Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30-45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented.

Clinical and virological outcomes are variable following acute infection with hepatitis C virus (HCV): there is successful viral clearance in some individuals, while persistent infection is established in the majority of cases (35). Chronic infection is characterized by attenuated and narrowly focused CD4 ϩ and CD8 ϩ T-cell responses (reviewed in reference 14) in conjunction with reduced proliferative, cytokine, and cytolytic capacity of HCV-specific T cells (10, 20, 40) and loss of antigen recognition (3). Conversely, CD4ϩ and CD8 ϩ T-cell responses are maintained in individuals who clear the virus (6,17,21,32,34,42). Therefore, effective T-cell-dependent immune responses to HCV-specific viral epitopes, restricted by the presence of host human leukocyte antigens (HLA) class I (CD8 ϩ cytotoxic T cells [CTL]) and class II (CD4 ϩ helper T cells), appear to make an important contribution to adaptive immunity and disease outcome. The high rate of HCV replication and mutation renders its genome susceptible to changes within and flanking HLA-restricted epitopes, thereby providing mutated HCV sequences with a means of "escaping" HLA-restricted immune responses. The relevance of HCV immune escape mutations in chronic infection (reviewed in reference 2) was first demonstrated in chimpanzees (7,9,41) and then subsequently demonstrated in humans (4, 33). Evidence for CTL escape and reversion has been recently demonstrated in acute infection (36) and 18 to 22 years after a common-source outbreak (24). As in human immunodeficiency virus (HIV) (1), flanking epitope escape mutations affecting proteasomal epitope processing provide an effective mechanism of immune escape in HCV (26). However, direct evidence for viral escape in humans is limited, as the sequence of the transmitted virus is rarely known and most previous studies have focused on a limited number of HLA alleles (38; www.hcv.lanl.gov).We have chosen a population-based approach to assess in * Corresponding author. Mailing address: Centre for Clinical Immu-

, including HCV protease and polymerase inhibitors, is limited by the presence of drugspecific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/ subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P < 0.05) at known drug resistance sites. Conclusion: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host "immune resistance" in the STAT-C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C.

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. Conclusion: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

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The host's immune response to hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, the fitness cost incurred by the mutation, and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leukocyte antigen (HLA)–specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and the ways in which the sequence of the incoming virus, including preexisting escape mutations, can influence subsequent mutations in recipients and infection outcomes. Conclusion: HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses, which likely represent new viral adaptations. These associations did not overlap with previously reported adaptations for genotypes 1a and 3a and possibly reflected a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites accounted for only a portion of the sites in which viral diversity was observed in the new hosts. Furthermore, preexisting viral adaptations in the incoming (source) virus likely influenced the outcomes in the new hosts. (Hepatology 2011;53:396-405)

Background and aimsCD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response.MethodsA sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants.ResultsA strong ‘HLA footprint’ in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03-positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A.ConclusionsIt is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.