Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort.

The clinical presentations of COVID-19 may range from an asymptomatic or mild infection to a critical or fatal disease. Several host factors such as elderly age, male gender, and previous comorbidities seem to be involved in the most severe outcomes, but also an impaired immune response that causes a hyperinflammatory state but is unable to clear the infection. In order to get further understanding about this impaired immune response, we aimed to determine the association of specific HLA alleles with different clinical presentations of COVID-19. Therefore, we analyzed HLA Class I and II, as well as KIR gene sequences, in 72 individuals with Spanish Mediterranean Caucasian ethnicity who presented mild, severe, or critical COVID-19, according to their clinical characteristics and management. This cohort was recruited in Madrid (Spain) during the first and second pandemic waves between April and October 2020. There were no significant differences in HLA-A or HLA-B alleles among groups. However, despite the small sample size, we found that HLA-C alleles from group C1 HLA-C*08:02, -C*12:03, or -C*16:01 were more frequently associated in individuals with mild COVID-19 (43.8%) than in individuals with severe (8.3%; p = 0.0030; pc = 0.033) and critical (16.1%; p = 0.0014; pc = 0.0154) disease. C1 alleles are supposed to be highly efficient to present peptides to T cells, and HLA-C*12:03 may present a high number of verified epitopes from abundant SARS-CoV-2 proteins M, N, and S, thereby being allegedly able to trigger an efficient antiviral response. On the contrary, C2 alleles are usually poorly expressed on the cell surface due to low association with ß2-microglobulin (ß2M) and peptides, which may impede the adequate formation of stable HLA-C/ß2M/peptide heterotrimers. Consequently, this pilot study described significant differences in the presence of specific HLA-C1 alleles in individuals with different clinical presentations of COVID-19, thereby suggesting that HLA haplotyping could be valuable to get further understanding in the underlying mechanisms of the impaired immune response during critical COVID-19.

Changes in the immune response against SARS-CoV-2 in individuals with severe COVID-19 treated with high dose of vitamin D.

Main cause of severe illness and death in COVID-19 patients appears to be an excessive but ineffectual inflammatory immune response that may cause severe acute respiratory distress syndrome (ARDS). Vitamin D may favour an anti-inflammatory environment and improve cytotoxic response against some infectious diseases. A multicenter, single-blind, prospective, randomized clinical trial was approved in patients with COVID-19 pneumonia and levels of 25-hydroxyvitamin D (25(OH)D) of 14.8 ng/ml (SD: 6.18) to test antiviral efficacy, tolerance and safety of 10,000 IU/day of cholecalciferol (vitamin D3) for 14 days, in comparison with 2000 IU/day. After supplementation, mean serum 25(OH)D levels increased to 19 ng/ml on average in 2000 IU/day versus 29 ng/ml in 10,000 IU/day group (p < 0.0001). Although levels of inflammatory cytokines were not modified by treatment with 10,000 IU/day, there was an increase of anti-inflammatory cytokine IL-10 and higher levels of CD4+ T cells, with predominance of T central memory subpopulation. Cytotoxic response against pseudotyped SARS-CoV-2 infected cells was increased more than 4-fold in patients who received 10,000 IU/day. Moreover, levels of IFNγ were significantly higher in this group. Beneficial effect of supplementation with 10,000 IU/day was also observed in participants who developed ARDS and stayed at the hospital for 8.0 days, whereas those who received 2000 IU/day stayed for 29.2 days (p = 0.0381). Administration of high doses of vitamin D3 as adjuvant of the standard care treatment during hospitalization for COVID-19 may improve the inflammatory environment and cytotoxic response against pseudotyped SARS-CoV-2 infected cells, shortening the hospital stay and, possibly, improving the prognosis.

Persistent Overactive Cytotoxic Immune Response in a Spanish Cohort of Individuals With Long-COVID: Identification of Diagnostic Biomarkers.

Long-COVID is a new emerging syndrome worldwide that is characterized by the persistence of unresolved signs and symptoms of COVID-19 more than 4 weeks after the infection and even after more than 12 weeks. The underlying mechanisms for Long-COVID are still undefined, but a sustained inflammatory response caused by the persistence of SARS-CoV-2 in organ and tissue sanctuaries or resemblance with an autoimmune disease are within the most considered hypotheses. In this study, we analyzed the usefulness of several demographic, clinical, and immunological parameters as diagnostic biomarkers of Long-COVID in one cohort of Spanish individuals who presented signs and symptoms of this syndrome after 49 weeks post-infection, in comparison with individuals who recovered completely in the first 12 weeks after the infection. We determined that individuals with Long-COVID showed significantly increased levels of functional memory cells with high antiviral cytotoxic activity such as CD8+ TEMRA cells, CD8±TCRγδ+ cells, and NK cells with CD56+CD57+NKG2C+ phenotype. The persistence of these long-lasting cytotoxic populations was supported by enhanced levels of CD4+ Tregs and the expression of the exhaustion marker PD-1 on the surface of CD3+ T lymphocytes. With the use of these immune parameters and significant clinical features such as lethargy, pleuritic chest pain, and dermatological injuries, as well as demographic factors such as female gender and O+ blood type, a Random Forest algorithm predicted the assignment of the participants in the Long-COVID group with 100% accuracy. The definition of the most accurate diagnostic biomarkers could be helpful to detect the development of Long-COVID and to improve the clinical management of these patients.

Impaired Antibody-Dependent Cellular Cytotoxicity in a Spanish Cohort of Patients With COVID-19 Admitted to the ICU.

SARS-CoV-2 infection causes COVID-19, ranging from mild to critical disease in symptomatic subjects. It is essential to better understand the immunologic responses occurring in patients with the most severe outcomes. In this study, parameters related to the humoral immune response elicited against SARS-CoV-2 were analysed in 61 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centres in Madrid, Spain, during the first pandemic peak between April and June 2020. Subjects were allocated as mild patients without hospitalization, severe patients hospitalized or critical patients requiring ICU assistance. Critical patients showed significantly enhanced levels of B cells with memory and plasmablast phenotypes, as well as higher levels of antibodies against SARS-CoV-2 with neutralization ability, which were particularly increased in male gender. Despite all this, antibody-dependent cell-mediated cytotoxicity was defective in these individuals. Besides, patients with critical COVID-19 also showed increased IgG levels against herpesvirus such as CMV, EBV, HSV-1 and VZV, as well as detectable CMV and EBV viremia in plasma. Altogether, these results suggest an enhanced but ineffectual immune response in patients with critical COVID-19 that allowed latent herpesvirus reactivation. These findings should be considered during the clinical management of these patients due to the potential contribution to the most severe disease during SARS-CoV-2 infection.

Impaired Cytotoxic Response in PBMCs From Patients With COVID-19 Admitted to the ICU: Biomarkers to Predict Disease Severity.

Infection by novel coronavirus SARS-CoV-2 causes different presentations of COVID-19 and some patients may progress to a critical, fatal form of the disease that requires their admission to ICU and invasive mechanical ventilation. In order to predict in advance which patients could be more susceptible to develop a critical form of COVID-19, it is essential to define the most adequate biomarkers. In this study, we analyzed several parameters related to the cellular immune response in blood samples from 109 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centers in Madrid, Spain, during the first pandemic peak between April and June 2020. Hospitalized patients with the most severe forms of COVID-19 showed a potent inflammatory response that was not translated into an efficient immune response. Despite the high levels of effector cytotoxic cell populations such as NK, NKT and CD8+ T cells, they displayed immune exhaustion markers and poor cytotoxic functionality against target cells infected with pseudotyped SARS-CoV-2 or cells lacking MHC class I molecules. Moreover, patients with critical COVID-19 showed low levels of the highly cytotoxic TCRγδ+ CD8+ T cell subpopulation. Conversely, CD4 count was greatly reduced in association to high levels of Tregs, low plasma IL-2 and impaired Th1 differentiation. The relative importance of these immunological parameters to predict COVID-19 severity was analyzed by Random Forest algorithm and we concluded that the most important features were related to an efficient cytotoxic response. Therefore, efforts to fight against SARS-CoV-2 infection should be focused not only to decrease the disproportionate inflammatory response, but also to elicit an efficient cytotoxic response against the infected cells and to reduce viral replication.

Role of ACE2 genetic polymorphisms in susceptibility to SARS-CoV-2 among highly exposed but non infected healthcare workers.

We aim to evaluate the role of single-nucleotide polymorphisms of the angiotensin-converting enzyme 2 in susceptibility to SARS-CoV-2 infection. We included 28 uninfected but highly exposed healthcare workers and 39 hospitalized patients with COVID-19. Thirty-five SNPs were rationally selected. Two variants were associated with increased risk of being susceptible to SARS-CoV-2: the minor A allele in the rs2106806 variant (OR 3.75 [95% CI 1.23-11.43]) and the minor T allele in the rs6629110 variant (OR 3.39 [95% CI 1.09-10.56]). Evaluating the role of genetic variants in susceptibility to SARS-CoV-2 infection could help identify more vulnerable individuals and suggest potential drug targets for COVID-19 patients.