From the beginning of the AIDS epidemic, Kaposi's sarcoma (KS) has been one of the defining conditions of the disease . Until the introduction of highly active antiretroviral therapy (HAART), it was the most common malignancy associated with HIV-1 infection . The high prevalence of HIV-related KS among men who have sex with men (MSM) compared to other groups of HIV patients infected by non-sexual routes, suggested that a sexually transmitted infectious agent could be responsible for KS . In 1994 a new herpes virus, Kaposi sarcoma associated herpesvirus/human herpes virus 8 (HHV-8) was identified in HIV-related KS tissue . HHV-8 has since been shown to be the causative agent of KS [5–9]. Cohort studies indicate that among HIV infected MSM, about half of those infected with HHV-8 develop KS between 5 and 10 years after contracting HHV-8 [10,11]. HHV-8 infection, once acquired, develops as a life-long chronic infection like other herpesviruses such as Epstein–Barr virus. In most cases, this infection is controlled through life by immune surveillance; however in a minority of individuals infection is not effectively controlled, leading to a proliferation of infected cells and, eventually, to malignancy. Thus, in addition to immunosuppression , host-related factors may also play a role in KS pathogenesis.
Because of the crucial role played by HLA in the development of the immune response, HLA could be one potential host-related cofactor. Binding of peptides from the infectious pathogens to HLA proteins is the first step in initiation of the host-specific immune response. The peptide-binding groove on HLA molecules contains pockets which are binding sites for anchor residues in peptides. Different HLA molecules have slightly different peptide-binding clefts and accordingly bind and present different sets of peptides to T cells. The differences are determined by the polymorphic residues of HLA molecules. Polymorphic amino acid residues located in the pockets have a dramatic effect on the spectrum of peptides that are bound by a particular HLA molecule. In accordance, immune response against a pathogen will vary among individuals.
Variability in HLA has been shown to influence response to infection with human pathogens and disease outcome. Strong evidence for HLA associations have been reported for malaria, hepatitis B, HTLV-I and HIV [13–16]. The association between HLA and classic KS has also been analysed and an increased incidence of HLA-DR5 has been reported [17,18]. However, when HLA typing was carried out by molecular techniques, this could not be confirmed . Several studies have reported significant associations between HLA genotype and AIDS-related KS [20–25]. In these studies, a positive association with DR5 has been described when population controls were used as the comparison group. When HIV-1 positive disease-free individuals were used as the control group, Mann et al.  found a positive association with DR1, DQ1, B35 and Cw4. An association between DR1 and AIDS-related KS was also described by Klein et al. . However, in these studies, the HHV-8 infection status of the controls was unknown.
The aim of this paper is to present the results of the HLA typing derived from a European case–control study, comparing patients with and without KS, and also to compare the HLA subtypes between HHV-8 positive and HHV-8 negative participants.
Materials and methods
In 1992 a multi-centre matched case–control study funded by the European Union (EURO-SHAKS: European Study on HIV Associated Kaposi's Sarcoma, DG XII), was designed to identify possible biological, behavioural and environmental risk factors for HIV associated KS. Participant countries were Spain, Belgium, UK, Italy and Greece. Cases were defined as any AIDS patient with a clinical diagnosis of KS and controls as any AIDS patient with an indicative disease other than KS or with CD4 cells counts < 200 × 106 cells/l, diagnosed at ± 4 months after case diagnosis. Each case was matched with two controls by sex, age and transmission category [26,27]. After informed consent, each study participant was interviewed for socio-demographic and behavioural data, and sera, blood clots, plasma and whole blood were obtained and stored at −20°C for later analysis. By the end of 1998, a total of 483 patients (161 triplets) had been recruited. In 1998, sera samples from both cases and controls were tested for HHV-8.
Because HLA typing was performed only among participants from Spain, for the purpose of this research, patients from other countries have been excluded from the analysis. Moreover, to decrease the number of potential confounders, only Caucasian MSM were included. Out of 354 Spanish participants, 308 fulfilled the above mentioned inclusion criteria. In addition, three patients were excluded from the study because of insufficient biological sample for HHV-8 analysis. Therefore, the study group consisted of 305 individuals: 103 (n1) cases with KS and 202 (n0) controls with AIDS (a CD4 cell count < 200 × 106 cells/l was accepted as AIDS criteria ) but without KS. Cases and controls were compared for both mean age (38.7 and 38.3 years, respectively) and CD4 cell count at entry (differences were not significant). Additionally, KS was the first indicative disease in 80% of the cases.
Sera were analysed for HHV-8 infection using three different assays: immunofluorescence assay for the latency associated antigen (LANA) encoded by Orf73, ELISA for Orf73 and ELISA for the lytic antigen Orf65 as described previously [6,8,12]. A sample was considered to be HHV-8 positive if reactive to any of the three assays.
DNA isolation and HLA typing
Peripheral blood samples were obtained and subjected to isolation of genomic DNA with a QIAamp blood kit (Qiagen, Hilden, Germany). HLA-DRB1* typing was performed by PCR with sequence-specific primers according to manufacturer's recommendations (Dynal3, Oslo, Norway).
Patients’ ages and CD4 cell counts were compared by Student's t test and the Mann–Whitney U test, respectively. Because the study population for this analysis comprised only homosexual men and no differences between cases and controls were found regarding age and CD4 cell count, an unmatched analysis was performed. HLA class II marker frequencies were analysed as described previously [29,30]. The odds ratio (OR) and its 95% confidence interval (CI) were computed to estimate the degree of association between the presence of the allele and the disease; when any observed value was equal to 0, Haldane's modification of Woolf's method was used . The significance of the OR deviation from unity was estimated by Fisher's exact test with two-tailed P-value. The corrected P-value is denoted by pc; the correction factor was 14 (given in parentheses), i.e., the number of alleles tested.
The overall HHV-8 seroprevalence was 70.8% (216/305), with 91.3% (94/103) and 60.4% (122/202) respectively for cases and controls (P < 0.001). There are no published data on susceptibility to HHV-8 infection in relation to HLA Class II molecules. To assess whether HHV-8 infected and non-infected patients were homogeneous for HLA Class II alleles, we first compared the results of HLA-DRB1 typing according to HHV-8 antibody status (Table 1). Slight differences in the frequency of some alleles were observed, but after correction these differences were not statistically significant. Although with our study design risk of infection cannot be estimated, the results could suggest that no HLA-DRB1 allele is a risk or protective factor for HHV-8 infection.
Given that HHV-8 is a necessary factor for KS development [5–9], for the purpose of assessing the potential role of HLA alleles in KS development we only used participants who were HHV-8 infected. Marker frequencies of HLA-DRB1 between KS AIDS patients (94 cases) and non-KS AIDS patients (122 controls) were compared among HHV-8 infected persons (Table 1). Several alleles exhibited a trend towards allelic frequency differences between the KS and control groups, with uncorrected P values < 0.05. Thus, the frequency of DRB1*01 was higher in KS patients than in the control group (DRB1*01: OR, 2.15; 95% CI, 1.13–4.08; P = 0.023; pc(14) = 0.322), suggesting that this allele could constitute a risk factor for KS development in HHV-8/HIV co-infected AIDS patients, in agreement with previously published data [21,25]. On the other hand, some alleles showed an inverse relationship, being more frequent among the AIDS patients who did not develop KS. There were two alleles with an OR < 0.3: DRB1*12 (OR, 0.12; 95% CI, 0.02–0.96) and DRB1*08 (OR, 0.25; 95% CI, 0.03–2.19). However, only DRB1*12 was statistically significant (P = 0.025), and this significance disappeared after correction for the number of comparisons [pc(14) = 0.350].
Interestingly, comparison of the amino acid sequence of the different DRB1 alleles shows that almost all the DRB1*08 and DRB1*12 alleles share an amino acid residue, glycine (G), at position 13 of the β chain. The other DRB1* alleles contain several other amino acids at position 13, but glycine seems to be restricted to DRB1*08 and DRB1*12. From analysis of the sequence we could not find any other polymorphic position that was common to DRB1*08 and DRB1*12 but was not present in all the other alleles. Comparison of the frequency of glycine's presence at position 13 among HHV-8 positive cases and controls shows an increased statistically significant difference (OR, 0.16; 95% CI, 0.03–0.70; P = 0.009).
Several reports describe susceptibility to KS pathogenesis associated with DR1 in AIDS patients [21,25]. Although in our group of patients the risk associated with the presence of DRB1*01 was not statistically significant, an increased frequency of this allele was observed in KS patients in comparison with the control group (Table 1). This fact is especially relevant taking into account that the amino acid at position 13 in the β chain of DRB1*01 alleles, with the exception of DRB1*0103, is phenylalanine (F). The presence of F at position 13 of the β chain shows a statistically significant relationship with the development of KS in AIDS patients (OR, 2.24; 95% CI, 1.19–4.20; P = 0.016). In this analysis, those patients displaying DRB*09 and DRB*10, alleles which also contained F at position 13 were included although they were very uncommon.
However, this comparison could possibly be influenced by the fact that the comparison group used also included those patients who were G13 and, as shown above, this residue's behaviour was the opposite to that of F13. Therefore, we reanalysed the sample (Table 2) by comparing cases against HHV-8 infected controls who typed as F13 with those who were neither F13 nor G13 and we found that frequency of F13 was still higher in cases (OR, 2.1; 95% CI, 1.09–3.97; P = 0.035). The same comparison was performed for the presence of G13, and similar results were found (OR, 0.20; 95% CI, 0.04–0.92; P = 0.029). Finally, we also analysed the effect of G13 and F13 polymorphism if cases were compared against controls, irrespective of serostatus. In this case, the differences were also statistically significant (data not shown).
One mechanism to account for HLA associations with susceptibility or resistance to KS pathogenesis is varying efficiency of HHV-8 antigen presentation by different HLA molecules. The different frequencies of several alleles could be interpreted in the context of the immune response against this infectious agent. It has been demonstrated that the natural polymorphism of HLA molecules can influence both the specificity and affinity of peptide binding. Therefore, it is possible that certain HLA molecules bind and present HHV-8 antigens more efficiently than others. Our data show that resistance to KS development was independently associated with a precise amino acid at position 13 of the DRβ chain. In this case, alleles DRB1*08 and DRB1*12 would have the capacity to present a certain peptide which would lead the immune response to control the pathogenic effect of HHV-8, thus suggesting that anti-HHV-8 class II restricted CD4 cell count may play an important role in protecting against viral challenge. In fact, strong T-helper cell responses to an HIV-1 p24 antigen have been correlated with a reduced virus load and risk of HIV-1 disease progression  and are likely to contribute to a sustained potent cytolytic CD8 response.
Position 13 of the β chain is especially significant because, together with amino acids at positions 70 and 71, it lines a pocket, named pocket 4 , which determines the binding characteristics of peptides to the cleft. Thus, we could relate the potential protective action of DRB1*08 and DRB1*12 more precisely to a specific sequence common to these alleles which is directly involved in determining the specificity of the peptide bound by the HLA molecule. Although this residue is also found in some other alleles, such as DRB1*1105 and DRB1*1404, these are extremely infrequent in Caucasians , and thus the possibility that they were present in our sample is negligible.
More interestingly, the side chain of F is especially large as it includes an aromatic group. This is in contrast with G, the amino acid present in DRB1*08/*12 alleles, which display the smallest side chain among the different amino acids. Thus, the size of the side chain of the amino acid at position 13 could determine that the peptide presented by the G13 containing DRB1*08/12 alleles, capable of eliciting a protective immune response, would not be presented by DRB1*01 alleles at all. An alternative explanation could be that DRB1*01 is responsible for the presentation of another peptide which could select a defective immune response and thus favour the development of KS.
Our results are constrained by some study design limitations that deserve further comment. Firstly, by studying patients participating in a case–control study, we are analysing disease outcomes at a given point of time and therefore we cannot assess either risk of HHV-8 infection or rate of KS development. Secondly, the original matching design scheme was not maintained in the analysis, and some confounders may have been introduced. Nevertheless, although no biological marker for disease stage was used as a matching factor, most of the non-KS patients were included because of a CD4 cell count < 200 × 106 cells/l inclusion criterion, and therefore both cases and controls were at a quite advanced stage of the infection. In this analysis we included only Spanish Caucasian men who acquired HIV infection through homosexual sex, so there was no need to match for these factors. The fact that all studied patients were included between 1994 and 1996, before HAART was widely introduced in Spain, ensures that the observed effect of HAART, decreasing the KS incidence, is not a confounder in our study [34,35]. Finally, since the ages of both KS and non-KS patients were similar, we believe that no confounders have affected the observed differences.
The HLA-DRB1 gene shows a close association with DRB3-5 loci as well as with HLA-DQ genes and the different alleles are found in linkage disequilibrium. Although we did not analyse these other genes, we believe it improbable that the observed association could be due to the effect of genes other than DRB1. Concerning the DRB3-5 loci, whereas the DRB1*01 or DRB1*08 haplotypes do not contain these genes, the DRB1*12 haplotypes contain the DRB3 gene. In addition, association with a common DQ allelic variant is quite unlikely, since DRB1*08 and DRB1*12 are usually linked to different DQ alleles.
In summary, we cannot conclude on the role of HLA in facilitating or protecting against HHV-8 infection; however, our findings of a statistically significant association between certain amino acids at position 13 of the β chain of DR molecules between HHV-8 infected patients with and without KS, suggest a relevant role of the immune response in the interaction of this agent and KS pathogenesis. To elucidate better the role of these molecules on HHV-8 acquisition and its interrelation with KS development, these associations should be confirmed in subsequent independent studies and, preferably, of longitudinal design.
We would like to acknowledge the technical assistance in DNA isolation and HLA typing of F. Isart, B. Suarez, V. Fabregat, M.T. Arias, and M. Massó, and the field work of R. Muñoz.
Sponsorship: Supported in part by DG XII of the European Commission (EURO-SHAKS), Fondo de Investigaciones Sanitarias (FIS) and Laboratories MSD (Madrid, Spain). We also acknowledge the support provided by CIRIT and the Health Department (Generalitat de Catalunya, Barcelona, Spain) and the U.K. Medical Research Council.
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EURO-SHAKS working group
Ma Mercè Alsina Gibert, Elisabeth Buira (Hospital Clínic i Provincial de Barcelona), Cristina Tural (Hospital Universitari Germans Trias i Pujol), Imma Ocaña, Isabel Ruiz, Marjori Díaz (Hospital General Universitari Vall d'Hebron), Montserrat Fuster, Lali Baselga, Marta Alegre (Hospital de la Santa Creu i Sant Pau), Ferran Bolao, Elena Ferrer, Daniel Podzamczer (Hospital de Bellvitge), Rafael Rubio (Hospital XII de Octubre), José Luís López Colomes (Hospital del Mar), Martí Vall (Direcció d'Atenció Primària Ciutat Vella), José M Gatell (Hospital Clínic i Provincial de Barcelona), Bonaventura Clotet (Hospital Universitari Germans Trias i Pujol), Josep Cadafalch (Hospital de la Santa Creu i Sant Pau).