Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

HLA and other gene associations with dengue disease severity.

Large case control gene association studies have been performed on cohorts of dengue virus (DENV) infected patients identified in mainland Southeast Asia, South Asia and the Caribbean. Candidate genes that have shown statistically significant associations with DENV disease severity encode HLA molecules, cell receptors for IgG (FcGII), vitamin D and ICAM3 (DCSIGN or CD209), pathogen recognition molecules such as mannose binding lectin (MBL), blood related antigens including ABO and human platelet antigens (HPA1 and HPA2). In ethnic Thais with secondary infections a variety of HLA class I alleles (HLA-A 0203, 0207, A11, B 15, B 44, B 46, B 48, B 51, B 52), DCSIGN promoter polymorphisms and the AB blood group, independently associate with either susceptibility or resistance to dengue fever (DF) and the more severe dengue hemorrhagic fever (DHF). There is also evidence that some HLA associations with disease severity correlate with the DENV serotype inducing secondary infections. Taken together, there is now evidence that allelic variants of multiple gene loci involved in both acquired and innate immune responses contribute significantly to DENV disease outcome and severity. Further analysis of the genetic basis of severe DENV disease in different at risk populations may contribute to the development of new preventative and therapeutic interventions.

Two novel MICA alleles, MICA*054 and MICA*056.

We report the identification of two novel major histocompatibility complex (MHC) class I-related chain A (MICA) alleles. MICA*054 has a nucleotide substitution of A to G at position 871 (codon 268), encoding an amino acid change of serine to glycine in the alpha-3 domain. MICA*056 has a nucleotide substitution at position 758 of G to C resulting in the substitution of tryptophan for serine at codon 230, also in the alpha-3 domain.

Does the polymorphism of MHC class II promoters matter?

The promoters of genes of the major histocompatibility complex vary not only because of linkage disequilibrium with their coding sequences but also, we argue, because of natural selection that acts particularly strongly on MHC II gene promoters. Thus, the promoter of H2Eb varies more than that of H2K, to an extent that cannot be accounted for by coding variation, and the same applies to HLA.DRB1 in comparison with H2D. We discuss how transduction by lentivirus vectors followed by adoptive transfer of monoclonal T cells could be used to test the functional activity of variant mouse promoters in vivo, and how homologous recombination in suitable cell lines might provide a short cut to obtaining promoter knock-ins.

Analysis of Fc gamma receptor II (CD32) polymorphism in populations of African and South Asian ancestry reveals east-west geographic gradients of allele frequencies.

Analysis of FcgammaRIIA alleles in Pakistanis and in Trinidadians of South Asian, African and mixed ancestry revealed no significant differences between Trinidadian South Asians and Pakistanis. H131 homozygotes were more common among Trinidadian South Asians than among Africans and those of mixed ancestry. Comparison with other populations revealed east-west geographic gradients of allele frequencies.

HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.

Associations of HLA class II alleles with pulmonary tuberculosis in Thais.

Tuberculosis is an important infectious disease in Thailand. Susceptibility to tuberculosis is influenced not only by the environment but also by host genetic factors. In this study, we investigated HLA alleles in 82 patients with tuberculosis from Bangkok and in 160 normal controls. HLA-DRB1, DQA1 and DQB1 genotyping was performed by the PCR-SSO method. The frequency of HLA-DQB1*0502 was increased in tuberculosis patients compared to the normal controls (P = 0.01, OR = 2.06). In contrast, the frequencies of DQA1*0601 and DQB1*0301 were decreased in tuberculosis patients compared to the controls (P = 0.02 and P = 0.01, respectively). Our results suggest that HLA-DQB1*0502 may be involved in the development of pulmonary tuberculosis, whereas HLA-DQA1*0601 and DQB1*0301 may be associated with protection against tuberculosis.

Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations.

Natural killer (NK) and some T cells express killer cell immunoglobulin-like receptors (KIRs), which interact with HLA class I expressed by target cells and consequently regulate cytolytic activity. The number of KIR loci can vary and so a range of genetic profiles is observed. We have determined the KIR genetic profiles from one African (n = 62) and two South Asian (n = 108, n = 78) populations. Several of the KIRs are present at significantly different frequencies between the two major ethnic groups (eg KIR2DS4 gene frequency 0.82 African, 0.47 S Asian. Pc < 1 x 10(-6)) and this is due to uneven distribution of two KIR haplotype families 'A' and 'B'. All three populations described here displayed a greater degree of diversity of KIR genetic profiles than other populations investigated, which indicates further complexity of underlying haplotypes; in this respect we describe two individuals who appear homozygous for a large deletion including the previously ubiquitous 2DL4. We have also reanalysed three populations that we studied previously, for the presence of a KIR which is now known to be an indicator of the 'B' haplotype. South Asians had the highest overall frequencies of all KIR loci characteristic of 'B' haplotypes (Pc < 0.0001 to < 0.004). Furthermore, gene frequency independent deviances in the linkage disequilibrium were apparent between populations.

Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations

Natural killer (NK) and some T cells express killer cell immunoglobulin-like receptors (KIRs), which interact with HLA class I expressed by target cells and consequently regulate cytolytic activity. The number of KIR loci can vary and so a range of genetic profiles is observed. We have determined the KIR genetic profiles from one African (n = 62) and two South Asian (n = 108, n = 78) populations. Several of the KIRs are present at significantly different frequencies between the two major ethnic groups (eg KIR2DS4 gene frequency 0.82 African, 0.47 S Asian. Pc < 1 x 10(-6)) and this is due to uneven distribution of two KIR haplotype families 'A' and 'B'. All three populations described here displayed a greater degree of diversity of KIR genetic profiles than other populations investigated, which indicates further complexity of underlying haplotypes; in this respect we describe two individuals who appear homozygous for a large deletion including the previously ubiquitous 2DL4. We have also reanalysed three populations that we studied previously, for the presence of a KIR which is now known to be an indicator of the 'B' haplotype. South Asians had the highest overall frequencies of all KIR loci characteristic of 'B' haplotypes (Pc < 0.0001 to < 0.004). Furthermore, gene frequency independent deviances in the linkage disequilibrium were apparent between populations.

T cell receptor Vbeta gene usage in Thai children with dengue virus infection.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand.

Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.

MICA and MICB genes: can the enigma of their polymorphism be resolved?

The human MHC class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. Their organization, expression and products differ considerably from classical HLA class I genes. MIC proteins are considered to be markers of "stress" in the epithelia, and act as ligands for cells expressing a common activatory natural killer-cell receptor (NKG2D). Molecular models are now available for the MICA protein, both bound and complexed with NKG2D. MICA molecules appear to be highly flexible and polymorphic, although the functional relevance and implications of their polymorphism have yet to be fully discerned.

Distribution of natural killer cell immunoglobulin-like receptor sequences in three ethnic groups.

Killer cell immunoglobulin-like receptors (KIRs) are members of a group of molecules that specifically recognize HLA class I ligands and are found on subsets of human lymphopoetic cells. The number of KIR loci can vary between individuals, resulting in a heterogeneous array of possible KIR genes. The range of observed profiles has been explained by the occurrence of two haplotype families termed A and B which can be distinguished on the basis of certain KIR sequences. Here we attempted to determine whether the frequencies of putative KIR loci and the two haplotype groups vary in three ethnically defined, healthy, and unrelated control populations, namely UK Caucasoid (n=136), Palestinian (n=105) and Thai (n=119). We molecularly typed genomic DNA for the presence of 12 putative KIR loci, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1, using modified PCR sequence-specific primers. The patterns of KIR locus frequencies combined with the similar linkage disequilibrium values suggest that there was a distinction in the distribution of the two broad haplotype groups between the populations studied. The A haplotype was always the most prevalent, but the ratio of A to B varied between populations. The frequency of B haplotype was highest in the Palestinians and lowest in the Thais (Pc<0.0001).

Genes of the class II and class III major histocompatibility complex are associated with typhoid fever in Vietnam.

The influence of genes of the major histocompatibility complex (MHC) class II and class III loci on typhoid fever susceptibility was investigated. Individuals with blood culture-confirmed typhoid fever and control subjects from 2 distinct geographic locations in southern Vietnam were genotyped for HLA-DRB1 and HLA-DQB1 alleles, the gene that encodes tumor necrosis factor (TNF)-alpha (TNFA [-238] and TNFA [-308]), the gene that encodes lymphotoxin-alpha, and alleles of the TNF-alpha microsatellite. HLA-DRB1*0301/6/8, HLA-DQB1*0201-3, and TNFA*2 (-308) were associated with susceptibility to typhoid fever, whereas HLA-DRB1*04, HLA-DQB1*0401/2, and TNFA*1 (-308) were associated with disease resistance. The frequency of all possible haplotypes of the 3 individually associated loci were estimated and were found to be significantly different in typhoid case patients and control subjects (chi2=55.56, 32 df; P=.006). Haplotypes that were either protective (TNFA*1 [-308].DRB1*04) or predisposed individuals to typhoid fever (TNFA*2 [-308].DRB1*0301) were determined. This report identifies a genetic association in humans between typhoid fever and MHC class II and III genes.

A comparison of molecular HLA-DR and DQ allele profiles forming DR51-, DR52-, and DR53-related haplotypes in five ethnic Thai populations from mainland southeast Asia.

Using PCR-SSOP typing we have deduced the composition and frequency of HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, and -DQB1 alleles present in DR51-, DR52-, and DR53-related haplotypes, in 519 individuals representative of five ethnic Thai populations recruited in central, northeastern and northern Thailand. In total, we have unequivocally detected at varying frequencies, 17 DR51-related haplotypes, 24 DR52 haplotypes, and 12 DR53 haplotypes in the study groups. We document evidence of north-south gradients of DR51-related haplotypes, whereby the overall frequency of DR51-containing haplotypes is relatively more common in the northern Thai groups. Similarly, within DR53-related haplotypes the frequency of DRB1*0901-containing haplotypes increases in the more northerly groups, and an inverse effect was observed with DRB1*0701-containing haplotypes that were relatively more common in the northeastern and central Thais. We have also compared the class II haplotype profiles of the Thais with the equivalent profiles reported in other non-Thai ethnic groups from mainland and insular SE Asia. One DR51-related haplotype DRB1*1502x, DRB5*0102x, DQA1*0101/4, DQB1*0501, would appear to be characteristic of Thai populations, as it was the most common DR2 haplotype in all five study groups and is also prevalent in other mainland southeast Asians, but is much less evident in the more northern populations of eastern Asia or China.

MIC-A allele profiles and HLA class I associations in Behçet's disease.

Recently a new family of non-classical MHC molecules, the MHC class I chain-related protein (MIC), encoded by genes located in the major histocompatability complex have been identified. On the basis of the location of MIC genes and the structure and expression of MIC molecules it has been postulated that MIC may be a susceptibility factor in Behçet's disease (BD). We investigated the association of the 16 described external domain alleles and the transmembrane triplet repeats of MIC-A with BD in a Middle Eastern population. DNA from ninety-five patients and 102 age- and sex-matched controls were analyzed by polymerase chain reaction using allele specific primers. Our results show an increase of MIC-A*009 in the BD patient group 44/95 (46%) compared with controls 24/102 (24%) (chi2=11.3, OR=2.8, P=0.00078). MIC-A*009 was also found to be strongly associated with HLA-B51 in the patients 39/44 (88%) when compared with controls 10/24 (42%) (chi2=4, P=0.04). MIC-A*009 was also found in linkage disequilibrium with HLA-B52, but only in controls. The A6 form of a MIC-A transmembrane triplet repeat was found to be significantly raised in the patients (80/95; 84%;) compared with controls (58/102, 57%) (chi2=17.5, OR=4, P=0.000028). Although the MIC-A associations described are highly significant, the association with HLA-B51 independently remains the most significant factor (chi2=56.8, P<10(-6)). The data suggests that as both MIC-A*009 and A6 are in strong linkage disequilibrium with HLA-B51, they are unlikely to be the susceptibility gene for BD but may be markers for additional risk factors.

Towards a molecular phototyping system for allelic variants of MICA, encoded by polymorphisms in exons 2, 3 and 4 of MHC class I chain-related genes.

In this study we have developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) or phototyping system to analyse the polymorphism of the human MICA gene locus. By scrutinising the reported MICA sequence variations in exons 2, 3 and 4 which encode the extracellular protein domains, we have selected and tested 20 MICA-specific primer mixes that should discriminate between the majority of homozygous and heterozygous combinations of MICA alleles 001-016. We have tested this scheme on DNA prepared from a large number of well-characterised tissue culture cell lines with previously reported MICA nucleotide sequences and found an excellent correlation with the observed PCR-SSP phototypes. We believe that this scheme can also be modified to detect new MICA alleles when they are characterised, as well as be incorporated into standard phototyping protocols to generate allele and haplotype profiles of both classical and non-classical HLA gene loci in test DNA samples.

Serological and molecular analysis of HLA class I and II alleles in Thai patients with psoriasis vulgaris.

The HLA class I and class II alleles in 67 patients with type I psoriasis vulgaris, 23 patients with type II psoriasis vulgaris and 140 healthy individuals were analyzed. The frequencies of HLA-A2, -B46, -B57 and DQB1*0303 were significantly increased in type I psoriasis compared to the controls (Pc<0.05). Molecular analysis of HLA-A2 alleles showed an increase in HLA-A*0207 and a decrease in HLA-A*0203 in type I psoriasis. HLA-DQB1*0301 was significantly decreased in type I psoriasis compared to the normal controls (Pc<0.05). No association of any alleles with type II psoriasis was observed. This data demonstrated two susceptible haplotypes: HLA-A1-B57-DRB1*0701-DQA1*0201-DQB1*0303 (AH57.1) and HLA-A2-B46-DRB1*0901-DQA1*0301-DQB1*0303 (AH46.1) for type I psoriasis in the Thai population. Besides, the haplotype AH46.1 was also associated with type II psoriasis.

HLA-DR and -DQ associations with melioidosis.

Melioidosis is an important infectious disease of southeast Asia caused by an intracellular bacterium, Burkholderia pseudomallei. Cellular immunity is postulated to play important roles in immunity to melioidosis that may influence the severity and clinical outcome of the disease. The present study was undertaken to investigate possible associations of melioidosis with HLA class II alleles. HLA typing of HLA-DRB1, -DQA1, and -DQB1 was performed using polymerase chain reaction and sequence-specific oligonucleotide hybridization (PCR-SSO). Seventy-nine melioidosis patients and 105 healthy, ethnically and geographically matched controls were studied. Among 24 DRB1 alleles, 7 DQA1 alleles, and 13 DQB1 alleles identified in this population, an association with melioidosis was observed with DRB1*1602 which was increased in melioidosis patients (10.1%) compared to normal controls (4.8%), p = 0.047 (odds ratio (OR) = 2.25). In addition, significant increase of DRB1*1602 allele frequency and decrease of DQA1*03 were also observed in septicemic melioidosis patients, the most severe form of the disease (p = 0.01, OR = 3.10; and p = 0.047, respectively). Furthermore, a trend of association of DRB1*0701, DQA1*0201, and DQB1*0201 with relapse cases of melioidosis was also noted. In contrast, no HLA association was observed in localized melioidosis or melioidosis with diabetes mellitus. These findings provide the suggestive evidence of an immunogenetic basis of certain aspects of melioidosis.

Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection.

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.