We included a total of 361 patients (median age: 69 years, 45% female), who were confirmed positive for SARS-CoV–2 by PCR from respiratory swabs 8 between the 17th February and 17th April 2020 at the Center of Virology, Medical University of Vienna. A total of 92/361 (25.5%) patients showed only minor symptoms and stayed in home quarantine (‘non-hospitalized’), 190/361 (52.6%) patients were hospitalized with severe COVID–19 symptoms but never required intensive care (‘hospitalized, non-ICU’), and 79/361 (21.9%) patients were severely affected and needed intensive care as defined earlier 9 (‘hospitalized-ICU’). In addition, we included 100 healthy control individuals (median age: 63 years, 50% female), from whom plasma samples were obtained at the Center for Virology between 2014–2016.
Patient characteristics are presented in Table 1. As shown, COVID–19 patients requiring ICU treatment were significantly older and were more likely to be males compared to non- hospitalized and hospitalized COVID–19 patients. This confirms previous data that male gender and higher age are risk factors for severe COVID–19 1.
From SARS-CoV–2 infected patients and controls, NKG2Cwt/del variants were determined by touchdown-PCR as described before 6. Overall, 65% of control persons encoded for the NKG2Cwt/wt variant, 33% for the NKG2Cwt/del and 2% for the NKG2Cdel/del variant. This is consistent with the NKG2C variant distribution in previously published European cohorts 10.
Among the COVID–19 patients in this study, hospitalized patients and patients requiring intensive care showed a significantly lower frequency of the NKG2Cwt/wt variant compared to non-hospitalized COVID–19 patients and controls (Fig.1A). Presence of the NKG2Cdel allele was significantly associated with severe COVID–19 requiring hospitalization and with hospitalization at ICU (p = 0.0006 and p<0.0001, respectively, χ2-Test).
We further analyzed the patients’ HLA-E*0101/0103 variants by a recently published Taqman assay 11. Overall, 33% and 35% of the controls were homozygous for the HLA-E*0101/0101 and HLA-E*0103/0103, respectively, while 32% showed the heterozygous HLA-E*0101/0103 variant. This distribution is similar to that shown in another European study cohort 11. In Fig.1B, the distribution of the HLA-E variant between controls and the different patient groups is shown. Hospitalized and ICU patients more often carried the HLA-E*0101/0101 and HLA- E*0101/0103 variants, compared to patients with mild COVID–19. The presence of an HLA- E*0101 allele was significantly associated with hospitalization and hospitalization in ICU (both: p = 0.01, χ2-Test).
We then analysed the association between combined HLA-E and NKG2C variants and COVID-19 severity. As shown in Figure 1C, the presence of combined NKG2Cwt/wt and HLA- E*0103/0103 alleles was significantly underrepresented in the hospitalized and ICU COVID–19 patients, and the combination of the hetero- and homozygous NKG2Cdel and HLA-E*0101 variants was associated with a higher risk for severe COVID–19 (both: p<0.0001, χ2-Test). We further performed a multi-variable analysis including the presence of the individual NKG2C alleles, HLA-E alleles, as well as age and gender of the different patient groups. Age >60 years (p>0.001 and p = 0.001, respectively), NKG2Cdel (p = 0.001 and p>0.001, respectively) and HLA- E*0101 (p = 0.006 and p = 0.006, respectively) alleles contributed significantly and independently to the overall probability for hospitalization without and with intensive care treatment.