Characterization of a novel respiratory syncytial virus-specific human cytotoxic T-lymphocyte epitope

Respiratory syncytial virus (RSV) infection is a major cause of morbidity in childhood worldwide. The first human RSV-specific cytotoxic T-lymphocyte epitope to be defined is described. This HLA B7-restricted epitope in nucleoprotein (NP) was detectable in four healthy, B7-positive adult subjects using B7-RSV-NP tetrameric complexes to stain CD8(+) T cells.     

Naturally occurring TAP-dependent specific T-cell tolerance for a variant of an immunodominant retroviral cytotoxic T-lymphocyte epitope

Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorous H-2K(b)-restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice.     

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.     

Recent advances in B-cell epitope prediction methods

Identification of epitopes that invoke strong responses from B-cells is one of the key steps in designing effective vaccines against pathogens. Because experimental determination of epitopes is expensive in terms of cost, time, and effort involved, there is an urgent need for computational methods for reliable identification of B-cell epitopes. Although several computational tools for predicting B-cell epitopes have become available in recent years, the predictive performance of existing tools remains far from ideal. We review recent advances in computational methods for B-cell epitope prediction, identify some gaps in the current state of the art, and outline some promising directions for improving the reliability of such methods.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.     

Benchmarking B cell epitope prediction: underperformance of existing methods

Sequence profiling is used routinely to predict the location of B-cell epitopes. In the postgenomic era, the need for reliable epitope prediction is clear. We assessed 484 amino acid propensity scales in combination with ranges of plotting parameters to examine exhaustively the correlation of peaks and epitope location within 50 proteins mapped for polyclonal responses. After examining more than 10(6) combinations, we found that even the best set of scales and parameters performed only marginally better than random. Our results confirm the null hypothesis: Single-scale amino acid propensity profiles cannot be used to predict epitope location reliably. The implication for studies using such methods is obvious.     

Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A(222-230), from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-alpha subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A(222-230)-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A(222-230) epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A(222-230) is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.     

Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost

Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.     

Abrogation of AIDS vaccine-induced cytotoxic T-lymphocyte efficacy in vivo due to a change in viral epitope flanking sequences

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.     

Dynamics of cytotoxic T-lymphocyte exhaustion.

We examine simple mathematical models to investigate the circumstances under which the dynamics of cytotoxic T-lymphocyte (CTL) activation and differentiation may result in the loss of virus specific CD8+ cells, a process known as CTL exhaustion. We distinguish between two general classes of viruses: (i) viruses infecting cells that are not involved in the immune response; and (ii) viruses infecting antigen presenting cells (APCs) and helper cells. The models specify host and viral properties that lead to CTL exhaustion and indicate that this phenomenon is only likely to be observed with viruses infecting APCs and helper cells. Moreover, it is found that for such viruses, a high rate of replication and a low degree of cytopathogenicity promote the exhaustion of the CTL response. In addition, a high initial virus load and a low CD4+ cell count promote the occurrence of CTL exhaustion. These conclusions are discussed with reference to empirical data on lymphocytic choriomeningitis virus and on human immunodeficiency virus.     

Mathematical models of cytotoxic T-lymphocyte killing

By killing infected host cells, cytotoxic T lymphocytes (CTL) mediate an important defense mechanism against viruses and other intracellular pathogens. Quantitative aspects of this killing process have been studied for several decades in vitro. More recently, methods have been developed to measure the timescales of CTL killing in vivo. Here, we review the estimates of kinetic rates involved in CTL killing which were obtained in these studies, and elaborate on the differences between them.     

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.     

Quantitative study of cytotoxic T-lymphocyte immunotherapy for nasopharyngeal carcinoma

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding.     

Compensatory substitutions restore normal core assembly in simian immunodeficiency virus isolates with Gag epitope cytotoxic T-lymphocyte escape mutations

The evolution of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) as they replicate in infected individuals reflects a balance between the pressure on the virus to mutate away from recognition by dominant epitope-specific cytotoxic T lymphocytes (CTL) and the structural constraints on the virus' ability to mutate. To gain a further understanding of the strategies employed by these viruses to maintain replication competency in the face of the intense selection pressure exerted by CTL, we have examined the replication fitness and morphological ramifications of a dominant epitope mutation and associated flanking amino acid substitutions on the capsid protein (CA) of SIV/simian-human immunodeficiency virus (SHIV). We show that a residue 2 mutation in the immunodominant p11C, C-M epitope (T47I) of SIV/SHIV not only decreased CA protein expression and viral replication, but it also blocked CA assembly in vitro and virion core condensation in vivo. However, these defects were restored by the introduction of upstream I26V and/or downstream I71V substitutions in CA. These findings demonstrate how flanking compensatory amino acid substitutions can facilitate viral escape from a dominant epitope-specific CTL response through the effects of these associated mutations on the structural integrity of SIV/SHIV.     

Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD(50)), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD(50) to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD(50) for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD(50) required for CTL to achieve 50% efficiency of infected cell killing [KE(50)]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE(50) despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE(50), demonstrating epitope dependence of KE(50). Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE(50). Thus, defining KE(50) values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.     

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.     

Molecular analyses of a five-amino-acid cytotoxic T-lymphocyte (CTL) epitope: an immunodominant region which induces nonreciprocal CTL cross-reactivity

The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.     

构象性B细胞表位预测的免疫信息学方法及其网络资源

抗原表位预测是免疫信息学研究的重要方向之一,可以给实验提供重要的线索。B细胞表位或抗原决定簇是抗原中可被B细胞受体或抗体特异性识别并结合的部位。实际上,近90%的B细胞表位是构象性的。即使抗原蛋白质三级结构已知,B细胞表位预测仍然是一大挑战。该文结合实例阐述当今主要的构象性B细胞表位预测方法和算法:机器学习预测、非机器学习的计算预测、基于噬菌体展示数据的识别方法,以及一些也可用于构象性B细胞表位预测的通用蛋白质-蛋白质界面预测方法;介绍最新相关预测软件和Web服务资源,说明未来的研究趋势。