Relationship between KIR genotypes and HLA-ligands with SARS-CoV-2 infection in the Saudi population

1 Background

Coronavirus disease 2019 (COVID-19) is an infectious disease which is caused by the most recently detected strain, SARS-CoV-2. This highly contagious infection is associated with severe manifestations such as acute respiratory syndrome, cytokine storms and multisystem inflammatory syndrome (Castagnoli et al., 2020; Salata et al., 2020).

The World Health Organization declared the novel coronavirus as a pandemic in March 2020. On a global scale, by the end of April 2021, over 153,000,000 infected people and over 3,206,000 have been reported (Muhareb and Giacaman, 2020). Since the first Saudi Arabian case was reported in March 2020, the total number of confirmed cases had reached 420,000 by the end of April. Among these, 6992 patients were deceased (COVID19 statistics, Ministry of Health, Kingdom of Saudi Arabia. MOH (2021) May https://covid19.moh.gov.sa).

In the course of viral infections, both innate and adaptive immune cells work in unison to promote an effective immune response. Natural killer (NK) cells are cytotoxic lymphocytes. These have the capacity to identify and eliminate tumor and virus-infected cells without prior sensitization using a panel of receptors that interact with ligands, mainly the Human Leukocyte Antigens (HLA), which are expressed at the surface of target cells (Ahlenstiel et al., 2008; Béziat et al., 2013). The KIR gene family, located on human chromosome 19q13.4, includes 15 functional genes (2DL1-4, 2DL5A, 2DL5B, 2DS1-5, 3DL1-3 and 3DS1), and 2 pseudogenes (2DP1, 3DP1). They are organized within a multi-gene family and characterized by their genetic diversity including gene content, copy number variation and also allelic polymorphism (Yawata et al., 2006). According to the number of external immunoglobulin domains (2D or 3D) genes have been divided into the following distinct groups: the inhibitory KIRs (2DL, 3DL) characterized by the presence of a long cytoplasmatic tail with two immune tyrosine-based inhibitory motifs (ITIM), and activating KIR receptors (2DS, 3DS) allowing the transmission of an inhibitory signal and characterizes by the presence of short cytoplasmatic tails corresponds (Selvakumar et al., 1997; McVicar and Burshtyn, 2001).

The genetic diversity of KIRs includes variations in gene content and copy number in addition to allelic polymorphisms + – (Middleton and Gonzelez, 2010).

These receptors did not undergo somatic modifications and remain at the germinal state. For that, they are placed at the interface between innate and adaptive immunity (Vivier et al., 2011).

Molecules of the HLA (or human major histocompatibility complex; MHC), mainly those of class I, play a crucial role in the control of activation or inhibition of the NK cells killing process through a mechanism of missing or altered self (Ljunggren and Kärre, 1990; Sivori et al., 2019).

Interactions with HLA ligands at the surface of target cells implies some surface receptors at the surface of NK cells (Jamil and Khakoo, 2011a; Pierce et al., 2020). The combination of HLA class-I and KIR receptor could influence NK cells and induce immune tolerance (Cao et al; Xu et al., 2020). Studies have revealed that patients with specific HLA alleles are at risk for viral infections including SARS (Sun and Xi, 2014). The relationship between HLA–KIR and cytokine storm have been detected in different diseases caused by viruses including: Spanish flu (Kobasa et al., 2007), severe influenza virus infections (La et al., 2011), dengue virus (Björkström et al., 2011) and hepatitis C infection (Fitzmaurice et al., 2015). Consequently, the diversity of HLA-KIR and their selection of ligand and pathogen-mediated activity have a strong influence on the efficiency of the immune response and then switch of disease toward clearance or severity explaining the occurrence of high mortality rate (Jamil and Khakoo, 2011b; Jost and Altfeld, 2013). It is worth mentioning that in the Saudi population, the associations between KIRs and some cancer or autoimmune diseases have recently been reported (Al Omar et al., 2015; Alomar et al., 2017; Osman et al., 2017).

Many studies have reported that NK cell frequency can fluctuate with moderate and severe disease in peripheral blood and at the site of infection (Wang et al., 2020). Nevertheless, a comprehensive understanding of the NK cell landscape during the infection with SARS-CoV-2 has not yet been determined. While they were widely explored for their roles in many viral infections, their involvement in the host reaction against SARS-CoV-2, has not been sufficiently investigated (Maucourant et al., 2020).

Due to the major role of NK cells in the early immune response against viruses, in the present study, the relationship between some individual KIR receptors is investigated in point of fact as the most important receptor expressed on NK cells, KIR genotype polymorphism and their corresponding ligand of its combinations with the HLA-G and HLA-C1 and -C2 with Covid-19 infection in Saudi Arabia.

2 Materials and methods

3 Results

The present study is the first to attempt the characterization of the KIR, HLA-C1/C2 and HLA-G 14 bp indel polymorphism patterns and its effects on COVID-19 infection among the Saudi population. In this study, 87 COVID-19 positive patients and 114 healthy controls were successfully genotyped for 16 KIR genes, HLA-C1/C2 allotypes and HLA-G 14 bp insertion deletion using sequence-specific PCR amplification method. All framework genes were detected in both patients and control groups. A significant increase of 2DL1 and 2DS4 genes among patients was observed (OR = 12.07; P = 0.0022) and (OR = 10.15; P = 0.0076) respectively. These genes occur in all patients and with lower frequency in healthy control. For the rest of genes, no significant differences between groups were detected (Table 1).

In this study, the frequency of HLA-C allotypes, which are the main ligands of KIR receptors, were also examined. The two main allotypes, HLA-C1 and -C2 frequency were determined for both COVID-19 patients and healthy controls. Distributions of HLA-C1C1 genotypes in control ad patients show slightly deviated from HWE (χ2 = 5.48, p = 0.019). Table 2, shows the relationship between HLA-C1C2 allotypes and genotype for codominant, dominant, recessive, over-dominant and additive genetic models with SARS-CoV-2. Significant association were observed for C1C1 genotype in codominant model and the protective effect against SARS-Cov-2 (OR-0.36; P = 0.024 ). A negative relationship with the disease were observed in the dominant model C1C2 + C1C1 (OR = 0.43; P = 0.0099) and for the Log additive model (OR = 0.61; P = 0.0082). According to AIC, the dominant models could the best fitted one (AIC = 262.4). The allotype C2 was more frequent in patients than in control and could be associated with the bad outcome of the disease (OR = 1.79; P = 0.0047).

Concerning the 14 bp indel polymorphism in the 3′UTR of the HLA-G gene, no significant difference for any genotype was observed between the two groups for all the tested models (Table 3). Noting that for this polymorphism, the genotypes are in HWE (P > 0.05).

In order to study the prevalence of KIR gene in a context of their HLA-C1/C2 ligands, a combinatory analysis was performed considering both KIR receptor and ligand. Taking into account the specific ligand yet reported for some KIR receptors, 15 combinatory analyses for five KIR receptors (3 combination for each) were performed.

The frequency of KIR2DL1-HLA-C2 was significantly higher in patients with COVID-19 (80.5 % versus 66.7 %; P = 0.037; OR = 2.058). A slightly higher protective effect was observed for people sharing KIR2DL2 genes with homozygous C2C2 (Table 4).

4 Discussion and conclusion

The involvement of Killer Immunoglobulin-like Receptor (KIR) polymorphisms and the Human Immune response against viral infection has been reported by a significant number of researchers (Wang et al., 2018; Chaisri et al., 2019; van Erp et al., 2019).

The mechanism by which these NK cells react against SARS-CoV2 could be comparable to that reported against other common viruses. To achieve its antiviral action, NK cells involve extraordinarily diverse interactions between the family of KIR and MHC class I molecules (Vivier et al., 2008; Boyton and Altmann, 2007). HLA-A, -B, and -C molecules are the most important molecules which are reported to interact with KIR receptors. Additionally, they impact on the diversity and individualized human immune response between individuals (Wilson et al., 2000; González-Galarza Faviel et al., 2014).

In the present study, for the first time, the relationship between KIR genotypic polymorphism and their cognate ligand HLA-C1 and -C2 ligands with COVI-19 infection in Saudi Arabia has been reported. We did not find a strong association of KIR genes when considered each KIR gene, except for 2DL1 and 2DS4 which were significantly increased in patients with COVID-19. These results suggest that inhibitory 2DL1 and the activating 2DS4 might be associated with the outcome of patients with COVID-19. However, this association should be retained take with great care because of the extended confidence interval range. Also, negative association between HLA-C1/C1 genotypes and COVID-19 were identified specially in codominant, dominant and Log additive models. This association should be taken with great care specially because of the slightly deviation from the HW equilibrium for this polymorphism. A large number of samples from other Saudi subpopulation should be considered later to confirm such association.

Conversely, a clear association was not identified between genotypes of the HLA-G 14 bp indel polymorphism and the explored disease.

Considering the interaction between some KIR receptors and their specific HLA-C allotype ligand, a significant positive association could be reported for the 2DL1 gene with its C2 ligand with infection. It is worth noting that 2DL1 was initially suspected to be positively associated with the infection. The associated was confirmed when considering its ligand at homozygote status. Overall, the study should be confirmed and consolidated with a use of a large and well-characterized cohort. This could facilitate the stratification of the patients and the identification of the effects of KIR in association with the clinical outcome of the SARS-CoV2 infected Saudi population.

In conclusion, KIR2DL1, and KIR2DS4, and the combination of 2DL1 + HLA-C2 + are associated with either susceptibility to be infected with SARS-CoV2 or protection in the Saudi population.

A full examination of the frequency, phenotype, and function of NK cells in COVI-19 patients is required in order to identify the precise mechanism of this association in the Saudi population.