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1.
Hum Immunol ; 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32173028

RESUMO

Abacavir hypersensitivity syndrome (ABC HSS) is strongly associated with carriage of human leukocyte antigen (HLA)-B*57:01, which has a 100% negative predictive value for the development of ABC HSS. However, 45% of individuals who carry HLA-B*57:01 can tolerate ABC. We investigated immune and non-immune related genes in ABC HSS (n = 95) and ABC tolerant (n = 43) HLA-B*57:01 + patients to determine other factors required for the development of ABC HSS. Assignment of phenotype showed that ABC HSS subjects were significantly less likely than tolerants to carry only ERAP1 hypoactive trimming allotypes (p = 0.02). An altered self-peptide repertoire model by which abacavir activates T cells is in keeping with observation that endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes that favour efficient peptide trimming are more common in ABC HSS patients compared to patients who tolerate ABC. Independently, non-specific immune activation via soluble cluster of differentiation antigen 14 (sCD14) may also influence susceptibility to ABC HSS.

2.
PLoS Pathog ; 15(12): e1008177, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31821379

RESUMO

Human immunodeficiency virus (HIV) can adapt to an individual's T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are specific to the host's human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child's HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child's T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs.


Assuntos
Adaptação Biológica/genética , Infecções por HIV/genética , HIV-1/genética , Transmissão Vertical de Doença Infecciosa , Adaptação Biológica/imunologia , Adulto , Criança , Pré-Escolar , Evolução Molecular , Feminino , Infecções por HIV/imunologia , Infecções por HIV/transmissão , HIV-1/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade
4.
Hum Immunol ; 79(12): 821-822, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30278218

RESUMO

DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, and -DRB1 loci was performed on 496 healthy adult donors from San Diego, California, to characterize allele frequencies in support of studies of T cell responses to common allergens. Deviations from Hardy Weinberg proportions were detected at each locus except A and C. Several alleles were found in more than 15% of individuals, including the class II alleles DPB1∗02:01, DPB1∗04:01, DQA1∗01:02, DQA1∗05:01, DQB1∗03:01, and the class I allele A∗02:01. Genotype data will be available in the Allele Frequencies Net Database (AFND 3562).


Assuntos
Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Antígenos HLA-DP/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Adolescente , Adulto , Alelos , California , Feminino , Frequência do Gene , Genótipo , Técnicas de Genotipagem/métodos , Teste de Histocompatibilidade/métodos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
5.
PeerJ ; 6: e4803, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29796347

RESUMO

Helicobacter pylori requires genetic agility to infect new hosts and establish long-term colonization of changing gastric environments. In this study, we analyzed H. pylori genetic adaptation in the Mongolian gerbil model. This model is of particular interest because H. pylori-infected gerbils develop a high level of gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. We analyzed the whole genome sequences of H. pylori strains cultured from experimentally infected gerbils, in comparison to the genome sequence of the input strain. The mean annualized single nucleotide polymorphism (SNP) rate per site was 1.5e-5, which is similar to the rates detected previously in H. pylori-infected humans. Many of the mutations occurred within or upstream of genes associated with iron-related functions (fur, tonB1, fecA2, fecA3, and frpB3) or encoding outer membrane proteins (alpA, oipA, fecA2, fecA3, frpB3 and cagY). Most of the SNPs within coding regions (86%) were non-synonymous mutations. Several deletion or insertion mutations led to disruption of open reading frames, suggesting that the corresponding gene products are not required or are deleterious during chronic H. pylori colonization of the gerbil stomach. Five variants (three SNPs and two deletions) were detected in isolates from multiple animals, which suggests that these mutations conferred a selective advantage. One of the mutations (FurR88H) detected in isolates from multiple animals was previously shown to confer increased resistance to oxidative stress, and we now show that this SNP also confers a survival advantage when H. pylori is co-cultured with neutrophils. Collectively, these analyses allow the identification of mutations that are positively selected during H. pylori colonization of the gerbil model.

6.
Hum Immunol ; 79(2): 87-88, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29289740

RESUMO

DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, and -DRB1 loci was performed on 714 healthy adult blood bank donors from Colombo, Sri Lanka, to characterize allele frequencies in support of studies on T cell immunity against pathogens, including Dengue virus. Deviations from Hardy Weinberg proportions were not detected at any locus. Several alleles were found in >30% of individuals, including the class II alleles DPB1 * 04:01, DPB1 * 02:01, DQB1 * 06:01 and DRB1 * 07:01, and the class I alleles A * 33:03 and A * 24:02. Genotype data will be available in the Allele Frequencies Net Database.


Assuntos
Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Antígenos HLA-DP/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Linfócitos T/imunologia , Adulto , Animais , Grupos Étnicos , Feminino , Frequência do Gene , Genótipo , Voluntários Saudáveis , Teste de Histocompatibilidade , Humanos , Masculino , Camundongos , Análise de Sequência de DNA , Sri Lanka
7.
Hum Immunol ; 79(1): 1-2, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29122684

RESUMO

DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, and -DRB1 loci was performed on anonymized samples provided by 339 healthy adult blood bank donors in Managua, Nicaragua. The purpose of the study was to characterize allele frequencies in the local population to support studies of T cell immunity against pathogens, including Dengue virus. Deviations from Hardy Weinberg proportions were detected for all class II loci (HLA-DPB1, -DQA1, -DQB1 and -DRB1), and at the class I C locus, but not at the class I A and B loci. The genotype data will be available in the Allele Frequencies Net Database.


Assuntos
Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Antígenos HLA-DP/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Adulto , Bases de Dados Genéticas , Dengue/imunologia , Frequência do Gene , Genética Populacional , Genótipo , Teste de Histocompatibilidade , Humanos , Desequilíbrio de Ligação , Nicarágua , Análise de Sequência de DNA , Linfócitos T/imunologia
8.
Gut ; 67(10): 1793-1804, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-28924022

RESUMO

OBJECTIVE: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. DESIGN: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. RESULTS: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). CONCLUSION: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.


Assuntos
Carcinogênese , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Proteínas de Bactérias/genética , Infecções por Helicobacter/patologia , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Técnicas In Vitro/métodos , Polimorfismo de Nucleotídeo Único/fisiologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/fisiopatologia
9.
Viral Immunol ; 30(7): 533-541, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28530508

RESUMO

Host hepatitis C virus (HCV)-specific T cell responses and the ability of the virus to escape this response are important correlates of infection outcome. Understanding this host-viral interplay has been difficult given the often asymptomatic nature of acute HCV infection. We studied a recent transmission case to determine whether adapted viral strains can be transmitted and influence the recipient's anti-HCV T cell response. The diversity of viral populations was examined using next-generation sequencing, and HCV-specific T cell interferon (IFN)-γ responses were assessed using a peptide panel representing the autologous viruses. HCV-specific T cell responses in the source were directed against peptides that did not match the dominant autologous virus but rather low-frequency variants, implying existing viral adaptation in the source strain. Most HCV T cell epitopes that elicited an IFN-γ response in the source did not in the recipient, despite the pair sharing human leukocyte antigen alleles that govern antigen presentation and similar autologous viruses. Intrahost HCV variation in the recipient fell within predicted T cell epitopes, suggesting alternative targets of the immune response. These data suggest that transmission of adapted viral species can direct the host's HCV-specific immune response profile during acute infection.


Assuntos
Epitopos de Linfócito T/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Adaptação Fisiológica/imunologia , Austrália , ELISPOT , Epitopos de Linfócito T/análise , Epitopos de Linfócito T/química , Evolução Molecular , Variação Genética/imunologia , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/virologia , Hepatite C Crônica/transmissão , Hepatite C Crônica/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Humanos , Uso Comum de Agulhas e Seringas
11.
Hum Immunol ; 78(4): 325-326, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28315719

RESUMO

One hundred healthy infants enrolled as controls in a tuberculosis vaccine study in Nyanza Province, Kenya provided anonymized samples for DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, -DRB1, and -DRB3/4/5 loci. The purpose of the study was to characterize allele frequencies in the local population, to support studies of T cell immunity against pathogens, including Mycobacterium tuberculosis. There are no detectable deviations from Hardy Weinberg proportions for the HLA-B, -C, -DRB1, -DPB1, -DQA1 and -DQB1 loci. A minor deviation was detected at the HLA-A locus due to an excess of HLA-A*02:02, 29:02, 30:02, and 68:02 homozygotes. The genotype data are available in the Allele Frequencies Net Database under identifier 3393.


Assuntos
Genética Populacional , Antígenos HLA/genética , Teste de Histocompatibilidade/métodos , Bases de Dados de Ácidos Nucleicos , Frequência do Gene , Genótipo , Humanos , Lactente , Quênia , Desequilíbrio de Ligação , Polimorfismo Genético , Análise de Sequência de DNA
12.
Retrovirology ; 14(1): 2, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-28086908

RESUMO

BACKGROUND: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. RESULTS: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. CONCLUSION: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.


Assuntos
HIV/fisiologia , Integração Viral , Latência Viral , Replicação Viral , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
13.
PLoS One ; 11(2): e0147567, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26849221

RESUMO

BACKGROUND: Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome. METHODS: Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins. RESULTS: EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes ('AEG': aa 481-496 and 'MVF': aa 562-577), and two putative epitopes between positions 502-543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis. CONCLUSIONS: This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1*15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Variação Genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Esclerose Múltipla/etiologia , Análise por Conglomerados , Reações Cruzadas/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/química , Feminino , Subtipos Sorológicos de HLA-DR/imunologia , Subtipos Sorológicos de HLA-DR/metabolismo , Herpesvirus Humano 4/classificação , Humanos , Masculino , Bainha de Mielina/imunologia , Peptídeos/imunologia , Filogenia , Ligação Proteica , Análise de Sequência de DNA
14.
Oncoimmunology ; 4(7): e1011492, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26140232

RESUMO

A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

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