JAIDS Journal of Acquired Immune Deficiency Syndromes:
Transition From Long-Term Nonprogression to HIV-1 Disease Associated With Escape From Cellular Immune Control
Kemal, Kimdar Sherefa PhD*; Beattie, Tara PhD†; Dong, Tao PhD†; Weiser, Barbara MD*‡; Kaul, Rupert MD, PhD§; Kuiken, Carla PhD∥; Sutton, Julian PhD†; Lang, Dorothy PhD∥; Yang, Hongbing PhD†; Peng, Yan Chun MS†; Collman, Ronald MD¶; Philpott, Sean PhD*; Rowland-Jones, Sarah MA, DM†; Burger, Harold MD, PhD*‡
From the *Wadsworth Center, New York State Department of Health, Albany, NY; †Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom; ‡Department of Medicine, Albany Medical College, Albany, NY; §Department of Medicine, University of Toronto, Toronto, Ontario, Canada; ∥Los Alamos National Laboratory, Los Alamos, NM; and ¶Department of Medicine, University of Pennsylvania, Philadelphia, PA.
Received for publication July 12, 2007; accepted January 30, 2008.
K. S. Kemal and T. Beattie made equal contributions to the study.
Supported in part by grant R01-AI42555 from the National Institute of Allergy and Infectious Diseases. T. Beattie, T. Dong, J. Sutton, H. Yang, Y. C. Peng, and S. Rowland-Jones were supported by the Medical Research Council, United Kingdom.
Data presented in part at the XVI International AIDS Conference, Toronto, Canada, August 13-18, 2006, and the XV International AIDS Conference, Bangkok, Thailand, July 11-16, 2004.
The authors have no conflict of interest.
Correspondence to: Harold Burger, MD, PhD, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208 (e-mail: email@example.com).
Transition from long-term nonprogressive infection to progressive HIV-1 disease presents an opportunity to investigate pathogenesis in a defined immunogenetic background. We studied a male long-term nonprogressor (LTNP) who remained asymptomatic and viremic and had normal CD4 T-cell counts without antiretroviral therapy for >18 years and then experienced a transition to disease progression. We analyzed the complete HIV-1 genomic RNA sequence from plasma and cellular immune responses to predefined human leukocyte antigen-matched autologous viral peptides spanning the viral genome, before and after progression. Serial viral sequences did not seem attenuated and consistently utilized coreceptor CCR5. LTNP status was associated with elongated V2 domains and broad low-level T-cell immune responses targeting several regions of the viral genome. The transition to progressive disease was associated with the acquisition of viral mutations conferring escape from CD8 T-cell responses. Multiple changes in HIV-1 sequence and loss of immune response over time most likely contributed to the transition from LTNP status to progressive disease. These data are relevant to vaccine design and identification of the correlates of protection from disease progression.
The natural course of HIV-1 infection varies widely among individuals. A small proportion of patients remain free of HIV-related disease without antiretroviral therapy (ART) and maintain normal CD4 T-cell counts for at least 10 years of infection, often with low viral loads. Such individuals are known as long-term nonprogressors (LTNPs) and represent 1% to 5% of HIV-1-infected subjects.1-3 Long-term nonprogressive infection presents an opportunity to study HIV-1 pathogenesis and the correlates of protection from disease progression.
Viral characteristics, host factors, immunogenetic background, viral coinfections, and age have been associated with variation in the rate of HIV-1 disease progression.4-13 Reports have linked attenuated HIV-1 strains with polymorphisms in viral genes, and the presence of a c-terminal env V2 region extension, with slowed disease progression.14-19 Many LTNPs are infected with HIV-1 strains that do not have the polymorphisms mentioned previously, however.20 Certain human leukocyte antigen (HLA) class I alleles, and polymorphisms in the human genes for chemokine receptors CCR5 and CCR2 have also been associated with a delay in progression to AIDS.5,21-24
Components of the cellular and humoral immune response, particularly cytotoxic T lymphocytes (CTLs), play major roles in control of replication.25,26 HIV-1-specific CTLs place considerable evolutionary pressure on the virus. Viral escape from HIV-1-specific T cells and subsequent disease progression have been described in humans and primates.27-31 The marked sequence variation characterizing HIV-1 influences the cellular immune response in an infected individual, because each step in the recognition of T-cell epitopes and development of virus-specific T-cell responses may be constrained by sequence specificity. Previous studies highlighted the need to assess virus-specific immune responses directed against the autologous virus in infected individuals to characterize the breadth and magnitude of the cellular immune response accurately.32,33 An important influence on the extent and kinetics of viral escape from epitope-specific CTL responses is the cost of escape to intrinsic viral fitness.34 The association of slow disease progression with heterozygosity at HLA class I loci suggests that the breadth of the CTL response is also important in viral control.35
Long-term nonprogressive infection presents an opportunity to investigate the correlates of protection from disease progression. Many LTNPs eventually experience immunologic and clinical decline. In this study, we present extensive longitudinal, virologic, and immunologic analyses of an LTNP who went on to progressive disease. The transition from an LTNP phenotype to progressive HIV-1 disease within an individual may provide a way to identify, in a defined immunogenetic environment, the viral and host factors that contribute to pathogenesis.
Clinical Laboratory Methods
The patient signed an informed consent form, and the institutional review board at each site approved the study. T-cell counts were determined at the Stanford Blood Center by standard flow cytometry. HIV-1 RNA was quantified by using NucliSens (BioMérieux, Durham, NC). Molecular HLA typing was performed using the Amplification Refractory Mutation System polymerase chain reaction (PCR).36
HIV-1 RNA Isolation, Long Reverse Transcriptase PCR, and Sequencing
HIV-1 RNA was extracted from plasma; primers, PCR conditions, and sequencing methods were used as described elsewhere.37-39 In addition to full-length sequencing of virus from 1998, we obtained sequences for gag, pol, env, and nef genes from plasma at multiple time points.
To determine HIV-1 coreceptor use of the patient's 1998 virus, gp160 was amplified by PCR and directly cloned into a TA vector (Invitrogen, Carlsbad, CA). Clones were used in a fusion assay.40 For other time points (1999 to 2004), we used the sequences of the V3 region to predict coreceptor use.41 CCR2, CCR5 genotypes, and CCR5 promoter polymorphisms were determined by PCR from peripheral blood mononuclear cells (PBMCs).8,22,24
Autologous Peptide Synthesis
Optimized autologous CD8 T-cell peptide epitopes were purchased (Sigma-Genosys, Suffolk, United Kingdom) or synthesized using F-moc chemistry and a Zinnser Analytical Synthesizer (Advanced Chemtech, Louisville, KY). Purity was established by high-performance liquid chromatography (HPLC).
Interferon-γ Enzyme Immunospot Assay
After thawing, all cryopreserved PBMCs were incubated at 37°C in 5% CO2 for at least 3 hours and then filtered through a cell sieve to remove dead cells prior to use. The viability of PBMCs after incubation and filtration was consistently >95%, as determined by Trypan blue exclusion. HIV-specific interferon-γ (IFNγ) release by PBMCs was assayed using enzyme immunospot (ELISpot) assay.42,43 PBMCs were plated at 1 × 105 viable cells per well with media alone (negative control; background), peptide at 10 μM, or phytohemagglutinin (PHA; positive control; Murex Biotech LTD, Dartford, Kent, United Kingdom). IFNγ-producing cells were counted using an automated ELISpot reader (Autoimmun Diagnostika GmbH, Strasburg, Germany). HIV-specific responses were reported in spot-forming units (SFUs)/1 million PBMCs, after subtraction of background (media alone). A response was considered positive if it was >50 SFUs/1 million PBMCs, at least twice the background, and the positive control (PHA) was >200 SFUs/1 million PBMCs. All peptides were tested against an HIV-uninfected laboratory control, with no positive responses seen (data not shown).
Patient Wadsworth Center 3 (WC3) was a white man, born in the United States in 1941, with >20 years' history of documented HIV-1 infection. He was exposed to HIV-1 in May 1982 by sexual contact with a man who later developed AIDS. After exposure to HIV-1 in 1982, he entered a long-term monogamous relationship with a seronegative partner. He was enrolled in the study in 1985 and found to be HIV-1-seropositive, with a CD4 T-cell count of 518 cells/mm3. From 1985 through 2003, he was clinically asymptomatic, maintaining a normal CD4 T-cell count (>400 cells/mm3) without ART. His PBMCs were positive by HIV-1 culture, with a titer of 25 tissue culture infectious doses on 4 occasions between 1992 and 1994. His plasma viral load was 7900 RNA copies/mL when it was first measured in 1996, and he remained consistently viremic. Between June 2003 and April 2004, his CD4 T-cell count fell from 426 to 216 cells/mm3, his HIV-1 RNA level increased to 67,000 copies/mL (Fig. 1), and he became fatigued. He was advised to start ART in 2001 and 2002 because of his viral load, but he declined. In October 2004, he initiated ART with emtricitabine, tenofovir, and efavirenz.
We obtained a near-full-length, 9118-base pair (bp), population-based HIV-1 genomic RNA sequence from plasma collected in April 1998. A complete HIV-1 RNA sequence was assembled, aligned, and analyzed by computational methods.37 In addition to the near-full-length sequence from 1998, we sequenced the gag, pol, env, and nef genes from plasma at multiple time points (10 gag, 9 pol, 6 env, and 9 nef) from 1992 to 2004. All sequences represent the predominant circulating variant and are deposited in GenBank (accession numbers: EF121385 to EF121419, EF175209 to EF175212). Phylogenetic tree analysis, including reference strains, in the Los Alamos HIV Sequence Database (available at: http://hiv-web.lanl.gov/content/hiv-db/mainpage.html) found that all the sequences were related, were clustered together, and were subtype B (data not shown). We analyzed sequences obtained at different time points and found no evidence of superinfection or recombination.37
HIV-1 sequences were examined for mutations that might contribute to attenuation. The length of the V2 region increased from 44 or 45 amino acids in 1998 to 2001 to 51 or 52 amino acids in 2002 to 2004 (Table 1). The R77Q mutation in the vpr gene and deletions in the nef gene were not present in the 1998 sequences.14,16 There were no other clearly attenuating mutations or deletions detected in HIV-1 from this patient. Nevertheless, it is possible that single-base mutations may have influenced the pathogenicity of the HIV-1 strains, and thereby played a role in the nonprogressive and progressive phases of his disease.
Coreceptor Utilization, Coinfection, and Host Genetic Polymorphisms
The patient's HIV-1 strains obtained in 1998 to 2004 utilized CCR5,40,41 and there was no evidence of GB virus-C (GBV-C) coinfection in plasma based on detection of E2, NS3, and 5′-NTR antibodies or viral RNA.13 The CCR2-64I and CCR5-Δ32 alleles were wild type; CCR5 promoter polymorphisms were heterozygous 59029-G (A/G) and 59353-C (C/T). These genotypes were not those associated with protection or slow progression.8,22,24 His HLA type was maximally heterozygous (A1, A6801, B8, B51, Cw7, Cw15, DR3, DR4, DQ2, DQ3), but he did not have alleles associated with delayed disease progression.5,21,23
T-Cell Responses to Autologous HIV-1 Peptide Epitopes
The detection of T-cell responses has been enhanced by the use of peptides based on autologous viral sequences.33 All predefined CD8 T-cell peptide epitopes predicted to be restricted by the patient's HLA alleles were synthesized to match the patient's autologous viral sequence from 1998 (Table 2). This included 37 HLA class I-restricted peptides spanning 7 HIV gene products (p17, p24, reverse transcriptase [RT], integrase, gp160, rev, and nef). Epitope predictions were made by using the Los Alamos HIV immunology database (available at: http://hiv-web.lanl.gov/content/immunology/index.html).
Decline in HIV-1-Specific IFNγ Releasing T Cells
In 1999, a limited number of cryopreserved PBMCs were available for HIV-1-specific T-cell analysis. Responses to 6 commonly described HLA-matched CD8 T-cell autologous epitopes (HLA-B*8 p17 GL8, HLA-B*8 p24 GI9, HLA-B*8 p24 VL10, HLA-B*8 p24 DL9, HLA-Cw*7 p24 QL8, and HLA-B*8 gp160 YL8) were screened using the IFNγ ELISpot assay, and the breadth and strength of the T-cell responses were compared with 2002 and 2004 time points (Fig. 2A). There were no responses for HLA-B*8 p24 DL9 (data not shown).
In 1999, an IFNγ response was detected to 5 of 6 autologous CD8 T-cell epitopes tested, with 2 of these responses (HLA-B*8 p24 VL10 and HLA-Cw*7 p24 QL8) not detectable at later time points. A significant reduction in IFNγ response frequencies to these HIV-1 epitopes was seen between 1999 and 2002 just before disease progression (paired t test, P = 0.03), with no further reduction between 2002 and 2004 (P = 0.64; see Fig. 2A).
Broad Low-Level Recognition of Autologous T-Cell Epitopes Before and After Disease Progression
As an LTNP in 2002 and after disease progression had occurred in 2004, the patient had broad low-level CD8 T-cell immune responses targeting multiple areas of the viral genome. Mean IFNγ responses were 182 (range: 50 to 540) SFUs/1 million PBMCs and 169 (range: 50 to 505) SFUs/1 million PBMCs for 2002 and 2004, respectively. In 2002, PBMCs had a detectable IFNγ response to 8 predicted class I-restricted autologous peptide epitopes representing 5 HIV-1 gene products (p17, p24, RT, gp160, and nef; see Fig. 2B). In 2004, a detectable IFNγ response was seen to 12 class I peptide epitopes spanning 5 gene products (p17, p24, RT, gp160, and nef; see Fig. 2B). There were insufficient cells to permit depletions or parallel intracellular staining (ICS) to confirm that class I epitope responses were CD8 T-cell mediated. The use of optimized 8- to 10-mer peptide epitopes should preclude major histocompatibility complex (MHC) class II presentation.
In 2004, compared with 2002, the patient had lost 1 CD8 T-cell response (gp160 YL8) but had gained responses to 5 new predicted class I-restricted epitopes (p17 EL9, p24 NL10, RT AK9, nef WM8, and nef VT15; see Fig. 2B). All lost or gained responses were close to the limits of detection for this assay (mean = 70 SFUs/106 PBMCs, range: 50 to 110 SFUs/106 PBMCs), suggesting that they may have been present but may have fallen lower than the limit of detection at other time points.
Screening for T-Cell Responses Using 15-Mer Peptides Overlapping by 11 Amino Acids Spanning gag
It is possible that by using autologous overlapping peptides spanning the entire genome in this study, additional immunodominant T-cell responses may have been detected.33 Because T-cell responses directed toward gag are important in immune control of HIV-1,44 we used consensus HXB2 15-mer peptides overlapping by 11 amino acids and spanning the whole of HIV-1 gag to screen for IFNγ T-cell responses using fresh PBMCs obtained before disease progression (2002), as previously described (data not shown).42 No additional responses to those seen using HLA-matched predefined gag epitopes were detected (see Fig. 2B), suggesting that the use of predetermined peptide epitopes did not miss significant T-cell responses.
Immunologic Associations With Transition to Disease Progression
To investigate if disease progression was associated with viral escape and subsequent loss of immune control, sequences from multiple time points between 1992 and 2004 were examined. Where the sequence had changed in positions containing known HLA-matched epitopes, the new autologous epitope variant was synthesized and screened for T-cell recognition (data not shown). In regions of the genome (RT and nef) in which sequence changes were seen in 1999 to 2004 but no T-cell epitopes had been defined for the HLA type of the patient, 18- to 20-mer peptides overlapping by 8 amino acids were synthesized and screened for T-cell recognition by using PBMCs from 2004. Responses were screened to early and emerging variants for a total of 5 predefined CD8 T-cell epitopes spanning 4 gene products (p17, rev, gp160, and nef). In addition, responses were screened to twelve 18- to 20-mer peptides overlapping by 8 amino acids spanning regions of RT and nef. Because of limited sample availability, the latter responses were tested at the latest time point only.
The emergence of novel env gp160 YL8 and nef WY20 epitope variants was associated with a dramatic loss in T-cell recognition of new and wild-type epitope variants (Fig. 3). The gp160 YL8[K3] variant was present in 1998 and 1999 but was replaced in 2001 by the YL8[K3R] variant. A progressive loss in response to the original variant and the emerging variant was seen between 1999 and 2004 such that by 2004, neither variant induced a detectable IFNγ response (see Fig. 3A). The loss of response to the emerging nef WY[H10R] 20-mer variant resulted from a loss of recognition to the B51 CD8 T-cell epitope, AL9, contained within it (see Fig. 3B).
Investigation of long-term nonprogressive infection and its eventual transition to progressive disease makes it possible to examine, in a defined immunogenetic environment, the pathogenic mechanisms involved in controlling HIV-1 disease progression. We focused on virologic, genetic, and immunologic aspects of long-term nonprogressive infection.
We investigated the known genetic polymorphisms associated with long-term nonprogressive infection and found that patient WC3 did not have any. He was maximally heterozygous at his HLA loci but did not have HLA alleles associated with nonprogressive infection. To the contrary, 3 of his HLA alleles (A1, B8, and DR3) have been associated with a poor HIV-1 clinical prognosis elsewhere.45
Near-full-length HIV-1 genomic RNA sequence analysis of plasma virus from 1998 revealed no apparent viral-attenuating mutations. However, longitudinal sequence analysis revealed the presence of V2 insertions in the env gp120 domain (see Table 1), as previously observed in slow progressors and LTNPs.17-19 Because of these insertions, the length of the V2 domain in our patient increased from 44 or 45 amino acids in 1998 to 2001 to 51 or 52 amino acids in 2002 to 2004. The average reported length of the V2 domain in rapid progressors was 40 amino acids.18 Unlike other patients in whom CXCR4 strains emerge at later stages of HIV-l infection, longitudinal HIV-1 sequence analysis revealed only CCR5 utilization throughout the infection. Although the relation between acquisition of elongated V2 and maintenance of CCR5 use among HIV-1-infected patients who did not develop AIDS has been described,18 it is not well established exactly how V2 insertions help to slow down disease progression. Moreover, the presence of V2 extension could be the consequence of prolonged HIV-1 infection. Disease progression is a multifactorial process. In this patient, the presence of V2 insertions may have contributed to delayed disease progression by maintaining CCR5 coreceptor use. As time went by, however, other factors such as the presence of escape variants may have contributed to disease progression.
Comprehensive screening of patient WC3's CD8 T-cell immune response used autologous peptide epitopes based on his 1998 viral sequence for all predefined CD8 T-cell peptide epitopes described for his HLA alleles. Before disease progression, he had a broad low-level CD8 T-cell response targeting 5 HIV-1 gene products (p17, p24, RT, gp160, and nef). It is possible, if autologous overlapping peptide epitopes spanning the entire genome had been used in this study, that additional T-cell responses would have been detected,33 although screening with HXB2 overlapping peptides spanning gag did not detect novel peptide epitopes. Studies of acute HIV-1 infection suggest that escape is less likely to occur if CTL pressure is codominantly directed against multiple viral epitopes instead of 1 or 2 epitope responses dominating the T-cell immune response.46 For viral escape to occur, the costs to intrinsic viral fitness incurred by mutations must outweigh the benefits of avoiding targeted T-cell recognition.30,32 Indeed, there have been reports of reversion of viral escape mutations back to wild type in instances in which virus has been transmitted to another individual who does not have the restricting HLA allele.47 A broadly directed T-cell immune response like that seen in patient WC3 is likely to restrict viral escape, because the virus is required to mutate in several locations and the cost to intrinsic viral fitness of multiple mutations may be too high. Although several of these responses were at a relatively low frequency, the fact that no such low-level responses were demonstrated in HIV low-risk controls in prior studies,48 or in a laboratory control in this study, and that they were consistently recognized over time before progression indicates that these represented true HIV-specific IFNγ responses. Thus, it is likely that the elongated V2 domain in env, along with the broad T-cell immune responses, were the main factors contributing to this patient's LTNP status.
To investigate virologic and immunologic factors that might have contributed to patient WC3's disease progression after >18 years of LTNP status, we analyzed sequences from multiple time points, spanning 1992 to 2004, before and after disease progression. Superinfection with another strain of HIV-1 has been associated elsewhere with a sudden change in immune status,49 but sequence analysis found no evidence of superinfection in patient WC3. We also investigated possible CD8 T-cell immune escape. Where the sequence had changed over time in positions containing known HLA-matched epitopes, the new autologous epitope variant was synthesized and screened for T-cell recognition. In regions that did not contain predefined epitopes but where sequence variation was frequent, 18- to 20-mer peptides overlapping by 8 amino acids for original and emerging variants were synthesized and screened.
During the period of disease progression, and before the transition, patient WC3 had a broad low-level CD8 T-cell immune response targeting 5 gene products (p17, p24, RT, gp160, and nef; see Fig. 2). The emergence over time of sequence variations within 2 epitopes (gp160 YL8 and nef WY20; see Fig. 3) was associated with a dramatic loss in recognition of new and wild-type epitopes, however, suggesting that immune escape in these regions may have contributed to disease progression in this patient.
In HIV-1 infection, as in other chronic viral infections, a decrease in CD4 T-cell help may contribute to CD8 T-cell dysfunction and disease progression.50,51 It is possible that persistent viremia, augmented by CTL escape in 2 epitopes, may have led to the depletion of CD4 T cells.52 Thus, although a broad CD8 T-cell immune response was still detectable after disease progression in 2004, the increasing viral load suggests that the quality of the HIV-1-specific CD8 T cells may have diminished and that these detectable CD8 T-cell responses may have been ineffective. The decrease in IFNγ T-cell responses over time (1999 to 2004) supports the hypothesis that CD8 T cells were not functioning as effectively as they had been before disease progression had occurred. Whether this relates to a loss of CD4 T-cell help, to the switching of CD8 T-cell response specificity attributable to accumulation of escape mutations, or to other mechanisms is not clear. The increase in T-cell epitopes targeted in 2004 compared with 2002 may have reflected the continued viral replication53 and/or the presence of minor HIV-1 variants that coexisted with the predominant virus population. All the newly recognized epitope responses were present at a relatively low frequency, however, near the detection limits of the IFNγ ELISpot assay; therefore, they may have been circulating but undetected before this time point.
Investigation of aspects of virology, genetics, and immunology that were beyond the scope of this study is also likely to be important in understanding pathogenesis.
This study is one of the first to perform extensive, longitudinal, genetic, virologic, and immunologic analyses on an LTNP who made a transition to progressive disease. A combination of factors likely contributed to his eventual disease progression, including advanced age, viral evolution, and CTL immune escape, which, in turn, may have led to the depletion of CD4 T cells and an ineffective CD8 T-cell immune response. The results help to identify correlates of protection from disease progression. Understanding the mechanisms involved is necessary for the development of vaccines and therapeutics.
The authors thank the subject, S.S., for his participation, M. Shudt and the Wadsworth Center Molecular Genetics Core for DNA sequencing, E. Engleman for T-lymphocyte analysis, J. Stapleton for GBV-C studies, C. Stevens and T. Rostrom for HLA typing, K. di Gleria for peptide synthesis, and M. Wren for graphics.
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This article has been cited 2 time(s).
RetrovirologyWithin-host and between-host evolutionary rates across the HIV-1 genomeRetrovirology
Clinical Infectious DiseasesSpontaneous Control of Viral Replication during Primary HIV Infection: When Is "HIV Controller" Status Established?Clinical Infectious Diseases
HIV-1 cellular immune control; HIV-1 complete genomic RNA sequence; HIV-1 correlates of protection; HIV-1 cytotoxic T-lymphocyte escape; HIV-1 long-term nonprogressor
© 2008 Lippincott Williams & Wilkins, Inc.
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