Escape Mutations In Epitopes Research Articles

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2.

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.

Frequencies of Gag-restricted T-cell escape “footprints” differ across HIV-1 clades A1 and D chronically infected Ugandans irrespective of host HLA B alleles

Objective(s)We evaluated relationships between critical Gag T-cell escape mutations and concomitant T-cell responses to determine whether HLA-restricted Gag mutations that confer protection, occur at similar rates in a population infected with mixed HIV-1 clades A1 and D viruses. MethodsAssessment of Gag selective pressure, and adaptive T-cell functions to KAFSPEVIPMF (KF11), ISPRTLNAW (ISW9) and TSTLQEQIGW (TW10) Gag epitopes were combined with host HLA to assess correlations with rates of critical epitope escape mutations in clades A1- (n=23) and D- (n=21) infected, untreated subjects. Infecting clades and selection pressure were determined from the gag sequences. ResultsOverall, Gag escape mutations A163X in KF11 were detected in 61% (14/23) A1- infected compared to 5% (1/21) in D-infected subjects (p=0.00015). Gag mutations I147X in the ISW9 epitope were seen in 43%: (10/23) clade A compared to 5%: (1/21) clade D infected subjects, p=0.007, Fisher's Exact test. Both mutations were more frequent in clade A1 infection. Frequencies of the measured epitope-specific T-cell responses were comparable across clades. Peptide binding affinities for the restricting HLA alleles did not differ across clades. Overall, selection pressure on the Gag protein was significantly greater in clade A than in clade D sequences. ConclusionsThese findings imply that HIV-1 vaccine strategies designed to target structurally constrained T-cell epitopes may be further challenged by clade-driven outcomes in specific HLA-restricted Gag epitopes. Equally, the data are line with slower HIV-1 disease progression in clade A infection; and raise hope that increased selective pressure on Gag may be protective irrespective of host HLA alleles.

Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation.

Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.

Loss of HIV-1-derived cytotoxic T lymphocyte epitopes restricted by protective HLA-B alleles during the HIV-1 epidemic

HIV-1 is known to adapt to the human immune system, leading to accumulation of escape mutations during the course of infection within an individual. Cross-sectional studies have shown an inverse correlation between the prevalence of human leukocyte antigen (HLA) alleles in a population and the number of cytotoxic T lymphocyte (CTL) escape mutations in epitopes restricted by those HLA alleles. Recently, it was demonstrated that at a population level HIV-1 is adapting to the humoral immune response, which is reflected in an increase in resistance to neutralizing antibodies over time. Here we investigated whether adaptations to cellular immunity have also accumulated during the epidemic. We compared the number of CTL epitopes in HIV-1 strains isolated from individuals who seroconverted at the beginning of the HIV-1 epidemic and from individuals who seroconverted in recent calendar time. The number of CTL epitopes in HIV-1 variants restricted by the most common HLA alleles in the population did not change significantly during the epidemic. In contrast, we found a significant loss of CTL epitopes restricted by HLA-B alleles associated with a low relative hazard of HIV-1 disease progression during the epidemic. Such a loss was not observed for CTL epitopes restricted by HLA-A alleles. Despite the large degree of HLA polymorphism, HIV-1 has accumulated adaptations to CTL responses within 20 years of the epidemic. The fact that such adaptations are driven by the HLA-B molecules that provide best protection against HIV-1 disease progression has important implications for our understanding of HIV evolution.

Epitope-Specific CD8+T Lymphocytes Cross-Recognize Mutant Simian Immunodeficiency Virus (SIV) Sequences but Fail To Contain Very Early Evolution and Eventual Fixation of Epitope Escape Mutations during SIV Infection

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) evade containment by CD8(+) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true representation of the plasticity of these viruses or the intensity of CD8(+) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8(+) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8(+) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8(+) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-specific CD8(+) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-specific CD8(+) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and fixation of the mutant epitope sequences in the viral quasispecies.

Macaque Long-Term Nonprogressors Resist Superinfection with Multiple CD8 + T Cell Escape Variants of Simian Immunodeficiency Virus

Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.

Broadly increased sensitivity to cytotoxic T lymphocytes resulting from Nef epitope escape mutations.

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8(+) CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.

Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes.

CD8+ class I–restricted cytotoxic T lymphocytes (CTLs) usually incompletely suppress HIV-1 in vivo, and while analogous partial suppression induces antiretroviral drug-resistance mutations, epitope escape mutations are inconsistently observed. However, escape mutation depends on the net balance of selective pressure and mutational fitness costs, which are poorly understood and difficult to study in vivo. Here we used a controlled in vitro system to evaluate the ability of HIV-1 to escape from CTL clones, finding that virus replicating under selective pressure rapidly can develop phenotypic resistance associated with genotypic changes. Escape varied between clones recognizing the same Gag epitope or different Gag and RT epitopes, indicating the influence of the T cell receptor on pressure and fitness costs. Gag and RT escape mutations were monoclonal intra-epitope substitutions, indicating limitation by fitness constraints in structural proteins. In contrast, escape from Nef-specific CTL was more rapid and consistent, marked by a polyclonal mixture of epitope point mutations and upstream frameshifts. We conclude that incomplete viral suppression by CTL can result in rapid emergence of immune escape, but the likelihood is strongly determined by factors influencing the fitness costs of the particular epitope targeted and the ability of responding CTL to recognize specific epitope variants.

The Outcome of Hepatitis C Virus Infection Is Predicted by Escape Mutations in Epitopes Targeted by Cytotoxic T Lymphocytes

CD8 + cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.

Delayed emergence of bovine leukemia virus after vaccination with a protective cytotoxic T cell-based vaccine.

In a previous study eight MHC class I-matched sheep were vaccinated with a minimal cytotoxic T lymphocyte (CTL) peptide epitope vaccine and were challenged with the retrovirus, bovine leukemia virus (BLV). Half the vaccinated animals remained PCR negative after challenge, whereas the remaining half and the placebo group became PCR positive within 4 weeks postchallenge (Hislop AD, Good MF, Mateo L, Gardner J, Gatei MH, Daniel RCW, Meyers BV, Lavin MF, and Suhrbier A: Nat Med 1998;4:1193). Here we show that neither epitope mutations nor processing differences explained why half the peptide-vaccinated animals failed to resist the BLV challenge. However, in these animals the development of BLV-induced lymphosarcomas was significantly delayed compared with the placebo group, suggesting a role for CTLs in preventing retrovirus-induced cancers. Importantly, two of the initially protected animals become PCR positive after approximately 1.5 years, indicating extended suppression but not elimination of challenge virus by vaccine-induced CTLs. The late emergence of virus could not be explained by epitope escape mutations or the loss of memory CTL responses. We speculate that high levels of effector CTL may be needed to protect animals from a postchallenge viremia and maintenance of such effector CTLs, rather than memory CTLs, may be required to prevent subsequent emergence of virus from latent pools.

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.

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We evaluated relationships between critical Gag T-cell escape mutations and concomitant T-cell responses to determine whether HLA-restricted Gag mutations that confer protection, occur at similar rates in a population infected with mixed HIV-1 clades A1 and D viruses.Assessment of Gag selective pressure, and adaptive T-cell functions to KAFSPEVIPMF (KF11), ISPRTLNAW (ISW9) and TSTLQEQIGW (TW10) Gag epitopes were combined with host HLA to assess correlations with rates of critical epitope escape mutations in clades A1- (n = 23) and D- (n = 21) infected, untreated subjects. Infecting clades and selection pressure were determined from the gag sequences.Overall, Gag escape mutations A163X in KF11 were detected in 61% (14/23) A1- infected compared to 5% (1/21) in D-infected subjects (p = 0.00015). Gag mutations I147X in the ISW9 epitope were seen in 43%: (10/23) clade A compared to 5%: (1/21) clade D infected subjects, p = 0.007, Fisher's Exact test. Both mutations were more frequent in clade A1 infection. Frequencies of the measured epitope-specific T-cell responses were comparable across clades. Peptide binding affinities for the restricting HLA alleles did not differ across clades. Overall, selection pressure on the Gag protein was significantly greater in clade A than in clade D sequences.These findings imply that HIV-1 vaccine strategies designed to target structurally constrained T-cell epitopes may be further challenged by clade-driven outcomes in specific HLA-restricted Gag epitopes. Equally, the data are line with slower HIV-1 disease progression in clade A infection; and raise hope that increased selective pressure on Gag may be protective irrespective of host HLA alleles.