients attending clinics for the first time were HLA typed by sequence-specific primer methods 22. Patients with HLA-B2705 had PBMCs taken whenever the patient attended clinic. Patients without HLA-B27 who were recruited to other studies had samples collected on at least one occasion. All studies were approved by the appropriate local Research Ethics committees.
PBMCs were isolated from heparinized blood by standard density centrifugation on Lymphoprep (Nycomed). Cells were washed twice in RPMI 1640 (Sigma-Aldrich) and then either cryopreserved or set up in culture.
2–3 x 106 PBMCs were resuspended in RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin (GIBCO BRL), and glutamine (GIBCO BRL), and stimulated for 36–48 h with a 1:200 dilution of PHA (Murex). DNA was isolated from PBMCs using the Puregene kit (Gentra Systems) and stored at –20°C. A nested PCR was performed as described which resulted in the amplification of a 335-bp sequence from p24 7. The products were ligated into a thymidine/adenosine (T/A) vector and used to transform competent cells (Invitrogen). Positive colonies were identified by blue/white selection and grown up over night in Luria-Bertani (LB) media in the presence of ampicillin. Plasmid DNA was isolated using either QIAGEN or Hybaid miniprep kits and cycle sequencing reactions were performed using 7-deaza-dGTP (Amersham Pharmacia Biotech) and Cy5-labeled T7 primer (Amersham Pharmacia Biotech). Reactions were loaded onto a 5.75% acryl amide gel and run out on an automated sequencer (Amersham Pharmacia Biotech) and analyzed using the ALF/WIN Express software. A minimum of 20 clones were sequenced at each time point in the HLA-B27–positive patients.
Phylogenetic trees were reconstructed using the maximum likelihood (ML) available in PAUP* (v4) provided by D.L. Swofford (Sinauer Associates, Sunderland, MA). The HKY85 model of DNA substitution was used in all cases with the maximum likelihood transition/transversion ratio (Ts/Tv) and alpha (
), the shape parameter of a discrete approximation to a gamma distribution of rate heterogeneity among sites (here assumed to contain eight rate categories), determined using an iterative procedure in which these parameters were continually adjusted until the tree of highest likelihood was found. These parameter values are available from the authors on request.
The effective population size (Ne) of HIV-1 within patients was estimated by rearranging the relation 
= 2Neµ, where , a measure of genetic diversity assuming neutral evolution, was estimated using a Metropolis-Hastings sampling method (program Fluctuate, v1.3; reference 23), and the mutation rate, µ, was set to 3 x 10–5 per site, per generation 24.
Viral loads were determined on sterile plasma separated within 6 h of collection into EDTA, and stored at –70°C by the Amplicor kit (Roche). CD4+ counts were determined by standard methodology.
HLA-B27, HLA-A2, HLA-A11, HLA-B35, HLA-B7, and HLA-B8 tetramers were synthesized as reported previously 22. The following HLA class I–peptide complexes were synthesized: B2705 (C67S) gag 263–272 (KRWIILGLNK), B2705 gag 263–272 (KRWIIMGLNK), A201 gag 77–85 (SLYNTVATL), A201 pol 476–484 (ILKEPVHGV), B35 nef 78–85 (VPLRPMTY), B35 gag 260–268 (PPIPVGDIY), B35 gp120 42–52 (vpvwkeatttl), B7 nef 128–137 (TPGPGVRYPL), B7 gag 148–156 (SPRTLNAWV), B8 gag 24–31 (GGKKKYKL), B8 nef 89–97(FLKEKGGL), B8 nef 13–20 (WPTVRERM), A11 pol 325–333 (AIFQSSMTK), and A11 nef 75–86 (QVPLRPMTYK). For staining, PBMCs were resuspended in PBS and 1% BSA, washed, and then incubated for 60 min at 4°C in the presence of the tetramer. The cells were then washed twice in PBS plus 1% BSA and then incubated for another 20 min at 4°C in the presence of an FITC-conjugated CD8 antibody (Dako). Cells were washed twice as above and then resuspended in PBS plus 1% BSA plus 4% formaldehyde and stored at 4°C for up to 48 h before analysis on a FACScanTM (Becton Dickinson) using CELLQuestTM (Becton Dickinson) software. Lymphocyte gates were set on forward versus side scatter. CD8+ gates were set by isotype control staining and cutoffs for positive staining were set by staining with an irrelevant tetramer.
Bulk culture and cell lines were set up as detailed previously. Chromium release assays were performed as detailed previously 79.
For each patient described, the date of identification, viral load, CD4+ cell count, and the percentage of total sequences at each time point with substitutions are shown in Table SI. CTL escape mutations are shown in bold. Available at http://www.jem/org/cgi/content/full/193/3/375/DC1.
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HLA-B27–positive Patients
12 HIV-infected patients with HLA-B27 were studied. All were male. Eight had acquired HIV through sexual contact. The others were hemophiliacs who acquired HIV from blood products (Table ). Three were studied during primary infection, five during the asymptomatic phase of the disease, and four during late stage disease. p24 sequences from each patient were subtype B.
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Late Stage Infection.
Patient SW presented with Pneumocystis carinii pneumonia, and was subsequently found to be HIV seropositive with a high viral load and a low CD4+ cell count. In the first sample available for sequencing, collected 12 wk after initial presentation, all proviral sequences showed the R264K mutation and M268 (Fig. 1 c). A HLA-B27–restricted T cell response to the M268 variant peptide was detectable by tetrameric staining (0.68% of CD8+ cells) at this and the next time point (0.28% of CD8+ cells) but not later (Fig. 1 c). No HLA-A11– or HLA-B35–restricted responses were detected in this patient at any time by tetramer staining or chromium release assays performed on bulk cultures. When effective antiretroviral therapy was started 16 wk after presentation, the viral load fell and the R264K mutation persisted.
As described previously 7, the hemophiliac patients 007 and 025 had w/t epitope sequences with R264 and L268; however, L268M occurred relatively early in the course of infection (online supplemental Table SI). Subsequently, each developed the R264K mutation as disease progressed with increasing viral loads and falling CD4+ counts (7; online supplemental Table SI, and Fig. 1). Patient 007 had exhibited a sustained immunodominant CTL response to the HLA-B27–restricted gag 263–272 epitope 7926. This was confirmed by tetramer staining but responses became undetectable as measured by tetrameric complexes, bulk culture, and peptide generated cell lines 1 yr after the rise to fixation of the R264K mutation (Fig. 1 b). Patient 025 also had detectable HLA-B27 gag-specific CTLs before the appearance of the R264K mutation.
Because of disease progression, patient 007 was commenced on dual nucleoside therapy, the only drug therapy available at the time. This had a transient effect on viral load and little effect on his low CD4+ count. Later, CAC was instituted but this had no effect on viral load or CD4+ cell count (Fig. 1 b). When the viral load was high and the CTL response was undetectable, the epitope sequences partially reverted from K264 to w/t (K264R). In the sequences where this occurred, but not in the escape sequences, there was an increasing frequency of the other reversion mutation M268L (online supplemental Table SI, and Fig. 1 b).
Patient RT presented with Pneumocystis carinii pneumonia and was then found to be infected with HIV. He received 8 wk zidovudine monotherapy, which resulted in a transient increase in CD4+ cell number, but this drug was stopped because of side effects. HLA-B27 gag epitope sequences 48 wk after AIDS was diagnosed were uniformly R264 and L268. 58 wk after diagnosis, zidovudine and didanosine, introduced because of falling CD4+ cell counts, resulted in a transient rise in CD4+ cell numbers. 6 mo later, a protease inhibitor was added resulting in a sustained rise in CD4+ cells and reduced viral loads. Approximately 20 mo after presentation the R264G mutation was found in all sequences (Fig. 1 d). This mutation was always associated with L268, and a E260D mutation in the NH2-terminal flanking region of the epitope, at this and at all subsequent time points. CD8+ T cell responses to the w/t epitope were detected by tetrameric staining at weeks 28 and 48, but disappeared when the mutant was present (Fig. 1 d). Tetramer staining for HIV-specific CD8+ T cells restricted through HLA-A2 and HLA-B8 was not found at any time.
Three other patients studied had progressive disease but no mutation at R264 was found during the course of the study. Patients CW and MH were recruited after an AIDS diagnosis. Patient CW has never received antiretroviral therapy but has had relatively stable clinical disease with an intermediate viremia (online supplemental Table SI). Patient MH commenced CAC shortly after being identified and since then has had a stable CD4+ cell count and undetectable viral load (online supplemental Table SI). Neither had HIV-specific CD8+ T cells detected either by traditional CTL assays or by tetramer staining with HLA-A2, HLA-B8, HLA-B7, or HLA-B27 constructs at any time point studied (data not shown). Both have proviral sequences that are w/t at the HLA-B27 gag epitope with L268. Patient 868 has been followed for over 10 yr. Over the last 2–3 yr he has had progressive disease and transiently effective dual nucleoside therapy, followed by CAC with a sustained virological response. Initially, he had robust CTL responses restricted through: HLA-A2 to gag (77–85) but not pol epitopes, HLA-B35 to nef (75–82), and HLA-B27 to gag (263–272; dominant response). After CAC, these responses were initially maintained but by 6 mo became undetectable by any technique. All sequences had w/t virus with M268. Despite high viral turnover before initiation of dual nucleosides and when therapy failed, mutation at position 264 was not detected (online supplemental Table SI).
Long-term Nonprogressors
Two patients (049 and 422) fulfil criteria for long-term nonprogression (at least 10 yr after diagnosis with CD4+ counts >500 cells/µl; online supplemental Table SI). Both have CD8+ T cells that recognize gag 263–272 restricted through HLA-B27 (data not shown), but there is no evidence for escape. Both have L268 and have been shown to have responses to HIV mediated through other HLA class I alleles, although in each case the HLA-B27 response appears to be dominant (reference 7, and Kelleher, A.D., unpublished results).
Context of Mutations at Codon 264
In each patient when the R264K mutant was first detected, a majority of the preceding viruses sampled had M268. R264 can be found with either M268 or L268, whereas K264 is strongly associated with M268. In only 2 out of 329 sequences were K264 and L268 found together. In neither case was this sequence detected in subsequent samples. Even in populations containing mixtures of R264 and K264, the latter always segregated with M268, whereas R264 associated with both M268 and L268 (Table ). Similarly, the R264G mutation appears only in the context of the amino acid residues L268 and E260D.
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2 test weighted for the different rates of substitution among bases) showing that dN > dS for this codon as expected under positive selection. In other words, codon 264 does not evolve quickly simply because it is subject to weaker selective constraints.
Support for positive selection also comes from a population genetic analysis of diversity. If, for example, the fixation of R264G in patient RT occurred between 0.9 and 2.4 yr after enrolment, as is compatible with the phylogenetic analysis, then this fixation event took no more than 
18 mo. For this process to occur by genetic drift alone would require an Neof only 100, assuming that the HIV generation time is 2.6 d 16 and that fixation of neutral mutations takes on average 2Ne generations 27. However, if Ne is estimated directly from the sequence data of patient RT using a neutral measure of genetic diversity (), then the average value of Ne across time points is 800 ( values from 0.025 to 0.076). Consequently, although the effective population size in patient RT is small, as might be expected to be the case after a selective sweep, it is still not small enough to allow fixation of a neutral mutation by genetic drift in such a short time period.
The phylogenetic analysis also supports the selection of R264K in patients 007 and 777. In patient 007, all sequences present at 11.8 yr after diagnosis were R264, but by 33 mo later (14.5 yr after diagnosis) all sequences were K264 (Fig. 2 b). Interestingly, R264 then reappeared after antiviral therapy, remained in the population for the last year of sampling, and reached a frequency of 25%. The phylogenetic tree reveals that the main source of this virus is persistence of a variant bearing R264, as those sequences which bore R264 later are clustered with those earlier samples which carry R264. However, it is also apparent from the phylogenetic tree that at least one K264R reversion mutation has occurred (Fig. 2 b).
A similarly complex evolutionary picture is seen in patient 777. In this patient, the R264K mutation may have arisen four times, but never reaches fixation (i.e., 100% frequency), perhaps because antiviral therapy was given after that time which altered the selection pressure on viral evolution. R264 then rises in frequency later on in infection seemingly because of the persistence of lineages with R264 across several time points.
The phylogenetic analysis also provides strong support for the compensatory relationship between the mutations at positions 264 and 268. In both patients 007 and 777, L268 only occurs in association with R264 (Fig. 2b and Fig. c). Moreover, the phylogenetic analysis reveals that changes at amino acid positions 264 and 268 occur on different branches of the tree, indicating that they are independent. Such observations suggest that although L268 and R264 are intimately related, they are not in direct linkage; the rise of M268L is not simply due to it being linked to a selectively favored K264R virus. For patient 777, the M to L change has actually occurred twice independently.
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The mutability of HIV and its ability to adapt to environmental pressures has been well demonstrated by the rapid emergence of drug resistance mutations in reverse transcriptase and protease (for reviews, see references 28 and 29). In the case of resistance to protease inhibitors, mutations that appear to have no direct effect on the active site of the enzyme have been described. These appear to compensate for critical mutations within the active site itself 303132.
Evidence supporting a role for CTLs in the control of HIV includes: depletion of CD8+ cells results in more rapid progression of SIV-related disease in macaques 12, the appearance of HIV-specific CTLs at primary infection coincides with control of viremia 253334, and CTL escape mutations allow immune evasion by HIV early in disease 56. These data indicate that CTLs can exert a significant selective force upon the virus. Later in the disease, the mutability of the virus allows it to generate escape mutations rapidly after artificial perturbations of the steady state which resulted in a highly focused CTL response 8. In animals infected with identical strains of SIV, nonsynonymous mutations in nef and env accumulate exclusively within antigenic sites subject to CTL pressure 3.
These studies suggest that several conditions need to be met to permit the evolution of CTL escape mutants at the HLA-B27 gag epitope. High viral turnover appears to be a requirement whether escape occurs early in the disease as with patient 777, or late in the disease as with patients 007, RT, SW, and 025. In those treated effectively and early in primary infection, escape did not occur for as long as 2.1 yr. Long-term nonprogressors and patient CW, whose viral load was constant at 4.5 logs, did not develop very high levels of viral turnover and did not generate escape mutants.
Why do these mutations, which arise in an epitope that is the target of a highly focused CTL response, not occur earlier in the disease? There are several possible explanations. These mutations may only arise in certain viral strains. SI phenotypes of HIV-1 are more pathogenic and usually occur later in the disease 353637. The only patient who had early escape developed an SI virus early in the infection. Those whose virus escaped late in infection are likely to have had SI virus. SI virus is not susceptible to inhibition by β-chemokines released from CD8+ T cells 38. In the absence of soluble factor inhibition, SI virus faces an immune selection pressure more clearly restricted to the CTL response than non-SI virus, and thus generation of escape mutants to lytic activity of CD8+ T cells may be favored.
A second explanation for constraint on evolution within gag involves the structure and function of this protein. The HLA-B27 epitope lies embedded in the NH2-terminal domain of the p24 capsid protein. Evidence for the need to conserve structure in this region of the protein comes from several sources. First of all, most mutations in this area result in nonviable virus 14, and deletion mutants indicate that this region is necessary for the conformational changes that allow p24 to form functional capsid after p24 multimerisation 2139. Second, the crystal structure of the NH2-terminal domain of p24 reveals that the epitope and its NH2-terminal flanking regions lie within helix 7 (amino acid residues 258–276), one of five helices within the NH2-terminal domain that form a coiled coil structure 404142. Residues 260, 264, and 268 all lie on the same aspect of helix 7 (Fig. 3 a). Furthermore, the packing of molecules into crystals of the NH2 terminus of p24 is dependent on the formation of two interfaces, and one of these interfaces is formed by parallel packing of helix 7 from adjacent molecules. Residues 264 and 268, along with 271 and 275, play a role in the formation of this interface, and R264 and L268 lose significant accessible surface area (Fig. 3 b). In this structure, the aliphatic portions of the two R264 side chains are in van der Waals contact with each other and guanidium groups participate in a charged interaction with the side chains of E260 41. Moreover, a model of core particle assembly predicts that antiparallel interactions between helix 7 and helix 2 are the basis for NH2-terminal dimerization and that helix 7 plays a role in the association of these dimers to form arrays of capsid molecules before the formation of the mature capsid 43. Taken together, these findings indicate that this region plays an important role in capsid self-association and conformational assembly. Furthermore, although this area is not the primary site of p55 association with cyclophilin 44, deletions in this area can abrogate cyclophilin binding to p24 45. Therefore, mutations in this area are likely to interfere with viral assembly and thus impair fitness. Accommodation of a new mutation in one part of this structure may require at least one compensatory mutation.
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R264G does not appear in the Los Alamos database. This mutation is probably disadvantageous in isolation. It may also indicate that in vivo the mutation is only tolerable in subtype B viruses, all of which on the database have E260, when E260D and R264G mutations occur in the presence of L268. Such requirements may explain why this mutation occurs infrequently. The observation that the R264G mutation can be tolerated by the laboratory-adapted LAI strain in vitro in the presence of E260 14 would argue that E260D is not an absolute requirement for the viability of R264G. However, the growth requirements of a mutant laboratory adapted strain of virus grown in isolation in vitro are likely to be different from in vivo requirements where a mutant must out compete all other strains to become dominant. The advantage of this dual mutation may only need to be small, as even minor enhancements of viral fitness can quickly lead to replacement of the less fit variants in a population 46. Standard in vitro culture techniques do not permit the definition of small differences in viral fitness that are revealed by viral competition and selective pressure 4748.
Further evidence that CTLs exert selective pressure on these escape mutations comes from the observation that when CTLs are absent and there is escalating viral replication, as in patient 007, the detectable virus partially reverts to w/t. Phylogenetic analysis suggests that although some of this recrudescence of w/t virus is derived from sequences laid down as provirus, other reversions arise de novo and do so in a manner where mutations occur in a certain order; that is, L268M appears to occur after reversion of K264R. This phenomenon of reversion on removal of selective pressure is reminiscent of the changes seen in drug resistant strains on drug cessation 49505152.
When antiretroviral therapy fails to control HIV replication well, evolution of the virus can continue. When therapy is effective proviral evolution appears to be slowed substantially 53, and sequences tend to reflect the virus population that was present when therapy started. The fact that an escape mutant can persist for many months after the initiation of therapy has therapeutic implications; any cessation or failure of therapy will result inevitably in recrudescence of a virus containing immune escape variants.
The exact causes of disease progression in HIV infection are still poorly understood. However, the analysis presented here and accumulating evidence from other work 345678 strengthens the early suggestion 54 that evasion of the CTL response through mutation of the virus is a significant mechanism of viral persistence.
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A.D. Kelleher was supported by grants from Royal College of Physicians of Australasia, the National Health and Medical Research Council (Australia), and Medical Research Council (UK). R. Phillips, C. Long, and K. Olson were supported by the Wellcome Trust. P. Goulder was supported by Medical Research Council (UK) and the Elizabeth Glaser Pediatric AIDS Foundation.
Submitted: 6 July 2000
Revised: 13 October 2000
Accepted: 30 November 2000
The online version of this article contains supplemental material.
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