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1.
Proc Natl Acad Sci U S A ; 109(25): 9959-64, 2012 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-22645359

RESUMEN

Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8(+) T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.


Asunto(s)
Hipersensibilidad a las Drogas , Complejo Mayor de Histocompatibilidad , Péptidos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares
2.
J Immunol ; 189(4): 1800-11, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22786768

RESUMEN

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).


Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito T/inmunología , Linfocitos T/inmunología , Citocinas/biosíntesis , Humanos , Hipersensibilidad/inmunología
3.
Immunogenetics ; 65(5): 371-86, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23417323

RESUMEN

Chinese rhesus macaques are of particular interest in simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) research as these animals have prolonged kinetics of disease progression to acquired immunodeficiency syndrome (AIDS), compared to their Indian counterparts, suggesting that they may be a better model for HIV. Nevertheless, the specific mechanism(s) accounting for these kinetics remains unclear. The study of major histocompatibility complex (MHC) molecules, including their MHC/peptide-binding motifs, provides valuable information for measuring cellular immune responses and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29 %. The peptide-binding specificity of two of these alleles, Mamu-A2*01:02 and Mamu-B*010:01, as well as the previously characterized allele Mamu-B*003:01 (and Indian rhesus Mamu-B*003:01), was found to be analogous to that of alleles in the HLA-B27 supertype family. Specific alleles in the HLA-B27 supertype family, including HLA-B*27:05, have been associated with long-term nonprogression to AIDS in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts and thereby warrants further studies to decipher the role of these alleles in the context of SIV infection.


Asunto(s)
Secuencias de Aminoácidos , Antígenos HLA/genética , Antígeno HLA-B27/genética , Fragmentos de Péptidos/genética , Alelos , Animales , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Antígeno HLA-B27/inmunología , Antígeno HLA-B27/metabolismo , Humanos , Macaca mulatta , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
4.
Immunogenetics ; 65(5): 357-70, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23392739

RESUMEN

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.


Asunto(s)
Variación Genética/genética , Genética de Población , Antígenos HLA-DP/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidad Clase II/genética , Alelos , Presentación de Antígeno , Linfocitos B/inmunología , Células Cultivadas , Epítopos/inmunología , Antígenos HLA-DP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Linfocitos T/inmunología
5.
Immunogenetics ; 64(6): 461-8, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22278177

RESUMEN

Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane-A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.


Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Macaca nemestrina/inmunología , Péptidos/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Virus de la Inmunodeficiencia de los Simios/inmunología
6.
Immunogenetics ; 64(6): 421-34, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22322672

RESUMEN

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the D(d) mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These "G2" alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the "G2" alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.


Asunto(s)
Evolución Molecular , Antígenos de Histocompatibilidad/química , Macaca mulatta/inmunología , Ratones/inmunología , Secuencias de Aminoácidos , Animales , Línea Celular , Antígenos H-2/química , Antígeno de Histocompatibilidad H-2D , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Alineación de Secuencia
7.
Immunogenetics ; 63(5): 275-90, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21274527

RESUMEN

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques' major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC-peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.


Asunto(s)
Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase I/inmunología , Macaca mulatta/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Frecuencia de los Genes , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A3/genética , Antígeno HLA-A3/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Macaca mulatta/genética , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología
8.
Immunogenetics ; 62(7): 451-64, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20480161

RESUMEN

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques.


Asunto(s)
Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/genética , Macaca mulatta/genética , Fragmentos de Péptidos/genética , Animales , China , Humanos , India , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético
9.
J Virol ; 82(1): 435-50, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17942551

RESUMEN

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.


Asunto(s)
Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Vacunas contra Hepatitis B/genética , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Inmunoterapia/métodos , Animales , Femenino , Vacunas contra Hepatitis B/uso terapéutico , Virus de la Hepatitis B/inmunología , Inmunización Secundaria , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plásmidos/genética , Plásmidos/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Vacunas Virales/genética , Vacunas Virales/inmunología
10.
J Urol ; 182(5): 2483-9, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19765754

RESUMEN

PURPOSE: A potential etiology of chronic prostatitis/chronic pelvic pain syndrome is autoimmunity. We determined whether T cells from men with chronic prostatitis/chronic pelvic pain syndrome would recognize peptides derived from the normal self-prostatic proteins prostate specific antigen and prostatic acid phosphatase. MATERIALS AND METHODS: CD4 T cells purified from peripheral blood of 31 patients with chronic prostatitis/chronic pelvic pain syndrome and from the buffy coat preparation of 27 normal male blood donors were stimulated in vitro with a panel of immunogenic peptides from prostate specific antigen and prostatic acid phosphatase, and assayed for reactivity with the peptides by interferon-gamma enzyme-linked immunosorbent spot assay. Intermediate resolution HLA typing was done by polymerase chain reaction. Peptides were also tested by binding assay against different class II alleles. RESULTS: Peptide PAP(173-192) was recognized more frequently by CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome than from healthy donors. The recognition of prostate specific antigen peptides was not statistically different when comparing cases to normal male blood donors individually. Peptide reactivity was more common in patients than in normal male blood donors for any prostate specific antigen peptide or any tested peptide. All peptides showed high promiscuity on binding assays. There was no association of cases with any specific HLA class II phenotype at intermediate resolution. CONCLUSIONS: CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome have a higher rate of recognizing the self-prostatic proteins prostatic acid phosphatase and prostate specific antigen compared to those from normal male blood donors. Data provide further evidence to support the role of autoimmunity in some men with chronic prostatitis/chronic pelvic pain syndrome.


Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Antígeno Prostático Específico/inmunología , Prostatitis/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Fosfatasa Ácida , Adulto , Anciano , Humanos , Masculino , Persona de Mediana Edad
11.
BMC Bioinformatics ; 7: 153, 2006 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-16545123

RESUMEN

BACKGROUND: T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. RESULTS: To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population. CONCLUSION: We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.


Asunto(s)
Mapeo Epitopo/métodos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Programas Informáticos , Vacunas/inmunología , Algoritmos , Frecuencia de los Genes , Genética de Población , Genotipo , Humanos , Inmunoensayo/métodos , Linfocitos T
12.
J Mol Biol ; 319(2): 449-61, 2002 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-12051920

RESUMEN

While most immunotherapies for cancer have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4+ T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4+ T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígeno HLA-DR1/química , Antígeno HLA-DR1/inmunología , Melanoma/inmunología , Mutación/genética , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos/química , Antígenos/genética , Antígenos/inmunología , Linfocitos T CD4-Positivos/citología , Cristalografía por Rayos X , Humanos , Activación de Linfocitos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Triosa-Fosfato Isomerasa/genética
13.
Clin Cancer Res ; 9(15): 5559-65, 2003 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-14654536

RESUMEN

PURPOSE: The purpose of this study was to immunize patients with HER-2/neu-overexpressing cancer with a multipeptide vaccine comprised of four class II HER-2/neu peptides that had been identified as the most immunogenic in a previous clinical trial. Furthermore, we questioned whether MHC binding affinity could predict the in vivo immunogenicity of the HER-2/neu helper peptides. EXPERIMENTAL DESIGN: Four putative class II HER-2/neu peptides, which were found to generate detectable specific T-cell responses (stimulation index > 2) in a majority of patients in a previous study, were used to formulate a single vaccine. The multipeptide vaccine was administered intradermally with granulocyte macrophage colony-stimulating factor as an adjuvant. Ten patients with HER-2/neu overexpressing breast or lung cancer were enrolled. HER-2/neu peptide-and protein-specific T cell and antibody immune responses were measured. Competitive inhibition assays were used to analyze the class II HER-2/neu peptides for their binding affinity to 14 common HLA-DR alleles. RESULTS: Twenty-five percent of patients developed HER-2/neu peptide-specific T-cell immunity, and 50% developed HER-2/neu peptide-specific antibody immunity. No patient developed HER-2/neu protein-specific T cell or antibody immunity. The majority of peptides exhibited high binding affinity, in vitro, to >/==" BORDER="0">3 of the 14 DR alleles analyzed. CONCLUSION: The group of peptides used in this study demonstrated high binding affinity to multiple DR alleles suggesting that in vitro binding affinity may be able to predict the in vivo immunogenicity of class II peptides. However, only a minority of patients immunized with the multipeptide vaccine developed HER-2/neu peptide-specific T cell or antibody immunity, and none developed HER-2/neu protein-specific immunity.


Asunto(s)
Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/uso terapéutico , Genes erbB-2/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Adulto , Anciano , Alelos , Vacunas contra el Cáncer/toxicidad , Femenino , Humanos , Inmunidad Celular/inmunología , Persona de Mediana Edad , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Linfocitos T/inmunología
14.
Curr Protoc Immunol ; Chapter 18: Unit 18.3., 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23392640

RESUMEN

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.


Asunto(s)
Antígenos/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Fragmentos de Péptidos/metabolismo , Ensayo de Unión Radioligante , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos/inmunología , Cromatografía en Gel , Humanos , Fragmentos de Péptidos/inmunología , Unión Proteica
15.
Hum Immunol ; 71(5): 468-74, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20156506

RESUMEN

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Virus de la Influenza A/inmunología , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Datos de Secuencia Molecular , Proteínas Virales/inmunología
16.
Vaccine ; 28(3): 664-72, 2010 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-19895924

RESUMEN

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.


Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antivirales/sangre , Células Cultivadas , Secuencia Conservada/genética , Secuencia Conservada/inmunología , Epítopos de Linfocito T/genética , Antígenos HLA-DR/metabolismo , Humanos , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Gripe Humana/prevención & control , Leucocitos Mononucleares/inmunología , Ratones , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Análisis de Supervivencia , Vacunas de ADN/genética
17.
J Gen Virol ; 88(Pt 7): 1986-1991, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17554032

RESUMEN

CD8(+) T-cell responses are central for the resolution of hepatitis C virus (HCV) infection, and viral escape from these CD8(+) T-cell responses has been suggested to play a major role in HCV persistence. However, the factors determining the emergence of CD8 escape mutations are not well understood. Here, the first identification of four HLA-A26-restricted CD8(+) T-cell epitopes is reported. Of note, two of these four epitopes are located in the NS3/4A and NS5A/5B cleavage sites. The latter epitope is targeted in all (three of three) patients with acute, resolving HCV infection and in a relatively high proportion (four of 14) of patients with chronic HCV infection. Importantly, the epitope corresponding to the NS5A/5B cleavage site is characterized by the complete absence of sequence variations, despite the presence of functional virus-specific CD8(+) T cells in our cohort. These results support previous findings that showed defined functional constraints within this region. They also suggest that the absence of viral escape may be determined by viral fitness cost and highlight an attractive target for immunotherapies.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Antígenos HLA-A/metabolismo , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Proteínas no Estructurales Virales/inmunología , Secuencia de Aminoácidos , Antígenos Virales/genética , Antígenos Virales/inmunología , Sitios de Unión/genética , Epítopos/química , Epítopos/genética , Hepatitis C/genética , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
18.
Cancer Immunol Immunother ; 55(11): 1358-66, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16435129

RESUMEN

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC50 <1,000 nM) and substituted analog peptides (IC50 <50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.


Asunto(s)
Antígenos HLA-A/química , Receptor ErbB-2/química , Linfocitos T Citotóxicos/química , Alelos , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Células Dendríticas/citología , Epítopos de Linfocito T/química , Antígeno HLA-A24 , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/citología , Péptidos/química
19.
J Virol ; 80(17): 8351-61, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16912286

RESUMEN

Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.


Asunto(s)
Mapeo Epitopo , Epítopos de Linfocito T/química , Antígeno HLA-A2/metabolismo , Virus Lassa/inmunología , Proteínas de la Nucleocápside/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Humanos , Inmunización , Fiebre de Lassa/inmunología , Fiebre de Lassa/prevención & control , Virus Lassa/genética , Virus Lassa/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Péptidos/administración & dosificación , Péptidos/química , Péptidos/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología
20.
Immunogenetics ; 57(6): 393-408, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16003466

RESUMEN

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing-to our knowledge-the first report of significant cross-reactivity among molecules belonging to different supertypes.


Asunto(s)
Antígenos HLA-A/química , Antígenos HLA-A/clasificación , Antígeno HLA-A1/química , Antígeno HLA-A1/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Reacciones Cruzadas , Antígenos HLA-A/metabolismo , Antígeno HLA-A1/metabolismo , Antígeno HLA-A24 , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo
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