Cytotoxic T Lymphocyte Escape Mutations Research Articles

A viral CTL escape mutation leading to immunoglobulin-like transcript 4–mediated functional inhibition of myelomonocytic cells

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide–major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705–restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1–specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1–specific CD8+ T cells, the HLA-B2705–KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I–specific receptor expressed on myelomonocytic cells. Binding of the B2705–KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversification.

Novel Cytotoxic T-Lymphocyte Escape Mutation by a Three-Amino-Acid Insertion in the Human Immunodeficiency Virus Type 1 p6 Pol and p6 Gag Late Domain Associated with Drug Resistance

Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8(+) CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6(Pol) protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6(Pol) epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6(Gag) late domain, PTAPP(APP). Interestingly, this p6(Pol) insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6(Gag) late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.

Evolution of Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitopes: Fitness-Balanced Escape

CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.

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.

Identification of HLA Class I-Associated Amino Acid Polymorphisms in the HIV-1C Proteome

Human immunodeficiency virus type 1 (HIV-1) evasion of host cytotoxic T lymphocyte (CTL) targeting is linked to the expression of variant amino acid residues, or escape mutations, in positions that alter the normal processing, presentation, or recognition of targeted epitopes. The combined genetic variability of HIV and the class I human leukocyte antigen (HLA) loci makes it difficult to characterize CTL escape mutations on a population level. However, a role in CTL escape may be inferred by identifying HIV amino acid polymorphisms that are specifically associated with particular HLA class I alleles. We describe here the results of a comprehensive analysis of HIV-1 subtype C (HIV-1C) to identify HLA class I-associated amino acid polymorphisms. We identified 94 HLA-associated amino acid polymorphisms distributed across the 15 major viral proteins analyzed. HLA-B alleles were involved in more associations (50%) than alleles from either the HLA-A (27%) or HLA-C (24%) loci. HLA-associated polymorphisms were identified in 18 of 26 previously described HIV-1C CTL immunoreactive regions including 7 of the 8 classified as immunodominant. Comparison to known HIV-1 CTL epitopes revealed that 19 of the HLA-associated polymorphisms were located in CTL epitopes restricted by the associated HLA allele. These results suggest that HIV-1C retains the potential for CTL escape across the entire proteome including regions that are broadly targeted on a population scale. The impact of CTL escape on natural and vaccine-induced CTL immunity warrants the continued characterization of the role of such HLA-associated polymorphisms in this process.

Maintenance of viral suppression in HIV-1–infected HLA-B*57+ elite suppressors despite CTL escape mutations

Rare human immunodeficiency virus 1–infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57–restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57–restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-γ responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57–restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations.

Estimating Costs and Benefits of CTL Escape Mutations in SIV/HIV Infection

Mutations that allow SIV/HIV to avoid the cytotoxic T lymphocyte (CTL) response are well documented. Recently, there have been a few attempts at estimating the costs of CTL escape mutations in terms of the reduction in viral fitness and the killing rate at which the CTL response specific to one viral epitope clears virus-infected cells. Using a mathematical model we show that estimation of both parameters depends critically on the underlying changes in the replication rate of the virus and the changes in the killing rate over time (which in previous studies were assumed to be constant). We provide a theoretical basis for estimation of these parameters using in vivo data. In particular, we show that 1) by assuming unlimited virus growth one can obtain a minimal estimate of the fitness cost of the escape mutation, and 2) by assuming no virus growth during the escape, one can obtain a minimal estimate of the average killing rate. We also discuss the conditions under which better estimates of the average killing rate can be obtained.

Waiting times for the appearance of cytotoxic T-lymphocyte escape mutants in chronic HIV-1 infection

The failure of HIV-1 to escape at some cytotoxic T-lymphocyte (CTL) epitopes has generally been explained in terms of viral fitness costs or ineffective or attenuated CTL responses. Relatively little attention has been paid to the evolutionary time required for escape mutants to be detected. This time is significantly affected by selection, mutation rates, the presence of other advantageous mutations, and the effective population size of HIV-1 in vivo (typically estimated to be ∼10 3 in chronically infected patients, though one study has estimated it to be ∼10 5). Here, we use a forward simulator with experimentally estimated HIV-1 parameters to show that these delays can be substantial. For an effective population size of 10 3, even highly advantageous mutants ( s = 0.5) may not be detected for a couple of years in chronically infected patients, while moderately advantageous escape mutants ( s = 0.1) may not be detected for up to 10 years. Even with an effective population size of 10 5, a moderately advantageous escape mutant ( s = 0.1) may not be detected in the population within 2 years if it has to compete with other selectively advantageous mutants. Stochastic evolutionary forces, therefore, in addition to viral fitness costs and ineffective or attenuated CTL responses, must be taken into account when assessing the selection of CTL escape mutations.

Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Reversion In Vivo after Inoculation of a Molecular Proviral DNA Clone of Simian Immunodeficiency Virus with a Cytotoxic-T-Lymphocyte Escape Mutation

Vaccine-based control of the replication of a simian immunodeficiency virus (SIV), SIVmac239, in macaques has recently been shown. In the process of the control, a mutant virus escaping from epitope-specific cytotoxic-T-lymphocyte (CTL) responses was rapidly selected and contained. In this study, we show that the wild-type virus appeared and became predominant in the absence of the epitope-specific CTL after inoculation of naive macaques with a molecular clone DNA of the CTL escape mutant SIV. This is the first report describing reversion in vivo from an inoculated, molecular proviral DNA clone of immunodeficiency virus with a CTL escape mutation.

HTLV-I viral escape and host genetic changes in the development of adult T cell leukemia

In the pathogenesis of adult T cell leukemia (ATL), an oncogenetic role of the human T cell lymphotropic virus type I (HTLV-I) Tax protein, viral escape from the host immune system, and host genetic changes have been proposed as contributory factors. We examined the premature stop codons in tax gene as one of the mutations that may lead to escape of HTLV-I from the cytotoxic T lymphocyte (CTL) response in HTLV-I carriers, to test whether a putative CTL escape mutant can emerge in the early stage of ATL development and whether HTLV-I infected cells with such a mutation can proliferate subsequently. We also examined deletion of cyclin-dependent kinase inhibitor 4 (INK4) genes and mutation of p53 gene in combination with changes in the HTLV-I genome in acute type ATL to test whether host genetic changes promoted the malignant transformation of ATL cells that carry putative CTL escape mutations. The premature stop codon in tax gene existed in many non-ATL HTLV-I carriers as a minor population but not in the commonest HTLV-I sequence of the individual. This minor population with a premature stop codon did not expand subsequently in 3 asymptomatic carriers tested. There were cases who had a mutation or deletion in HTLV-I who also have either deletion of INK4 genes or mutation in p53 gene. Our findings suggest that CTL escape mutation can occur at an early stage of ATL development, and that certain host genetic changes favor the development of the aggressive form of ATL.

A New Variant Cytotoxic T Lymphocyte Escape Mutation in HLA-B27-Positive Individuals Infected with HIV Type 1

The immune response in HIV-infected individuals who carry HLA-B27 is characterized by an immunodominant cytotoxic T lymphocyte (CTL) response to a conserved epitope corresponding to amino acids 263-272 of HIV-1 p24 gag. The arginine at position 264 is a crucial anchor residue. Amino acid substitution at 264 from arginine (R) to glycine (G), lysine (K), or threonine (T) results in a low affinity peptide that binds to HLA-B27 inefficiently and is poorly recognized by T cells that respond to the wild-type peptide. These mutants have been characterized as CTL escape mutations. We studied the plasma virus of 20 HLA-B27 longterm nonprogressors: 14 were wild type and 6 were found to be mutant. Five of these carried known escape mutations coding for K or G at position 264. One patient demonstrated a previously undescribed R264Q mutation in 30/31 clones. This altered epitope failed to elicit an IFN-gamma response from PBMC isolated from any of four HLA-B27-positive individuals with strong responses to wild-type peptide. A peptide binding assay confirmed that the R264Q mutant peptide had 30-fold lower binding affinity to HLA-B27 compared to wild type. Therefore, the R264Q variant is a likely novel escape mutation in HLA-B27-positive individuals.

Interactive Selective Pressures of Hla-Restricted Immune Responses and Antiretroviral Drugs on HIV-1

HIV-specific cytotoxic T lymphocyte (CTL) responses mediated by human leukocyte antigen (HLA) recognition and antiretroviral drugs exert selection pressure on HIV-1 in vivo. The selection of CTL escape mutations strongly underpins the failure of CTL control in most untreated infections whilst drug-resistance mutations predict failure of drug control. These two evolutionary forces share common target residues in HIV-1 at which their selection effects could be synergistic or antagonistic, such that the propensity to develop drug resistance and virological treatment failure may be influenced by HLA type. We examined HIV-1 reverse transcriptase (RT) and protease sequences in a large clinical observational cohort of 487 HIV-infected individuals and found evidence of site-specific interactions between specific antiretroviral drug exposures, HLA alleles and HIV sequence diversity at population level. Such interactions may have general and specific implications for explaining in vivo/in vitro discordance of drug resistance, host-specific susceptibility to drug resistance, individualization of therapy and therapeutic vaccine design.

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

Antibody-mediated protection against cytotoxic T-cell escape in coronavirus-induced demyelination.

C57BL/6 (B6) mice infected with mouse hepatitis virus (MHV) strain JHM develop a clinically evident, demyelinating encephalomyelitis. Infectious virus can be isolated from the spinal cords of these mice and is invariably mutated in the immunodominant CD8 T-cell epitope recognized in this strain. We showed previously that these persistently infected mice did not mount a measurable serum anti-MHV neutralizing antibody response. Here we show that cytotoxic T-lymphocyte (CTL) escape was not detected in MHV-infected BALB/b mice (H-2(b) haplotype), even though the same CD8 T-cell epitopes were recognized as in B6 mice. BALB/b mice had 25-fold more MHV-specific antibody-secreting cells in the central nervous system, the site of infection, than B6 mice, suggesting that local production of anti-MHV antibody contributed to this absence of CTL escape. Additionally, administration of anti-MHV neutralizing antibody to infected B6 mice suppressed the development of CTL escape mutants. These findings indicate a key role for the anti-MHV antibody response in suppressing virus replication, thereby minimizing the emergence and competitive advantage of CTL escape mutants.

Transmission of CTL Escape Mutants May Enhance HIV Disease Progression

Cytotoxic T lymphocyte (CTL) responses are important in initial control of adult HIV infection. HIV strains with structural mutations in key viral CTL

Evolution and transmission of stable CTL escape mutations in HIV infection

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.

Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses.

Resistance to HIV infection in a small group of Kenyan sex workers is associated with CD8+-lymphocyte responses to HIV cytotoxic T-lymphocyte (CTL) epitopes. Eleven prostitutes meeting criteria for HIV resistance seroconverted between 1996 and 1999. The occurrence and specificity of preexisting HIV-1 epitope-specific responses were examined using the IFN-gamma enzyme-linked immunospot assay, and any epitopes recognized were cloned and sequenced from the infecting viral isolate. Immunologic and behavioral variables were compared between late seroconverters and persistently uninfected sex worker controls. HIV-1 CTL epitope responses were present in four of six cases, 5-18 months before seroconversion, and their presence was confirmed by bulk CTL culture. A possible viral escape mutation was found in one of six epitopes. The key epidemiologic correlate of late seroconversion was a reduction in sex work over the preceding year. In persistently uninfected controls, a break from sex work was associated with a loss of HIV-specific CD8+ responses. Late seroconversion may occur in HIV-1-resistant sex workers despite preceding HIV-specific CD8+ responses. Seroconversion generally occurs in the absence of detectable CTL escape mutations and may relate to the waning of HIV-specific CD8+ responses due to reduced antigenic exposure.

Infection with cytotoxic T-lymphocyte escape mutants results in increased mortality and growth retardation in mice infected with a neurotropic coronavirus.

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.

HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.