Cytotoxic T-lymphocyte Pressure Research Articles

HIV-1 Group M Capsid Amino Acid Variability: Implications for Sequence Quality Control of Genotypic Resistance Testing.

With the approval of the HIV-1 capsid inhibitor, lenacapavir, capsid sequencing will be required for managing lenacapavir-experienced individuals with detectable viremia. Successful sequence interpretation will require examining new capsid sequences in the context of previously published sequence data. We analyzed published HIV-1 group M capsid sequences from 21,012 capsid-inhibitor naïve individuals to characterize amino acid variability at each position and influence of subtype and cytotoxic T lymphocyte (CTL) selection pressure. We determined the distributions of usual mutations, defined as amino acid differences from the group M consensus, with a prevalence ≥ 0.1%. Co-evolving mutations were identified using a phylogenetically-informed Bayesian graphical model method. 162 (70.1%) positions had no usual mutations (45.9%) or only conservative usual mutations with a positive BLOSUM62 score (24.2%). Variability correlated independently with subtype-specific amino acid occurrence (Spearman rho = 0.83; p < 1 × 10-9) and the number of times positions were reported to contain an HLA-associated polymorphism, an indicator of CTL pressure (rho = 0.43; p = 0.0002). Knowing the distribution of usual capsid mutations is essential for sequence quality control. Comparing capsid sequences from lenacapavir-treated and lenacapavir-naïve individuals will enable the identification of additional mutations potentially associated with lenacapavir therapy.

HLA-associated polymorphisms in the HIV-2 capsid highlight key differences between HIV-1 and HIV-2 immune adaptation.

HIV-1 frequently adapts in response to immune pressure from cytotoxic T-lymphocytes (CTL). Many HIV-2 infected individuals have robust capsid-specific CTL responses associated with viral control. Despite this CTL pressure, adaptive changes in this key immunogenic HIV-2 protein have not previously been described. We sought to compare selective pressure on HIV-1 and HIV-2 capsids and identify HLA-associated viral polymorphisms in HIV-2. Bioinformatic algorithms to identify sites under positive and negative selective pressure and a statistical model of evolution to identify HLA-associated polymorphisms in HIV-2 was applied to sequences from a community cohort in Guinea-Bissau. IFN-γ ELISpots were used to compare T-cell responses to wild-type and variant epitopes. We identified greater purifying selection and less sites under positive selective pressure in HIV-2 compared with HIV-1. Five HIV-2 codons with HLA-associated polymorphisms were detected all within or around known or predicted CTL epitopes. One site was within the HLA-B58 SuperType (ST)-restricted epitope (TSTVEEQIQW), the HIV-2 equivalent of the HIV-1 TW10 epitope. In contrast to HIV-1, where a T→N mutation at position 3 is associated with resulting loss of CTL control, an E→D mutation at position 5 was observed in HIV-2. Robust CTL responses to the variant HIV-2 epitope were seen, suggesting that HIV-2 adaptation may be at the level of T-cell receptor recognition. Greater constraints on evolution may exist in HIV-2, resulting in more purifying selection and different immune adaptation pathways in HIV-1 and HIV-2 capsids. This may allow CTL responses to persist in HIV-2.

Multiple T-cell responses are associated with better control of acute HIV-1 infection: An observational study.

Cytotoxic T lymphocyte (CTL) responses play pivotal roles in controlling the replication of human immunodeficiency virus type 1 (HIV-1), but the correlation between CTL responses and the progression of HIV-1 infection are controversial on account of HIV immune escape mutations driven by CTL pressure were reported.The acute HIV-1-infected patients from Beijing were incorporated into our study to investigate the effects of CTL response on the progression of HIV-1 infection.A longitudinal study was performed on acute HIV-1-infected patients to clarify the kinetic of T-cell responses, the dynamic of escape mutations, as well as the correlation between effective T-cell response and the progression of HIV infection.Seven human leukocyte antigen-B51+ (HLA-B51+) individuals were screened from 105 acute HIV-1 infectors. The detailed kinetic of HLA-B51-restricted CTL responses was described through blood sampling time points including seroconversion, 3 and 6 months after HIV-1 infection in the 7 HLA-B51+ individuals, by using 16 known HLA-B51 restricted epitopes. Pol743–751 (LPPVVAKEI, LI9), Pol283–289 (TAFTIPSI, TI8), and Gag327–3459 (NANPDCKTI, NI9) were identified as 3 dominant epitopes, and ranked as starting with LI9, followed by TI8 and NI9 in the ability to induce T-cell responses. The dynamics of escape mutations in the 3 epitopes were also found with the same order as T-cell response, by using sequencing for viral clones on blood sampling at seroconversion, 3 and 6 months after HIV-1 infection.We use solid evidence to demonstrate the correlation between T-cell response and HIV-1 mutation, and postulate that multiple T-cell responses might benefit the control of HIV-1 infection, especially in acute infection phase.

Rapid, complex adaptation of transmitted HIV-1 full-length genomes in subtype C-infected individuals with differing disease progression

There is limited information on full-length genome sequences and the early evolution of transmitted HIV-1 subtype C viruses, which constitute the majority of viruses spread in Africa. The purpose of this study was to characterize the earliest changes across the genome of subtype C viruses following transmission, to better understand early control of viremia. We derived the near full-length genome sequence responsible for clinical infection from five HIV subtype C-infected individuals with different disease progression profiles and tracked adaptation to immune responses in the first 6 months of infection. Near full-length genomes were generated by single genome amplification and direct sequencing. Sequences were analyzed for amino acid mutations associated with cytotoxic T lymphocyte (CTL) or antibody-mediated immune pressure, and for reversion. Fifty-five sequence changes associated with adaptation to the new host were identified, with 38% attributed to CTL pressure, 35% to antibody pressure, 16% to reversions and the remainder were unclassified. Mutations in CTL epitopes were most frequent in the first 5 weeks of infection, with the frequency declining over time with the decline in viral load. CTL escape predominantly occurred in nef, followed by pol and env. Shuffling/toggling of mutations was identified in 81% of CTL epitopes, with only 7% reaching fixation within the 6-month period. There was rapid virus adaptation following transmission, predominantly driven by CTL pressure, with most changes occurring during high viremia. Rapid escape and complex escape pathways provide further challenges for vaccine protection.

Mathematical modeling of escape of HIV from cytotoxic T lymphocyte responses

Human immunodeficiency virus (HIV-1 or simply HIV) induces a persistent infection, which in the absence of treatment leads to AIDS and death in almost all infected individuals. HIV infection elicits a vigorous immune response starting about 2–3 weeks postinfection that can lower the amount of virus in the body, but which cannot eradicate the virus. How HIV establishes a chronic infection in the face of a strong immune response remains poorly understood. It has been shown that HIV is able to rapidly change its proteins via mutation to evade recognition by virus-specific cytotoxic T lymphocytes (CTLs). Typically, an HIV-infected patient will generate 4–12 CTL responses specific for parts of viral proteins called epitopes. Such CTL responses lead to strong selective pressure to change the viral sequences encoding these epitopes so as to avoid CTL recognition. Indeed, the viral population ‘escapes’ from about half of the CTL responses by mutation in the first year. Here we review experimental data on HIV evolution in response to CTL pressure, mathematical models developed to explain this evolution, and highlight problems associated with the data and previous modeling efforts. We show that estimates of the strength of the epitope-specific CTL response depend on the method used to fit models to experimental data and on the assumptions made regarding how mutants are generated during infection. We illustrate that allowing CTL responses to decay over time may improve the model fit to experimental data and provides higher estimates of the killing efficacy of HIV-specific CTLs. We also propose a novel method for simultaneously estimating the killing efficacy of multiple CTL populations specific for different epitopes of HIV using stochastic simulations. Lastly, we show that current estimates of the efficacy at which HIV-specific CTLs clear virus-infected cells can be improved by more frequent sampling of viral sequences and by combining data on sequence evolution with experimentally measured CTL dynamics.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8(+) cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo.

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient’s blood when acute graft-versus-host disease developed, recognized the mismatched B∗51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B∗51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.

CTL Escape and Viral Fitness in HIV/SIV Infection

Cytotoxic T lymphocyte (CTL) responses exert a suppressive effect on HIV and simian immunodeficiency virus (SIV) replication. Under the CTL pressure, viral CTL escape mutations are frequently selected with viral fitness costs. Viruses with such CTL escape mutations often need additional viral genome mutations for recovery of viral fitness. Persistent HIV/SIV infection sometimes shows replacement of a CTL escape mutation with an alternative escape mutation toward higher viral fitness. Thus, multiple viral genome changes under CTL pressure are observed in the chronic phase of HIV/SIV infection. HIV/SIV transmission to HLA/MHC-mismatched hosts drives further viral genome changes including additional CTL escape mutations and reversions under different CTL pressure. Understanding of viral structure/function and host CTL responses would contribute to prediction of HIV evolution and control of HIV prevalence.

Viral CTL Escape Mutants Are Generated in Lymph Nodes and Subsequently Become Fixed in Plasma and Rectal Mucosa during Acute SIV Infection of Macaques

SIVmac239 infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIVmac239-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIVmac239 infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.

Molecular Evolution of Human Immunodeficiency Virus Type 1 upon Transmission between Human Leukocyte Antigen Disparate Donor-Recipient Pairs

BackgroundTo address evolution of HIV-1 after transmission, we studied sequence dynamics in and outside predicted epitopes of cytotoxic T lymphocytes (CTL) in subtype B HIV-1 variants that were isolated from 5 therapy-naive horizontal HLA-disparate donor-recipient pairs from the Amsterdam Cohort Studies on HIV-1 infection and AIDS.Methodology/Principal FindingsIn the first weeks after transmission, the majority of donor-derived mutations in and outside donor-HLA-restricted epitopes in Gag, Env, and Nef, were preserved in the recipient. Reversion to the HIV-1 subtype B consensus sequence of mutations in- and outside donor-HLA-restricted CTL epitopes, and new mutations away from the consensus B sequence mostly within recipient-HLA-restricted epitopes, contributed equally to the early sequence changes. In the subsequent period (1–2 years) after transmission, still only a low number of both reverting and forward mutations had occurred. During subsequent long-term follow-up, sequence dynamics were dominated by forward mutations, mostly (50–85%) in recipient-HLA-restricted CTL epitopes. At the end of long-term follow-up, on average 43% of the transmitted CTL escape mutations in donor-HLA-restricted epitopes had reverted to the subtype B consensus sequence.Conclusions/SignificanceThe relatively high proportion of long-term preserved mutations after transmission points to a lack of back selection even in the absence of CTL pressure, which may lead to an accumulating loss of critical CTL epitopes. Our data are supportive for a continuous adaptation of HIV-1 to host immune pressures which may have implications for vaccine design.

Escape and Compensation from Early HLA-B57-Mediated Cytotoxic T-Lymphocyte Pressure on Human Immunodeficiency Virus Type 1 Gag Alter Capsid Interactions with Cyclophilin A

Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.

Vaccine-based, long-term, stable control of simian/human immunodeficiency virus 89.6PD replication in rhesus macaques

The X4-tropic simian/human immunodeficiency virus (SHIV) 89.6P (or 89.6PD) causes rapid CD4(+) T-cell depletion leading to an acute crash of the host immune system, whereas pathogenic R5-tropic simian immunodeficiency virus (SIV) infection, like HIV-1 infection in humans, results in chronic disease progression in macaques. Recent pre-clinical vaccine trials inducing cytotoxic T lymphocyte (CTL) responses have succeeded in controlling replication of the former but shown difficulty in control of the latter. Analysis of the immune responses involved in consistent control of SHIV would contribute to elucidation of the mechanism for consistent control of SIV replication. This study followed up rhesus macaques that showed vaccine-based control of primary SHIV89.6PD replication and found that all of these controllers maintained viraemia control for more than 2 years. SHIV89.6PD control was observed in vaccinees of diverse major histocompatibility complex (MHC) haplotypes and was maintained without rapid selection of CTL escape mutations, a sign of particular CTL pressure. Despite the vaccine regimen not targeting Env, all of the SHIV controllers showed efficient elicitation of de novo neutralizing antibodies by 6 weeks post-challenge. These results contrast with our previous observation of particular MHC-associated control of SIV replication without involvement of neutralizing antibodies and suggest that vaccine-based control of SHIV89.6PD replication can be stably maintained in the presence of multiple functional immune effectors.

Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8+ Cytotoxic T Lymphocyte Response

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication.

Early and late cytotoxic T lymphocyte responses in HIV infection

Early and late cytotoxic T lymphocyte responses in HIV infection

Macrophage Tropism of Human Immunodeficiency Virus Type 1 Facilitates In Vivo Escape from Cytotoxic T-Lymphocyte Pressure

Macrophage Tropism of Human Immunodeficiency Virus Type 1 Facilitates In Vivo Escape from Cytotoxic T-Lymphocyte Pressure

Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure.

Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.

Selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine.

In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.

Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus.

The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.