Association between HLA and SARSCOV- 2 infection in Mexican Mestizos

The Acute Respiratory Syndrome caused by the new coronavirus described in Wuhan, China in 2019 is a viral, respiratory multifactorial infectious disease, which presents different stages depending on genetic and environmental factors that influence severity. As December 19, 2022, 653,192,573 COVID-19 cases worldwide and over six million deaths;330,795 occurred in Mexico, were reported. Our aim was to analyze the contribution of HLA in Mexican patients infected with COVID-19, categorized in different clinical subgroups. A total of 114 COVID-19 patients and 164 healthy controls, all of them Mexican Mestizos from the highlands, were included in the study;RNA columns were used for extraction, and real-time PCR method was performed for the virus identification. DNA was isolated with the Maxwell16 system and 11 HLA loci were typed using NGS (CareDx, Immucor, and One Lambda). The subjects included: 22 asymptomatic, 86 symptomatic and 109 who were previously vaccinated. We compared controls versus positive patients;versus symptomatic;vaccinated negative versus vaccinated positive;controls versus asymptomatic;asymptomatic versus symptomatic individuals. The significant high risk alleles were A*29:02 (OR = 3.95), B*45:01 (OR = 6.92), C*03:04 (OR = 2.24). DPB1*03:01(OR = 3.17) is a susceptibility marker in vaccinated and unvaccinated patients. The latter is prevalent in Hispanics, Russia, Finland, Spain and the United Kingdom. DQA1*02:01 (p = 0.009, OR = 1.96;DQB1*02:02 (p = 0.009, OR = 2.13) was a susceptibility marker in infected patients who were vaccinated. This is prevalent in Argentina, Brazil, Algeria, Australia, Canada, and China, while high-risk B*45:01 and C*03:04 are prevalent in India, Israel, Eastern Europe, and Mediterranean countries. Protective alleles where DRB1*04:01, A*02:01, DQB1*03:01 and DPB1*02:01. These data are relevant to prioritize vaccination, according to the HLA profile in Mexicans, therefore these data are relevant for the epidemiology of COVID-19.

Immunogenetics and SARS-CoV-2 infection

Since the beginning of the SARS-Cov-2 pandemic, in 2020, numerous data with respect to the influence of immunogenetics to the predisposition and infection severity have been reported worldwide. It is well accepted that immunogenetics plays a pivotal role in infection and vaccination, as well as vaccination failures and/or breakthrough. Factors of the major histocompatibility complex and the common ABO blood group system have been so far discussed. Here, we describe the association of HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, -DPB1, and HLA-E, -F, -G, -H on the results of molecular detection of COVID-19 or in some cases on antibody detection upon first testing. Furthermore, we defined molecularly 22 blood group systems comprising 26 genes and 5 platelet antigen genes. We observed 37% COVID-19 PCR negative individuals and 63% positive. Within the negative subjects HLA-B*57:01, HLA-B*55:01, DRB1*13:01, DRB1*01:01, were enriched, and in the positive group homozygosity for DQA1/DQB1, DRB1*09:01 and DRB1*15:01. For HLA-DQA1 we observe an enrichment for DQA1*01:01, DQA1*02:01 and DQA1*01:03. For HLADQB1 we found HLA-DQB1*06:02 was enriched in the positive group while HLA-DQB1*05:01 and HLA-DQB1*06:03 in the negative group. We observed a significant enrichment of homozygosity for DQA1/DQB1 in the positive group. The homozygous platelet antigen HPA-1a was significantly enriched in the negative group, contrasting the result of HPA-1ab that was enriched in the COVID-19 infected group. Despite limitations of our study, the data presented here show clearly that COVID-19 infection and all the consequences of that are multifactorial and multigenetic. The virus is in a continuous mutation/selection process leading to escape possibilities. Therefore, associations are a momentum in science.

HLA allele association studies with the kinetics of SARS-CoV-2 spike protein-specific IgG antibody responses to BNT162b2 mRNA vaccine

BNT162b2, an mRNA-based SARS-CoV-2 vaccine (Pfizer- BioNTech), is one of the most effective COVID-19 vaccines and has been approved by more than 130 countries worldwide. However, several studies have reported that the COVID-19 vaccine shows high interpersonal variability in terms of humoral and cellular responses, such as those with respect to SARS-CoV-2 spike protein immunoglobulin (Ig)G, IgA, IgM, neutralizing antibodies, and CD4+ & CD8+ T cells. The objective of this study is to investigate the kinetic changes in anti-SARS-CoV-2 spike IgG (IgG-S) profiles and adverse reactions and their associations with HLA profiles among 100 hospital workers from the Center Hospital of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan. DQA1*03:03:01 (p = 0.017;OR 2.80, 95% CI 1.05-7.25) was significantly associated with higher IgG-S production after two doses of BNT162b2 while DQB1*06:01:01:01 (p = 0.028, OR 0.27, 95% CI 0.05-0.94) was significantly associated with IgG-S declines after two doses of BNT162b2. No HLA alleles were significantly associated with either local symptoms or fever. However, C*12:02:02 (p = 0.058;OR 0.42, 95% CI 0.15-1.16), B*52:01:01 (p = 0.031;OR 0.38, 95% CI 0.14-1.03), DQA1*03:02:01 (p = 0.028;OR 0.39, 95% CI 0.15-1.00) and DPB1*02:01:02 (p = 0.024;OR 0.45, 95% CI 0.21-0.97) appeared significantly associated with protection against systemic symptoms after two doses of BNT162b2 vaccination. Further studies with larger sample sizes are clearly warranted to determine HLA allele associations with the production and long-term sustainability of IgG-S after COVID-19 vaccination.

Impact of HLA diversity on humoral response to SARS-Cov-2 and HBV vaccines in liver transplant recipients

Organ transplant recipients show weaker immune responses to vaccines than immunocompetent individuals, which may be related to the repertoire of HLAbound vaccine antigens presented to T lymphocytes. The HLA evolutionary divergence (HED) metric, which quantifies pairwise allele divergence at each HLA locus, provides a primary measure of the breadth of the immunopeptidome. We recently showed that high class I HED of the donor is a strong and independent driver of allograft rejection in a large cohort of liver transplant recipients. Here, in the same cohort, we explored the relation between HED, the size of the predicted immunopeptidome derived from vaccine antigens, and the quality of vaccine responses. We analyzed humoral response to the SARS-CoV-2 BNT162b2 vaccine (n = 310 patients;undetectable anti-spike IgG titers considered as no response, <=250 BAU/mL as moderate and >250 BAU/mL as strong response) and Hepatitis B virus (HBV) vaccine (n = 424 patients;anti-HBs IgG <10 mIU/mL considered as no response, 10-100 mIU/mL as moderate and >=100 mIU/mL as strong response). HED at HLA-A, -B, -C, -DRB1, -DQA1 and -DQB1 loci were measured using the Grantham distance. NetMHCIIpan-4.0 was used to predict the binding to HLA-DQ molecules of all possible 15mer peptides derived from the Spike and HBS sequences. For each vaccine, HED at the DQB1 locus, but not at the other loci, was significantly higher in responders than in non-responders (p = 0.0003), independent of response-associated covariates (age, time since transplant, immunosuppression). Moreover, for both vaccines, there was a strong relationship between DQB1 HED, the diversity of the immunopeptidome and the quality of the vaccine response. In conclusion, DQB1 HED is a critical determinant of humoral response to vaccines in liver transplant recipients. This metric could guide the design of future vaccines as it predicts the magnitude of the repertoire of vaccine-derived peptides presented to CD4 helper T cells.

An Unusual Case of Autoantibody-Negative KetosisProne Diabetes (KPD) Secondary to Marked Insulin Resistance

KPD is classically regarded as an atypical form of diabetes caused by near-complete beta-cell failure. A 37-year-old Egyptian man (BMI: 27.7 Kg/m2) presented with hyperglycemia (362 mg/dL) and DKA (arterial pH 7.20, ketonemia 5.0 mmol/L, ketonuria 80 mg/dL) . He was afebrile, with recent polyuria, polydipsia and weight loss. HbA1c was 107 mmol/mol (11.9%) and blood tests excluded diabetes secondary to endocrinopathies. SARS-CoV-2 RT-PCR test was negative. IV insulin infusion (0.1 IU/kg/h) and IV fluid therapy were started. He was shortly transitioned to a sc basal-bolus insulin regimen (0.7 IU/kg/day) . Mixed-meal tolerance test (MMTT) revealed a peak 120-min stimulated C-peptide of 12.3 ng/mL, suggesting marked insulin resistance. Islet autoantibodies (ICA, IAA, GADA, IA-2A, ZnT8A) and insulin receptor autoantibodies (IgG/IgM) were negative. HLA genotyping detected the following haplotypes: DRB1∗01, ∗04;DQA1∗01:01P, ∗03:01P;DQB1∗03:02P, ∗05:01P. Insulin dose was gradually reduced and insulin therapy was discontinued after 4 months in favor of metformin (2550 mg/day) plus sc semaglutide (up to 1 mg/week) . After one year, MMTT revealed a peak 60-min stimulated C-peptide of 8.25 ng/mL. During the 18-month follow-up period, fasting capillary beta-hydroxybutyrate values were <0.2 mmol/L and HbA1c remained <48 mmol/mol (<6.5%) , indicating disease remission. This case suggests the existence of an autoantibody-negative KPD subtype driven by marked insulin resistance rather than by insulinopenia.

SINGLE-CELL IMMUNE PROFILING AND REPERTOIRE ANALYSIS OF CONVALESCENT PATIENTS WITH INFLAMMATORY BOWEL DISEASE AND SARS-COV-2 ANTIBODY RESPONSE

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by immune dysregulation and decreased T cell receptor (TCR) repertoire diversity. Patients with immune-mediated disorders such as IBD have attenuated convalescent antibody responses after COVID-19 infection. We sought to understand the immune configuration associated with high versus low convalescent SARS-CoV- 2 antibodies in patients with IBD using single-cell immunophenotyping. Methods: We performed a study of 9 patients with IBD who were SARS-CoV-2 convalescent (recovered from COVID-19 and converted RNA positive to negative) and 9 matched SARS-CoV-2 naïve controls (no prior COVID-19, confirmed RNA negative). We measured plasma SARS-CoV- 2 antibody (N protein IgG, S1RBD IgG, S1RBD IgA) levels from patients with IBD two months after recovering from COVID-19 (RNA negative). We selected three patients with the highest SARS-CoV-2 antibodies and three matched (for age, sex, IBD subtype and disease activity, medications, COVID-19 severity) patients with the lowest antibodies and performed their peripheral blood mononuclear cell (PBMC) single-cell transcriptomics with paired TCR and BCR sequencing using 10X Genomics. Normalization, dimensionality reduction, and clustering were performed using Seurat. TCR and BCR immune repertoire analyses were performed using Immunarch. Results: SARS-CoV-2 convalescent patients with IBD had detectable but variable SARS-CoV-2 antibody levels (range 0-469 U/mL), whereas SARSCoV- 2 naïve IBD patients had no detectable antibodies. The mean SARS-CoV-2 antibody concentration among the three IBD patients with the highest and three patients with the lowest groups differed by more than 10-fold (206.0 vs 17.5 U/mL, P<0.001). PBMC singlecell immunophenotyping revealed decreased naïve CD4+ T cell and increased CD14+ monocyte and memory CD4+ T cell proportions in IBD patients in the low versus high SARSCoV- 2 antibody group. There were higher numbers of HLA-DQA1+ B cells and CD8 T cells and lower GPR183+ B cells and CD8 T cells in the high SARS-CoV-2 antibody group. There was a trend towards decreased TCR and BCR repertoire diversity in the low SARS-COV-2 antibody group. Finally, we identified immunoglobulin gene signatures (IGHV1-69D/IGLV3- 25, IGHV3-48, IGHV3-7/IGKV41/IGLV1-47, IGHV3-7/IGKV4-1, IGHV3-7/IGKV4-44) that were enriched only in the high SARS-CoV-2 antibody group. Conclusions: Single-cell immunophenotyping of PBMC from convalescent patients with IBD reveal differences in CD4+ T cell, CD14+ monocyte, and HLA-DQA1+ and GPR183+ B and CD8 T cell immunophenotypes, immune repertoire diversity, and immunoglobulin gene signatures in patients with high versus low SARS-CoV-2 antibody levels.(Figure Presented)Figure 1. SARS-COV-2 Antibodies in Convalescent Patients with IBD and Single-Cell Immunophenotypes. A) SARS-COV-2 antibody levels in COVID-19 convalescent versus SARS-CoV-2 naïve patients with IBD B) T-SNE plot of PBMC immunophenotypes in all convalescent patients with IBD C) Differences in proportion of single-cell PBMC immunophenotypes in high versus low SARS-COV-2 antibody patients D) Differences in HLA-DQA1 and GPR183 immunophenotypes in high versus low SARS-COV-2 antibody patients.