Human-Leukocyte Antigen Class I Cw 1502 and Class II DR 0301 Genotypes Are Associated with Resistance to Severe Acute Respiratory Syndrome (SARS) Infection

Introduction

S evere acute respiratory syndrome (SARS) is caused by SARS coronavirus (SARS-CoV), which is transmitted via respiratory droplets or close contact (5,6,11). In 2003, Taiwan experienced a series of SARS outbreaks and many hospitals suffered severe outbreaks of SARS-CoV nosocomial infections (5,11). According to the World Health Organization (WHO) there were 664 probable cases, and 364 cases were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and/or neutralizing antibody tests (5).

Infectious diseases, which often cause high morbidity and mortality in humans, exert a selective pressure that triggers genetic evolution and diversity, especially in the immune system, in response to a wide range of infectious pathogens (1,22,23). Early in the 1970s, the classical HLA loci were proved to be associated with infectious diseases (28). HLA class I or II gene products present antigenic peptides to T cells, initiating an immune response and the removal of foreign material via neutralizing antibodies, cytokines, and activated cytotoxic T cells. Previous studies indicated that both HLA and non-HLA loci were mostly responsible for the genetic component of the immune responses to infection or vaccination, and were closely associated with persistent antibody development (19,29). The HLA system is presently considered to be associated with the etiology of infectious diseases and autoimmune disorders. Researchers have demonstrated that specific HLA alleles are associated with susceptibility to and outcomes from viral infections, such as human immunodeficiency virus type 1, human T-cell leukemia virus type 1, hepatitis C virus, and SARS-CoV (1,5,17,18).

Serological assays are often used to diagnose SARS-CoV infection, and they mainly detect the anti-sera against spike (S) or nucleocapsid (N) proteins of SARS-CoV (4,5,7). The spike proteins mediate membrane fusion and induce protective antibodies in hosts (4,7,31). Reports indicate that the antibodies against S proteins may be a good means of early detection and neutralization of SARS-CoV infection (4,14,30). The N protein of coronavirus has been reported to participate in the replication and transcription of viral RNA and viral pathogenesis (7,18). However, the N protein in many coronaviruses is highly conserved, immunogenic, and abundantly expressed during infection (7). Previous studies indicated that antibodies against N protein were also useful in the early diagnosis and neutralization of the infection (5,7,18).

In order to follow-up the health status of SARS-infected patients, the Department of Health of the Taipei City Government held a physical examination exclusively for confirmed SARS cases in September 2006. Of these cases, 130 confirmed SARS cases were recruited for this study, and signed informed consent was obtained from all study participants. Peripheral blood mononuclear cells (PBMCs) from participants were purified and isolated as previously described (24). Genomic DNA was extracted with a DNA extraction kit (Qiagen, Valencia, CA). The class II HLA genotypes of these subjects were determined by a two-step protocol. In the first step, low-resolution class II typing was performed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP) (Dynal Biotech, Ltd., Wirral, U.K.). In the second step, high-resolution class II typing was performed using DNA amplification and sequencing with allele-specific primers (5). Western blot (WB) assays were performed to measure the anti-SARS antibodies in the sera (5). The N and S proteins of SARS-CoV expressed in a bacterial system and purified by glutathione-agarose beads were used for WB detection. Fisher's exact and chi-square (X²) tests were performed as part of univariate analyses to determine the statistical significance of all comparisons between SARS cases and control participants. Data were analyzed using SAS software (version 8.2). A conditional logistic regression was used to analyze the odds ratios of different HLA alleles and susceptibility to SARS infection. To confirm the statistical significance of the HLA allele in the case-control study, the age, gender, and occupation were included in the analysis (5).

We first compared the sensitivity and specificity between the neutralization assay (the gold standard for viral infection diagnosis) and WB analysis. The results showed a high correlation between these two assays (kappa=0.9283) (Supplementary Table 1; see online supplementary material at http://www.liebertonline.com). WB was used for evaluation of anti-SARS neutralization antibody and SARS-CoV infection. The results from WB indicated that 9 (6.9%) subjects had antibodies against both SARS nucleocapsids and spikes, 51(39.2%) had anti-N antibody, and 70 (53.8%) had none of these antibodies in their sera (Table 1).

Previous studies reported that the HLA-B, HLA-Cw, and HLA-DR alleles were highly associated with SARS infection and development (5,17,20). We focused on these alleles to determine their association with SARS infection. Our results showed that HLA-B 4001 (19.4%), 4601 (12.5%), 1301, and 5801 (8.5%); HLA-Cw 0102 (20.6%), 0702 (19.8%), and 0304 (14.1%); and HLA-DR 0901 (16.5%), 1501 (12.9%), and 1202 (8.9%), were highly prevalent in confirmed SARS cases (Table 2). Our subjects included health care workers and community residents in direct or indirect contact with SARS patients via respiratory droplets or contaminated sewage. For this study we chose health care workers as our cohort because (1) the outbreaks mainly occurred in hospitals, and health care workers are usually the front-line workers and the first to come into contact with SARS patients, and (2) their risk factors were easily identified and investigated. We conducted a case-control study among these health care workers to determine the association between HLA and SARS infection.

Fifty-six SARS-confirmed health care workers and 41 SARS-negative health care workers were recruited (these cases were diagnosed by both real-time RT-PCR and WB assays) (Table 3 and Supplementary Table 4; see online supplementary material at http://www.liebertonline.com). Our results showed that HLA-Cw 1502 and DR 0301 were significantly associated with SARS infection or development (p<0.05), and both alleles were associated with resistance to SARS infection (OR 0.17 and 0.3, respectively) (Table 3).

Analysis of the association between HLA type and prolonged neutralizing antibody expression showed that HLA-B 3802 (18.7%, 3/16) and 4601 (18.7%, 3/16); HLA-Cw 0102 (25%, 4/16) and 0702 (18.7%, 3/16); and HLA-DR 0901 (31.3%, 5/16) and 1501 (25%, 4/16) were highly prevalent in prolonged neutralizing antibody expression (Supplementary Table 2; see online supplementary material at http://www.liebertonline.com). However, a low prevalence of HLA Cw1502 and DR 0301 (0% and 6.25%) was seen in these groups (Supplementary Table 2; see online supplementary material at http://www.liebertonline.com).

Analysis of the association of acute respiratory distress syndrome (ARDS) with prolonged neutralizing antibody expression and HLA type showed that 51.8% (14/27) of SARS-seronegative cases developed ARDS, compared to 3.7% (1/27) cases in the prolonged neutralizing antibody groups (Supplementary Table 3; see online supplementary material at http://www.liebertonline.com). HLA-B 4001 (27%, 13/48) and 4601 (8.3%, 4/48); HLA-Cw 0304 (22.9%, 11/48) and 0702 (20.8%, 10/48); and HLA-DR 0901(18.8%, 9/48), 1101, and 1501 (12.5, 6/48) were highly prevalent in ARDS development. In these SARS-confirmed cases, the prevalence of SARS resistance-related HLA-Cw1501 and DR 0301 was lower in those with ARDS development (Supplementary Table 3; see online supplementary material at http://www.liebertonline.com). However, the associations between prolonged antibody expression and HLA type, and between ARDS and HLA type, in all confirmed SARS cases were not statistically significant (p>0.05, data not shown).

After studying the HLA types A, B, DR, and DQ alleles among SARS patients in Hong Kong, Ng et al. (20) reported that the HLA-B 0703 and DRB1 0301 genotypes conferred susceptibility and resistance to the development of SARS, respectively. Our results also indicated that subjects carrying the HLA-DR 0301 genotype conferred resistance against SARS infection. As for HLA-B 0703, none of our subjects carried this genotype (Table 2). The HLA-DR 0301 genotype has also been associated with pulmonary sarcoidosis (12), diabetes (13), and HCV infection and clearance (8).

Previously, Lin et al. (17) indicated that HLA-B 4601 was associated with increased severity of SARS infection in Asian populations. In our study, the prevalence of HLA-B 4601 in our SARS cases (12.5%; 31/248), and controls (10.7%; 12/112), was high. Although no significant association was seen due to the high prevalence of this allele in our control group, we still believe that HLA-B 4601 is an important HLA genotype associated with increased SARS infections.

In this study, the HLA-Cw 1502 genotype was found to be associated with resistance against SARS infection. A recent study (25) indicated that HLA-Cw 1502 was significantly increased among lepromatous leprosy patients, and played vital roles in disease association and pathogenesis. Ovsyannikova et al. (21) indicated that HLA-Cw 1502 was positively associated with lymphoproliferative responses to rubella virus antigens after rubella vaccine inoculation (p=0.035).

The mechanism behind HLA type and SARS pathogenesis remains elusive and needs further investigation. The interactions among HLA-restricted T lymphocytes, B lymphocytes, cytokines, and NK cells contribute to the immune response to viral infections. Effective viral antigen presentation to CD8⁺ and CD4⁺ T cells by HLA class I and II complexes play an important role in the regulation of an optimal immune response against viral infections. Previous studies of the HLA types in HCV and human papillomavirus patients suggested that some HLA types were involved in the host's ability to clear these viruses (3,9,26,27). HLA-DR 0301 was reported to provide protective antiviral effects in enhancing the function of CD4⁺ T-helper cells (9,20). The HLA class II-restricted response is important for immunological control of viral disease progression. HLA-C was reported to serve as a ligand for killer immunoglobulin receptors (KIRs) expressed on NK cells, and induces the cytotoxic CD8+ T-cell (CTL) response (2,10,26). Reports indicated that HLA-Cw 04 and 08 elicited protection against HIV-1 and HTLV-1 infection (2,16). The mechanism may be a result of a stronger HLA-C-restricted NK or CTL response (2). Recent studies reported that HLA-Cw 1502 was associated with the lymphoproliferative and CTL responses to viral antigens (15,21). Taken together, these data suggest that HLA Cw1502 and DR 0301 expression may facilitate viral antigen presentation, and thereby enhance the function or activity of NK and CD8⁺/CD4⁺ T cells during SARS-CoV clearance.

In summary, the HLA system plays an important role in the immune system and is associated with the response to viral infections. The results of this study indicate that some SARS patients still had neutralizing antibodies 3 y after infection, and that the HLA-Cw 1502 and DR 0301 genotypes conferred resistance against SARS infection.