Association of SARS-COV-2 viral RNAemia, IL- 6 gene polymorphism, serum IL-6 and peripheral blood lymphocytes and monocytes with disease severity in COVID-19 patients

Coronavirus disease 2019 (COVID-19) is a fatal pandemic viral disease caused by the severe acute respiratory syndrome corona virus type-2 (SARS-CoV-2). The aim of this study is to observe the associations of IL-6, SARS-COV-2 viral load (RNAemia), IL- 6 gene polymorphism and lymphocytes and monocytes in peripheral blood with disease severity in COVID-19 patients. This study was carried out from March 2021 to January 2022. RT-PCR positive 84 COVID-19 patients and 28 healthy subjects were enrolled. Blood was collected to detect SARS-COV-2 viral RNA (RNAemia) by rRT-PCR, serum IL-6 level by chemiluminescence method, SNPs of IL-6 by SSP-PCR, immunophenotyping of lymphocytes and monocyte by flow cytometry. Serum IL-6 level (pg/ml) was considerably high among critical patients (102.02 +/- 149.7) compared to severe (67.20 +/- 129.5) and moderate patients (47.04 +/- 106.5) and healthy controls (3.5 +/- 1.8). Serum SARS-CoV-2 nucleic acid positive cases detected mostly in critical patients (39.28%) and was correlated with extremely high IL-6 level and high mortality (R =.912, P < 0.001). Correlation between IL-6 and monocyte was statistically significant with disease severity (severe group, p < 0.001, and 0.867*** and critical group p < 0.001 and 0.887***). In healthy controls, moderate, severe and critically ill COVID-19 patients, IL-6 174G/C (rs 1800795) GG genotype was 82.14%, 89.20%, 67.85% and 53.57% respectively. CC and GC genotype had strong association with severity of COVID-19 when compared with GG genotype. Significant statistical difference found in genotypes between critical and moderate groups (p < 0.001, OR-10.316, CI-3.22-23.86), where CC genotype was associated with COVID-19 severity and mortality. The absolute count of T cell, B cell, NK cell, CD4+ T cells and CD8+ T cells were significantly decreased in critical group compared to healthy, moderate and severe group (P < 0.001). Exhaustion marker CD94/NKG2A was increased on NK cells and CD8+ cytotoxic T cell among critical and severe group. Absolute count of monocyte was significantly increased in critical group (P < 0.001). Serum IL-6, IL-6 174 G/C gene and SARS-CoV-2 RNAaemia can be used in clinical practice for risk assessment;T cell subsets and monocyte as biomarkers for monitoring COVID-19 severity. Monoclonal antibody targeting IL-6 receptor and NKG2A for therapeutics may prevent disease progression and decrease morbidity and mortality.Copyright © 2023 Elsevier Inc.

SIGLEC-9 RESTRAINS ANTIBODY-DEPENDENT NK CELL CYTOTOXICITY AGAINST SARS-CoV-2

Background: SARS CoV 2 infection alters the immunological profiles of natural killer (NK) cells. However, whether NK anti-viral functions (direct cytotoxicity and/or antibody-dependent cell cytotoxicity (ADCC)) are impaired during severe COVID-19 and what host factors modulate these functions remain unclear. Method(s): Using functional assays, we examined the ability of NK cells from SARS-CoV-2 negative controls (n=12), mild COVID-19 patients (n=26), and hospitalized COVID-19 patients (n=41) to elicit direct cytotoxicity and ADCC [NK degranulation by flow] against cells expressing SARS-CoV-2 antigens. SARS-CoV- 2 N antigen plasma load was measured using an ultra-sensitive Simoa assay. We also phenotypically characterized the baseline expression of NK activating (CD16 and NKG2C), maturation (CD57), and inhibitory (NKG2A and the glyco-immune negative checkpoint Siglec-9) by flow cytometry. Finally, an anti-Siglec-9 blocking antibody was used to examine the impact of Siglec-9 expression on anti-SARS-CoV-2-specific ADCC [degranulation and target cell lysis]. Result(s): NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2-antigen-expressing cells (in direct cytolytic and ADCC assays) than did cells from mild COVID-19 patients or negative controls (Fig. 1A). The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 N-antigen (P<=0.02). Phenotypic and functional analyses showed that NK cells expressing Siglec-9 elicited higher ADCC than Siglec-9- NK cells (P<0.05;Fig. 1B). Consistently, Siglec-9+ NK cells expressed an activated and mature phenotype with higher expression of CD16, CD57, and NKG2C, and lower expression of NKG2A, than Siglec-9- NK cells (P<=0.03). These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells (P<=0.05;Fig. 1C). Conclusion(s): These data support a model (Fig. 1D) in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity can further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. (Figure Presented).

HLA-E*01:01 + HLA-E*01:01 genotype confers less susceptibility to COVID-19, while HLA-E*01:03 + HLA-E*01:03 genotype is associated with more severe disease.

BACKGROUND: HLA-E interaction with inhibitory receptor, NKG2A attenuates NK-mediated cytotoxicity. NKG2A overexpression by SARS-CoV-2 exhausts NK cells function, whereas virus-induced down-regulation of MHC-Ia reduces its derived-leader sequence peptide levels required for proper binding of HLA-E to NKG2A. This leads HLA-E to become more complex with viral antigens and delivers them to CD8+ T cells, which facilitates cytolysis of infected cells. Now, the fact that alleles of HLA-E have different levels of expression and affinity for MHC Ia-derived peptide raises the question of whether HLA-E polymorphisms affect susceptibility to COVID-19 or its severity. METHODS: 104 COVID-19 convalescent plasma donors with/without history of hospitalization and 18 blood donors with asymptomatic COVID-19, all were positive for anti-SARS-CoV-2 IgG antibody as well as a group of healthy control including 68 blood donors with negative antibody were subjected to HLA-E genotyping. As a privilege, individuals hadn't been vaccinated against COVID-19 and therefore naturally exposed to the SARS-CoV-2. RESULTS: The absence of HLA-E*01:03 allele significantly decreases the odds of susceptibility to SARS-CoV-2 infection [p = 0.044; OR (95 %CI) = 0.530 (0.286 - 0.983)], suggesting that HLA-E*01:01 + HLA-E*01:01 genotype favors more protection against SARS-CoV-2 infection. HLA-E*01:03 + HLA-E*01:03 genotype was also significantly associated with more severe COVID-19 [p = 0.020; 2.606 (1.163 - 5.844) CONCLUSION: Here, our observation about lower susceptibility of HLA-E*01:01 + HLA-E*01:01 genotype to COVID-19 could be clinical evidence in support of some previous studies suggesting that the lower affinity of HLA-E*01:01 to peptides derived from the leader sequence of MHC class Ia may instead shift its binding to virus-derived peptides, which then facilitates target recognition by restricted conventional CD8+ T cells and leads to efficient cytolysis. On the other hand, according to other studies, less reactivity of HLA-E*01:01 with NKG2A abrogates NK cells or T cells inhibition, which may also lead to a greater cytotoxicity against SARS-CoV-2 infected cells compared to HLA-E*01:03. Taken together given HLA-E polymorphisms, the data presented here may be useful in identifying more vulnerable individuals to COVID-19 for better care and management. Especially since along with other risk factors in patients, having HLA-E*01:03 + HLA-E*01:03 genotype may also be associated with the possibility of severe cases of the disease.

Implications of NKG2A in immunity and immune-mediated diseases.

In recent studies, NKG2A is revealed to be a key immune checkpoint for both natural killer (NK) cells and CD8+ T cells. It form heterodimer receptors with CD94, and targets the peptide-presenting human leukocyte antigen-E (HLA-E) molecules. Upon crosslinking, NKG2A/CD94 delivers inhibitory signals for NK cells and CD8+ T cells, while blocking NKG2A can effectively unleash functions of these cytotoxic lymphocytes. The interaction between NKG2A and HLA-E contributes to tumor immune escape, and NKG2A-mediated mechanisms are currently being exploited to develop potential antitumor therapeutic strategies. In addition, growing evidence shows that NKG2A also plays important roles in other immune-related diseases including viral infections, autoimmune diseases, inflammatory diseases, parasite infections and transplant rejection. Therefore, the current work focuses on describing the effect of NKG2A on immune regulation and exploring its potential role in immune-mediated disorders.

NKG2A Expression among CD8 Cells Is Associated with COVID-19 Progression in Hypertensive Patients: Insights from the BRACE CORONA Randomized Trial.

Cardiovascular comorbidities and immune-response dysregulation are associated with COVID-19 severity. We aimed to explore the key immune cell profile and understand its association with disease progression in 156 patients with hypertension that were hospitalized due to COVID-19. The primary outcome was progression to severe disease. The probability of progression to severe disease was estimated using a logistic regression model that included clinical variables and immune cell subsets associated with the primary outcome. Obesity; diabetes; oxygen saturation; lung involvement on computed tomography (CT) examination; the C-reactive protein concentration; total lymphocyte count; proportions of CD4+ and CD8+ T cells; CD4/CD8 ratio; CD8+ HLA-DR MFI; and CD8+ NKG2A MFI on admission were all associated with progression to severe COVID-19. This study demonstrated that increased CD8+ NKG2A MFI at hospital admission, in combination with some clinical variables, is associated with a high risk of COVID-19 progression in hypertensive patients. These findings reinforce the hypothesis of the functional exhaustion of T cells with the increased expression of NKG2A in patients with severe COVID-19, elucidating how severe acute respiratory syndrome coronavirus 2 infection may break down the innate antiviral immune response at an early stage of the disease, with future potential therapeutic implications.

IMMUNE SYSTEM PROFILE and CYTOKINE LEVELS of SARS-CoV-2 in PREGNANCY and NEWBORNS

Background: Knowledge about SARS-CoV2 infection in pregnancy and exposed newborns is deficient. We performed a longitudinal analysis of innate immune system status and determined soluble cytokines of women infected with SARS-CoV2 during pregnancy and their newborns Methods: Women with confirmed SARS-CoV2 infection (RT-PCR+ or SARS-CoV2 anti-IgM/IgG+) (COVID MOTHER group, CM n=29, median age of 31 years) and their SARS-CoV2 exposed uninfected newborns were recruited from Hospital Gregorio Marañón, Spain. Peripheral blood mononuclear cells (PBMCs), cord cells and plasma were collected at birth and 6 months later (n=15). The immunophenotyping of innate components (natural killer cells [NK] and monocytes) was studied on cryopreserved PBMCs and cord cells by multiparametric flow cytometry. Up to 4 soluble pro/anti-inflammatory cytokines were assessed in plasma and cord plasma by ELISA assay. CM was compared to a healthy non-SARS-CoV2 infected mothers' group matched by age (SARS-CoV2 PCR-and SARS-CoV2 anti-IgM/IgG-)(Uninfected Mothers, UM n=16) and their newborns (n=16) Results: On NK cell assays, CM show at baseline lower percentage of CD16++ subset, higher NKG2D and lower NKG2A expression on CD16++ and CD56++ subsets and reduced CD57 expression compared to UM;proportion of CD16++ subset and percentage of NKG2D reverted after 6 months(A). Regarding monocytes, CM show increased levels of CD62L and decreased CD49d expression on classical subset, elevated intermediate monocytes proportion and decreased CD40 expression on patrolling subset(B). No differences were found 6 months later. No newborn was infected by SARS-CoV2 and the phenotype analyzed on cord cells shows lower frequency of NK subsets compared with unexposed children and increased CD16++ subset after 6 months(C). In monocytes distribution, exposed children present lower frequency of total monocytes and its subsets than unexposed. Classical monocytes show significant changes at follow-up time-point(D). Increased TNFα and IL10 levels were found on CM compared to UM. Strong and direct correlations were observed between the age and IL6(E). No differences were observed in soluble cytokine levels comparing both groups of newborns Conclusion: SARS-CoV2 infection during pregnancy shows differences in activation, maturation and endothelial markers on innate immune system that could lead newborns clinical implications at birth. However, altered cell proportions and phenotypes found at SARS-CoV2 at birth time and on their exposed newborns is later reverted.

Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19.

The kinetics of NKG2C+ adaptive natural killer (ANK) cells and NKG2A+inhibitory NK (iNK) cells with respect to the incidence of SARS-CoV-2 infection were studied for 6 months in a cohort of healthcare workers following the administration of the heat-killed Mycobacterium w (Mw group) in comparison to a control group. In both groups, corona virus disease 2019 (COVID-19) correlated with lower NKG2C+ANK cells at baseline. There was a significant upregulation of NKG2C expression and IFN-γ release in the Mw group (p=0.0009), particularly in those with a lower baseline NKG2C expression, along with the downregulation of iNK cells (p<0.0001). This translated to a significant reduction in the incidence and severity of COVID-19 in the Mw group (incidence risk ratio-0.15, p=0.0004). RNA-seq analysis at 6 months showed an upregulation of the ANK pathway genes and an enhanced ANK-mediated antibody-dependent cellular cytotoxicity (ADCC) signature. Thus, Mw was observed to have a salutary impact on the ANK cell profile and a long-term upregulation of ANK-ADCC pathways, which could have provided protection against COVID-19 in a non-immune high-risk population.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

Puzzling out the COVID-19: Therapy targeting HLA-G and HLA-E.

SARS-CoV2 might conduce to rapid respiratory complications challenging healthcare systems worldwide. Immunological mechanisms associated to SARS-CoV2 infection are complex and not yet clearly elucidated. Arguments are in favour of a well host-adapted virus. Here I draw a systemic immunological representation linking actual SARS-CoV2 infection literature that hopefully might guide healthcare decisions to treat COVID-19. I suggest HLA-G and HLA-E, non classical HLA class I molecules, in the core of COVID-19 complications. These molecules are powerful in immune tolerance and might inhibit/suppress immune cells functions during SARS-CoV2 infection promoting virus subversion. Dosing soluble forms of these molecules in COVID-19 patients' plasma might help the identification of critical cases. I recommend also developing new SARS-CoV2 therapies based on the use of HLA-G and HLA-E or their specific receptors antibodies in combination with FDA approved therapeutics to combat efficiently COVID-19.

SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway.

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

Consideration of Severe Coronavirus Disease 2019 As Viral Sepsis and Potential Use of Immune Checkpoint Inhibitors.

Taking into consideration the multisystemic clinical and autopsy findings in "severe" coronavirus disease 2019 patients, viral sepsis would be a more accurate term to describe the whole clinical picture. The most significant pathophysiological components of this picture are intense cytokine release, prolonged inflammation, immunosuppression with T cell exhaustion, and the development of organ dysfunctions. Currently, the optimal treatment for severe coronavirus disease 2019 is uncertain. Supportive treatment and immunomodulators have a critical place in the treatment of severe patients until effective antivirals are developed. Interleukin-6 antagonists, one of the immunomodulating agents, appears to be effective in the treatment of cytokine storm, but some patients continue to have severe lymphopenia and immunosuppression. We believe it can be useful as immunomodulator therapy in critical coronavirus disease 2019 patients because of the benefits of immune checkpoint inhibitors in cancer and sepsis patients.

Innate immunity in COVID-19 patients mediated by NKG2A receptors, and potential treatment using Monalizumab, Cholroquine, and antiviral agents.

Following the outbreak of a novel coronavirus (SARS-CoV-2), studies suggest that the resultant disease (COVID-19) is more severe in individuals with a weakened immune system. Cytotoxic T-cells (CTLs) and Natural Killer (NK) cells are required to generate an effective immune response against viruses, functional exhaustion of which enables disease progression. Patients with severe COVID-19 present significantly lower lymphocyte, and higher neutrophil, counts in blood. Specifically, CD8+ lymphocytes and NK cells were significantly reduced in cases of severe infection compared to patients with mild infection and healthy individuals. The NK group 2 member A (NKG2A) receptor transduces inhibitory signalling, suppressing NK cytokine secretion and cytotoxicity. Overexpression of NKG2A has been observed on CD8+ and NK cells of COVID-19 infected patients compared to healthy controls, while NKG2A overexpression also functionally exhausts CD8+ cells and NK cells, resulting in a severely compromised innate immune response. Blocking NKG2A on CD8+ cells and NK cells in cancers modulated tumor growth, restoring CD8+ T and NK cell function. A recently proposed mechanism via which SARS-CoV-2 overrides innate immune response of the host is by over-expressing NKG2A on CD+ T and NK cells, culminating in functional exhaustion of the immune response against the viral pathogen. Monalizumab is an inhibiting antibody against NKG2A which can restore the function of CD8 + T and NK cells in cancers, successfully ceasing tumor progression with no significant side effects in Phase 2 clinical trials. We hypothesize that patients with severe COVID-19 have a severely compromised innate immune response and could be treated via the use of Monalizumab, interferon α, chloroquine, and other antiviral agents.