RESUMEN

Effective T-cell surveillance of antigen-presenting cells is dependent on the expression of an array of antigenic peptides bound to major histocompatibility complex (MHC) class I (MHC-I) or class II (MHC-II) molecules. Pathogens co-evolving with their hosts exploit crucial translational regulatory mechanisms in order to evade host immune recognition and thereby sustain their infection. Evasion strategies that downregulate viral protein synthesis and thereby restrict antigen presentation to cytotoxic T-cells through the endogenous MHC-I pathway have been implicated in the pathogenesis of viral-associated malignancies. An understanding of the mechanisms by which messenger RNA (mRNA) structure modulates both viral mRNA translation and the antigen processing machinery to escape immune surveillance, will stimulate the development of alternative therapeutic strategies focused on RNA-directed drugs designed to enhance immune responses against infected cells. In this review, we discuss regulatory aspects of the MHC-I pathway and summarize current knowledge of the role attributed by mRNA structure and other translational regulatory mechanisms in immune evasion. In particular we highlight the impact of recently identified G-quadruplex structures within virally encoded transcripts as unique regulatory signals for translational control and antigen presentation.

Asunto(s)

Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/biosíntesis , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Inmunidad Adaptativa , Animales , Humanos , Evasión Inmune , Tolerancia Inmunológica , Virus/inmunología

RESUMEN

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

Asunto(s)

Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , ARN Mensajero/genética , Animales , Antígeno CD11c/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Genes MHC Clase I/inmunología , Ratones Endogámicos C57BL , Biosíntesis de Proteínas/genética

RESUMEN

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

Asunto(s)

Antígenos Nucleares del Virus de Epstein-Barr/genética , G-Cuádruplex , Biosíntesis de Proteínas , ARN Mensajero/genética , Secuencia de Bases

RESUMEN

T-box transcription factors T-bet (Tbx21) and Eomesodermin (Eomes) are critical players in CD8(+) cytotoxic T lymphocyte effector function and differentiation, but how the expression of these transcription factors is regulated remains poorly defined. Here we show that dominant T cells directed toward human CMV, expressing significantly higher levels of T-bet with graded loss of Eomes expression (T-bet(hi)Eomes(hi/lo)), are more efficient in recognizing endogenously processed peptide-major histocompatibility complexes (pMHC) compared with subdominant virus-specific T cells expressing lower levels of T-bet and high levels of Eomes (T-bet(int)Eomes(hi)). Paradoxically, the T-bet(hi)Eomes(hi/lo) dominant populations that efficiently recognized endogenous antigen demonstrated lower intrinsic avidity for pMHC, whereas T-bet(int)Eomes(hi) subdominant populations were characterized by higher pMHC avidity and less efficient recognition of virus-infected cells. Importantly, differential endogenous viral antigen recognition by CMV-specific CD8(+) T cells also correlated with the differentiation status and expression of perforin, granzyme B and K. Furthermore, we demonstrate that the expression of T-bet correlates with clonal expansion, differentiation status, and expression of perforin, granzyme B and K in antigen-specific T cells. These findings illustrate how endogenous viral antigen presentation during persistent viral infection may influence the transcriptional program of virus-specific T cells and their functional profile in the peripheral blood of humans.

Asunto(s)

Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Dominio T Box/metabolismo , Linfocitos T Citotóxicos/inmunología , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/microbiología , Citometría de Flujo , Granzimas/metabolismo , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Complejo Mayor de Histocompatibilidad/inmunología , Perforina/metabolismo , Proteínas de Dominio T Box/inmunología

RESUMEN

Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to therapeutic development through the use of anti-sense strategies or small molecules targeting EBNA1 mRNA structure.

Asunto(s)

Secuencia de Aminoácidos/genética , Presentación de Antígeno/inmunología , Secuencia de Bases/genética , Antígenos Nucleares del Virus de Epstein-Barr , ARN Mensajero/genética , Presentación de Antígeno/genética , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Mutación del Sistema de Lectura , Genes MHC Clase I , Células HEK293 , Humanos , Evasión Inmune , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Alineación de Secuencia , Linfocitos T Citotóxicos/inmunología

RESUMEN

EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.

Asunto(s)

Benzoquinonas/farmacología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Herpesvirus Humano 4 , Lactamas Macrocíclicas/farmacología , Trastornos Linfoproliferativos/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Benzoquinonas/uso terapéutico , Línea Celular Tumoral , Cartilla de ADN/genética , Dipéptidos/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Inmunoprecipitación , Lactamas Macrocíclicas/uso terapéutico , Trastornos Linfoproliferativos/virología , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/efectos de los fármacos

RESUMEN

The major ligands presented by MHC class I molecules after natural antigen processing are peptides of eight to ten residues in length, and it is widely accepted that the binding preferences of MHC class I molecules play a dominant role in dictating this classic feature of antigen presentation. In this report, we have reassessed the peptide size specificity of class I human leukocyte antigens (HLAs). By lengthening previously defined T cell epitopes by central amino acid insertion, we demonstrate that the peptide length specificity of some common HLA class I alleles (HLA-B*3501, B*0702 and A*2402) is very broad, and includes peptides of up to 25 residues. These data suggest that the length limitation of naturally processed MHC class I-associated peptides is primarily controlled by peptide availability after antigen processing rather than the binding specificity of MHC class I molecules. Furthermore, the findings provide an explanation for recent reports highlighting that epitopes of >10 amino acids play a minor but significant role in virus-specific immune surveillance by CD8(+) T cells.

Asunto(s)

Secuencia de Aminoácidos/fisiología , Antígenos HLA-B/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Sitios de Unión/fisiología , Células Cultivadas , Antígenos HLA-B/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica/genética , Especificidad por Sustrato

RESUMEN

Upon primary infection, EBV establishes a latent infection in B cells, characterized by maintenance of the viral genome in the absence of viral replication. The Epstein-Barr Nuclear Antigen 1 (EBNA1) plays a crucial role in maintenance of the viral DNA episome and is consistently expressed in all EBV-associated malignancies. Compared to other EBV latent gene products, EBNA1 is poorly recognized by CD8(+) T lymphocytes. Recent studies are discussed that shed new light on the mechanisms that underlie this unusual lack of CD8(+) T cell activation. Whereas the latent phase is characterized by the expression of a limited subset of viral gene products, the full repertoire of over 80 EBV lytic gene products is expressed during the replicative phase. Despite this abundance of potential T cell antigens, which indeed give rise to a strong response of CD4(+) and CD8(+) T lymphocytes, the virus can replicate successfully. Evidence is accumulating that this paradoxical situation is the result of actions of multiple viral gene products, inhibiting discrete stages of the MHC class I and class II antigen presentation pathways. Immediately after initiation of the lytic cycle, BNLF2a prevents peptide-loading of MHC class I molecules through inhibition of the Transporter associated with Antigen Processing, TAP. This will reduce presentation of viral antigens by the large ER-resident pool of MHC class I molecules. Synthesis of new MHC class I molecules is blocked by BGLF5. Viral-IL10 causes a reduction in mRNA levels of TAP1 and bli/LMP2, a subunit of the immunoproteasome. MHC class I molecules present at the cell surface are downregulated by BILF1. Also the antigen presenting capacity of MHC class II molecules is severely compromised by multiple EBV lytic gene products, including gp42/gH/gL, BGLF5, and vIL-10. In this review, we discuss how concerted actions of these EBV lytic proteins result in highly effective interference with CD8(+) and CD4(+) T cell surveillance, thereby providing the virus with a window for undisturbed generation of viral progeny.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Citocinas/inmunología , Citocinas/metabolismo , Desoxirribonucleasas/inmunología , Desoxirribonucleasas/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Herpesvirus Humano 4/inmunología , Humanos , Inmunidad , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo

RESUMEN

Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, beta-catenin, Glycogen Synthase Kinase 3ss (GSK3beta), axin and alpha-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and beta-catenin interaction and did not induce transcriptional activation of beta-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Wnt/metabolismo , Animales , Cadherinas/metabolismo , Membrana Celular/metabolismo , Perros , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Modelos Biológicos , Activación Transcripcional , alfa Catenina/metabolismo , beta Catenina/metabolismo

RESUMEN

Many viruses avoid immune surveillance during latent infection through reduction in the synthesis of virally encoded proteins. Although antigen presentation critically depends on the level of viral protein synthesis, the precise mechanism used to regulate the generation of antigenic peptide precursors remains elusive. Here, we demonstrate that a purine overloaded virally encoded mRNA lacking secondary structure significantly impacts the efficiency of protein translation and prevents endogenous antigen presentation. Reducing this purine bias through the generation of constructs expressing codon-modified sequences, while maintaining the encoded protein sequence, increased the stem-loop structure of the corresponding mRNA and dramatically enhanced self-synthesis of the viral protein. As a consequence, a higher number of HLA-peptide complexes were detected on the surface of cells expressing this viral protein. Furthermore, these cells were more efficiently recognized by virus-specific T cells compared with those expressing the same antigen expressed by a purine-biased mRNA. These findings delineate a mechanism by which viruses regulate self-synthesis of proteins and offer an effective strategy to evade CD8(+) T cell-mediated immune regulation.

Asunto(s)

Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Biosíntesis de Proteínas , ARN Mensajero/química , Secuencia de Bases , Línea Celular , Codón/genética , Epítopos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido , Estructura Terciaria de Proteína , Purinas , Estabilidad del ARN , ARN Mensajero/genética

RESUMEN

A significant proportion of endogenously processed CD8(+) T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8(+) T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus-encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1DeltaGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1DeltaGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1DeltaGA. As a consequence, a higher number of major histocompatibility complex-peptide complexes can be detected on the surface of cells expressing EBNA1DeltaGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8(+) T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins.

Asunto(s)

Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr/genética , Homología de Secuencia de Aminoácido , Presentación de Antígeno/genética , Línea Celular , Línea Celular Transformada , Dipéptidos/genética , Antígenos Nucleares del Virus de Epstein-Barr/química , Humanos , Secuencias Repetitivas de Aminoácido/genética , Eliminación de Secuencia

RESUMEN

Lymphocryptoviruses (LCV) that infect humans and Old World primates display a significant degree of genetic identity. These viruses use B lymphocytes as primary host cells to establish a long-term latent infection and express highly homologous latent viral proteins. Of particular interest is the expression of the EBV-encoded nuclear antigen-1 (EBNA1), which plays a crucial role in maintaining the viral genome in B cells. Using human and Old World primate homologues of EBNA1, we show that the internal repeat sequences differentially influence their in vitro translation efficiency. Although the glycine-alanine repeat domain of human LCV (EBV) EBNA1 inhibits its self-synthesis, the repeat domains within the simian LCV homologues of EBNA1 do not inhibit self-synthesis. As a consequence, simian LCV EBNA1-expressing cells are more efficiently recognized by virus-specific CTL when compared to human EBV EBNA1, even though both proteins are highly stable in B cells. Interestingly, we also show that similar to human EBNA1, CD8+ T cell epitopes from simian LCV EBNA1 are predominantly derived from newly synthesized protein rather than the long-lived pool of stable protein. These observations provide additional evidence that supports the theory that immune recognition of EBNA1 can occur without compromising the biological maintenance function of this protein.

Asunto(s)

Presentación de Antígeno/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Lymphocryptovirus/inmunología , Biosíntesis de Proteínas , Animales , Linfocitos T CD8-positivos/inmunología , Electroforesis en Gel de Poliacrilamida , Epítopos de Linfocito T/inmunología , Humanos , Immunoblotting , Transfección

RESUMEN

Recent studies on Hodgkin's lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-gamma production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L(high) and CD27(high), and CCR7(low), consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Enfermedad de Hodgkin/terapia , Inmunoterapia Adoptiva , Proteínas de la Matriz Viral/inmunología , Adenoviridae/genética , Secuencia de Aminoácidos , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Femenino , Vectores Genéticos , Herpesvirus Humano 4/inmunología , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/virología , Humanos , Técnicas In Vitro , Activación de Linfocitos/inmunología , Masculino , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Infecciones Tumorales por Virus/complicaciones , Proteínas de la Matriz Viral/genética

RESUMEN

Thousands of potentially antigenic peptides are encoded by an infecting pathogen; however, only a small proportion induce measurable CD8(+) T cell responses. To investigate the factors that control peptide immunogenicity, we have examined the cytotoxic T lymphocyte (CTL) response to a previously undefined epitope ((77)APQPAPENAY(86)) from the BZLF1 protein of Epstein-Barr virus (EBV). This peptide binds well to two human histocompatibility leukocyte antigen (HLA) allotypes, HLA-B*3501 and HLA-B*3508, which differ by a single amino acid at position 156 ((156)Leucine vs. (156)Arginine, respectively). Surprisingly, only individuals expressing HLA-B*3508 show evidence of a CTL response to the (77)APQPAPENAY(86) epitope even though EBV-infected cells expressing HLA-B*3501 process and present similar amounts of peptide for CTL recognition, suggesting that factors other than peptide presentation levels are influencing immunogenicity. Functional and structural analysis revealed marked conformational differences in the peptide, when bound to each HLA-B35 allotype, that are dictated by the polymorphic HLA residue 156 and that directly affected T cell receptor recognition. These data indicate that the immunogenicity of an antigenic peptide is influenced not only by how well the peptide binds to major histocompatibility complex (MHC) molecules but also by its bound conformation. It also illustrates a novel mechanism through which MHC polymorphism can further diversify the immune response to infecting pathogens.

Asunto(s)

Proteínas de Unión al ADN/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos HLA-B/metabolismo , Herpesvirus Humano 4/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Proteínas Virales/inmunología , Alelos , Células Clonales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Epítopos de Linfocito T/química , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/metabolismo , Antígeno HLA-B35 , Antígeno HLA-B38 , Herpesvirus Humano 4/metabolismo , Humanos , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virología , Transactivadores/química , Transactivadores/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo

RESUMEN

Stem cell transplantation (SCT) remains the most effective curative therapy for the majority of hematopoietic malignancies. Unfortunately, SCT is limited by its toxicity and infectious complications that result from profound immunosuppression. In particular, acquisition of exogenous or reactivation of endogenous human cytomegalovirus (HCMV) is common after SCT. More recently, reconstitution of host immunity through augmentation of anti-HCMV T cell responses has been proposed as an exciting candidate therapy to avoid the requirement for antiviral drug use. Here we have developed a novel antigen presentation system based on a replication-deficient adenovirus that encodes multiple HLA class I-restricted epitopes from eight different antigens of HCMV as a polyepitope (referred to as AdCMVpoly). Ex vivo stimulation of peripheral blood mononuclear cells with AdCMVpoly consistently showed rapid stimulation and expansion of multiple epitope-specific T cells that recognized endogenously processed epitopes presented on virus-infected cells. Interestingly, the AdCMVpoly expression system is capable of expanding antigen-specific T cells even in the absence of CD4(+) T cells. These studies show the effectiveness of a polyepitope antigen presentation system for reproducible expansion of antigen-specific T cells from immunocompetent and immunocompromised settings.

Asunto(s)

Antígenos Virales/inmunología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/inmunología , Línea Celular , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Técnicas Inmunológicas , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética

RESUMEN

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer (56LPQGQLTAY64), 11-mer (54EPLPQGQLTAY64), and 13-mer (52LPEPLPQGQLTAY64) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HLA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156Leucine; B*3508, 156Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Asunto(s)

Epítopos de Linfocito T , Antígeno HLA-B35/metabolismo , Herpesvirus Humano 4/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T/inmunología , Presentación de Antígeno , Línea Celular , Proteínas de Unión al ADN/inmunología , Humanos , Epítopos Inmunodominantes , Polimorfismo Genético , Transactivadores/inmunología , Proteínas Virales/inmunología

RESUMEN

The geographically constrained distribution of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in southeast Asian populations suggests that both viral and host genetics may influence disease risk. Although susceptibility loci have been mapped within the human genome, the role of viral genetics in the focal distribution of NPC remains an enigma. Here we report a molecular phylogenetic analysis of an NPC-associated viral oncogene, LMP1, in a large panel of EBV isolates from southeast Asia and from Papua New Guinea, Africa, and Australia, regions of the world where NPC is and is not endemic, respectively. This analysis revealed that LMP1 sequences show a distinct geographic structure, indicating that the southeast Asian isolates have evolved as a lineage distinct from those of Papua New Guinea, African, and Australian isolates. Furthermore, a likelihood ratio test revealed that the C termini of the LMP1 sequences of the southeast Asian lineage are under significant positive selection pressure, particularly at some sites within the C-terminal activator regions. We also present evidence that although the N terminus and transmembrane region of LMP1 have undergone recombination, the C-terminal region of the gene has evolved without any history of recombination. Based on these observations, we speculate that selection pressure may be driving the LMP1 sequences in virus isolates from southeast Asia towards a more malignant phenotype, thereby influencing the endemic distribution of NPC in this region.

Asunto(s)

Evolución Molecular , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virología , Selección Genética , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Asia Sudoriental , Línea Celular , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Linfocitos , Datos de Secuencia Molecular , Filogenia

RESUMEN

Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos B/inmunología , Línea Celular , Cicloheximida/farmacología , Epítopos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/genética , Humanos , Proteínas Recombinantes/genética , Linfocitos T Citotóxicos/inmunología , Transfección

RESUMEN

Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30-50% of HL cases are Epstein-Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus' role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy.

Asunto(s)

Infecciones por Virus de Epstein-Barr/complicaciones , Enfermedad de Hodgkin/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/terapia , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/terapia , Humanos , Tolerancia Inmunológica , Inmunoterapia/métodos , Pronóstico

RESUMEN

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Secuencia de Aminoácidos , Reacciones Antígeno-Anticuerpo , Línea Celular , Línea Celular Tumoral , Prueba de Histocompatibilidad , Humanos , Fragmentos de Péptidos/inmunología