NK cell receptor NKG2C deletion and HLA-E variants are risk factors for severe COVID-19

Patients infected with SARS-CoV-2 may show mild infection or may develop severe coronavirus disease 2019 (COVID-19). In the present study, we investigated whether there is an association between the severity of COVID-19 and the naturally occurring human genetic variants in the natural killer (NK) cell NKG2C receptor (NKG2C wt/del ) and its cellular ligand HLA-E (HLA-E*0101/0103). Both factors are essential components of the NKG2C + NK cell response and important parts of the defence against pulmonary viral infection. NKG2C del and HLA-E*0101 were signicantly overrepresented in hospitalized patients (p=0.0006 and p=0.01, respectively) and particularly in critically ill patients requiring intensive care (p<0.0001 and p=0.01, respectively), compared to patients with mild symptoms or healthy controls. Both genetic variants were found to be independent risk factors for severe COVID-19. The data highlight that specic NKG2C + NK cell responses play an important role against SARS-CoV-2 and that variations thereof may signicantly inuence the severity of disease.


Introduction
Patients infected with SARS-CoV-2 may either show mild or even asymptomatic infection or they may develop severe and potentially lethal coronavirus disease 2019 (COVID-19). So far, several risk factors for severe COVID-19 have been reported, including older age, male gender and co-morbidities, such as diabetes, cardiovascular or respiratory system diseases 1. However, these factors alone do not explain the differences in severity of COVID-19 observed between the individuals. Antiviral host immune responses limit viral infections and in uence the severity of viral diseases. Among these, natural killer (NK) cell responses are of major importance and signi cantly contribute to the early defense against pulmonary virus infections. NK cell activation depends on a panel of germline-encoded activating or inhibitory receptors. NKG2C+ cells are characterized by the elevated expression of the activating CD94/NKG2C receptor 2 and, in response to viral pulmonary infections, a subset of these highly potent NKG2C+ NK cells migrates into the lung 3. The interaction of NKG2C/CD94 and its cellular ligand HLA-E further mediates cytotoxic NK cell responses and the release of pro-in ammatory effector molecules of NKG2C+ cells against virus-infected cells 4.
The activation of NKG2C+ NK-cells varies depending on the presence of different genetic variants of the NKG2C receptor and of HLA-E, which naturally occur in the human population. Homozygous and heterozygous deletion of the NKG2C receptor was recently linked to decreased or absent NKG2C receptor expression 5 and was associated with the severity of speci c virus infections 6. HLA-E occurs in European populations mostly as HLA-E*0101 or HLA-E*0103, which exhibit signi cant differences in cell surface expression levels and peptide binding capacities 7.
In the present study, we therefore investigated whether NKG2C deletion and speci c HLA-E variants are signi cant factors associated with the severity of COVID-19 in the individual host.

Genotyping
Genomic DNA was isolated from respiratory swabs of COVID-19 patients and plasma of controls using NucliSens EasyMag extractor (BioMérieux, Marcy-l'Étoile, France). DNA was eluted in 50μl of nucleasefree H 2 O. HLA-E*0101/0103 genotypes were determined by a Taqman assay and NKG2C wt/del variants were determined by touchdown PCR as described before 6,11 .
Statistical analysis: The distribution of the patient's gender, genetic variants and donor and recipient serostatus was compared by the χ 2 -Test. For multiple comparisons, unpaired ANOVA and the Dunn's post-test were used. For multivariable analysis a general log-linear model with genetic variables, gender and age groups (<60, 60-70, 70-80, >80 years) was used to identify combined genetic variables associated with the risk for severe SARS-CoV-2 infections, which were hospitalized or hospitalized in an ICU. P-values<0.05 were considered signi cant. Statistical analyses were performed using IBM SPSS Statistics 24. The study was approved by the local ethics committee (EK No. 1389/2019).

Characteristics of the Study Cohort
We included a total of 361 patients (median age: 69 years, 45% female), who were con rmed 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 de ned 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 signi cantly older and were more likely to be males compared to non-hospitalized and hospitalized COVID-19 patients. This con rms previous data that male gender and higher age are risk factors for severe COVID-19 1.
Association between NKG2C and HLA-E genetic variants and severe COVID-19 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 signi cantly lower frequency of the NKG2Cwt/wt variant compared to non-hospitalized COVID-19 patients and controls (Fig.1A). Presence of the NKG2Cdel allele was signi cantly 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 signi cantly 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 signi cantly 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 signi cantly and independently to the overall probability for hospitalization without and with intensive care treatment.

Discusssion
Our data provide rst evidence that NKG2C/CD94-driven NK cell response might be a main factor limiting virus infection and severity of COVID-19. We demonstrate that distinct naturally occurring genetic variants in the pathway leading to activation of NKG2C+ cells via the NKG2C/CD94/HLA-E axis may be of high importance for antiviral defence. Limited data are so far available concerning the NK cell response against SARS-CoV-2. Recently it was shown that NK cell counts were signi cantly reduced in patients with severe compared to those with mild COVID-19, respectively 12. In addition, NK cells from patients with severe COVID-19 showed increased expression of the inhibitory NKG2A+ receptor 12, which is associated with functional exhaustion of NK cells. Our data, linking the deletion of the activating NKG2C receptor with severe COVID-19, further underline the important role of a potent antiviral NK-cell response against SARS-CoV-2, and highlight that the NKG2C+ mediated NK cell response may signi cantly contribute to limit SARS-CoV-2 infections and to prevent severe COVID-19.
Epithelial cells of the respiratory tract are main targets of viral entry due to their high-level expression of the cellular ACE2 receptor 13. These cells also show elevated expression of HLA-E on their cell surface and are therefore targets of the NKG2C response 14. It is thus reasonable that the NKG2C-mediated NKcell responses represent an important mechanism of immune defence against SARS-CoV-2 infected cells in the respiratory tract.
Furthermore, recently published in vitro studies demonstrated that cell surface expression levels are lower for HLA-E*0101/0101 than for HLA-E*0103/0103 7, which may result in a delayed or decreased NKG2C+ NK-cell response. Therefore both, deletion of the NKG2C receptor and HLA-E*0101 expression, may provide a disadvantage in antiviral defence.  Distributions of NKG2C (A) or HLA-E (B) variants between control patients and non-hospitalized, hospitalized non-ICU as well as hospitalized ICU COVID-19 patients. Bars represent the relative frequency