Assessment of SARS-CoV-2 neutralizing antibody titers in breastmilk from convalescent and vaccinated mothers.

Breastmilk contains antibodies that could protect breastfed infants from infections. In this work, we examined if antibodies in breastmilk could neutralize SARS-CoV-2 in 84 breastmilk samples from women that were either vaccinated (Comirnaty, mRNA-1273, or ChAdOx1), infected with SARS-CoV-2, or both infected and vaccinated. The neutralization capacity of these sera was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. We found that natural infection resulted in higher neutralizing titers and that neutralization correlated positively with levels of immunoglobulin A in breastmilk. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA-based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contains SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection.

A machine learning model for predicting serum neutralizing activity against Omicron SARS-CoV-2 BA.2 and BA.4/5 sublineages in the general population.

Supervised machine learning (ML) methods have been used to predict antibody responses elicited by COVID-19 vaccines in a variety of clinical settings. Here, we explored the reliability of a ML approach to predict the presence of detectable neutralizing antibody responses (NtAb) against Omicron BA.2 and BA.4/5 sublineages in the general population. Anti-SARS-CoV-2 receptor-binding domain (RBD) total antibodies were measured by the Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics) in all participants. NtAbs against Omicron BA.2 and BA4/5 were measured using a SARS-CoV-2 S pseudotyped neutralization assay in 100 randomly selected sera. A ML model was built using the variables of age, vaccination (number of doses) and SARS-CoV-2 infection status. The model was trained in a cohort (TC) comprising 931 participants and validated in an external cohort (VC) including 787 individuals. Receiver operating characteristics analysis indicated that an anti-SARS-CoV-2 RBD total antibody threshold of 2300 BAU/mL best discriminated between participants either exhibiting or not detectable Omicron BA.2 and Omicron BA.4/5-Spike targeted NtAb responses (87% and 84% precision, respectively). The ML model correctly classified 88% (793/901) of participants in the TC: 717/749 (95.7%) of those displaying ≥2300 BAU/mL and 76/152 (50%) of those exhibiting antibody levels <2300 BAU/mL. The model performed better in vaccinated participants, either with or without prior SARS-CoV-2 infection. The overall accuracy of the ML model in the VC was comparable. Our ML model, based upon a few easily collected parameters for predicting neutralizing activity against Omicron BA.2 and BA.4/5 (sub)variants circumvents the need to perform not only neutralization assays, but also anti-S serological tests, thus potentially saving costs in the setting of large seroprevalence studies.

Cumulative incidence of SARS-CoV-2 infection in the general population of the Valencian Community (Spain) after the surge of the Omicron BA.1 variant.

Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision-making. A cross-sectional study on 935 participants, conducted in the Valencian Community (VC), measuring anti-SARS-CoV-2-receptor binding domain-RBD-total antibodies and anti-Nucleocapsid (N)-IgGs via electrochemiluminescence assays. Quantitation of neutralizing antibodies (NtAb) against ancestral and Omicron BA.1 and BA.2 variants and enumeration of SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells was performed in 100 and 137 participants, respectively. The weighted cumulative incidence was 51.9% (95% confidence interval [CI]: 48.7-55.1) and was inversely related to age. Anti-RBD total antibodies were detected in 97% of participants; vaccinated and SARS-CoV-2-experienced (VAC-ex; n = 442) presented higher levels (p < 0.001) than vaccinated/naïve (VAC-n; n = 472) and nonvaccinated/experienced (UNVAC-ex; n = 63) subjects. Antibody levels correlated inversely with time elapsed since last vaccine dose in VAC-n (Rho, -0.52; p < 0.001) but not in VAC-ex (rho -0.02; p = 0.57). Heterologous booster shots resulted in increased anti-RBD antibody levels compared with homologous schedules in VAC-n, but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75%, and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively. For Omicron BA.2, the figures were 97%, 84%, and 40%, respectively. SARS-CoV-2-S-reactive IFN-γ T cells were detected in 73%, 75%, and 64% of VAC-ex, VAC-n and UNVAC-ex, respectively. Median frequencies for both T-cell subsets were comparable across groups. In summary, by April 2022, around half of the VC population had been infected with SARS-CoV-2 and, due to extensive vaccination, displayed hybrid immunity.

A performance comparison of two (electro) chemiluminescence immunoassays for detection and quantitation of serum anti-spike antibodies according to SARS-CoV-2 vaccination and infections status.

The information provided by SARS-CoV-2 spike (S)-targeting immunoassays can be instrumental in clinical-decision making. We compared the performance of the Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics) and the LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin) using a total of 1176 sera from 797 individuals, of which 286 were from vaccinated-SARS-CoV-2/experienced (Vac-Ex), 581 from vaccinated/naïve (Vac-N), 147 from unvaccinated/experienced (Unvac-Ex), and 162 from unvaccinated/naïve (Unvac-N) individuals. The Roche assay returned a higher number of positive results (907 vs. 790; p = 0.45; overall sensitivity: 89.3% vs. 77.6%). The concordance between results provided by the two immunoassays was higher for sera from Vac-N (Ï°: 0.58; interquartile ranges [IQR]: 0.50-0.65) than for sera from Vac-Ex (Ï°: 0.19; IQR: -0.14 to 0.52) or Unvac-Ex (Ï°: 0.18; IQR: 0.06-0.30). Discordant results occurred more frequently among sera from Unvac-Ex (34.7%) followed by Vac-N (14.6%) and Vac-Ex (2.7%). Antibody levels quantified by both immunoassays were not significantly different when <250 (p = 0.87) or <1000 BAU/ml (p = 0.13); in contrast, for sera ≥1000 BAU/ml, the Roche assay returned significantly higher values than the DiaSorin assay (p < 0.008). Neutralizing antibody titers (NtAb) were measured in 127 sera from Vac-Ex or Vac-N using a S-pseudotyped virus neutralization assay of Wuhan-Hu-1, Omicron BA.1, and Omicron BA.2. The correlation between antibody levels and NtAb titers was higher for sera from Vac-N than those from Vac-Ex, irrespective of the (sub)variant considered. In conclusion, neither qualitative nor quantitative results returned by both immunoassays are interchangeable. The performance of both assays was found to be greatly influenced by the vaccination and SARS-CoV-2 infection status of individuals.

Correction: The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

[This corrects the article DOI: 10.1371/journal.ppat.1010631.].

SARS-CoV-2-Spike Antibody and T-Cell Responses Elicited by a Homologous Third mRNA COVID-19 Dose in Hemodialysis and Kidney Transplant Recipients.

The effect of a third vaccine dose (3D) of homologous mRNA vaccine on blood levels of SARS-CoV-2-receptor binding domain (RBD)-total antibodies was assessed in 40 hemodialysis patients (HD) and 21 kidney transplant recipients (KTR) at a median of 46 days after 3D. Anti-RBD antibodies were detected in 39/40 HD and 19/21 KTR. Overall, 3D boosted anti-RBD antibody levels (median: 58-fold increase). Neutralizing antibodies (NtAb) against the Wuhan-Hu-1, Delta, and Omicron variants were detected in 14, 13, and 11 out of 14 HD patients, and in 5, 5, and 4 out of 8 KTR patients, respectively. The median fold increase in NtAb titers in HD patients was 77, 28, and 5 and 56, 37, and 9 in KTR patients for each respective variant. SARS-CoV-2-S S-IFN-γ-producing CD8+ and CD4+ T-cell responses were detected in the majority of HD (35 and 36/37, respectively) and all KTR (16/16) patients at 3D. Overall, the administration of 3D boosted T-cell levels in both population groups. In conclusion, a homologous mRNA COVID-19 vaccine 3D exerts a booster effect on anti-RBD antibodies, NtAb binding to Wuhan-Hu-1, Delta, and Omicron variants, and SARS-CoV-2-S-IFN-γ-producing T cells in both HD and KTR patients. The magnitude of the effect was more marked in HD than KTR patients.

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

Neutralizing antibodies against SARS-CoV-2 variants of concern elicited by the comirnaty COVID-19 vaccine in nursing home residents.

Immunosenescence may impact the functionality and breadth of vaccine-elicited humoral immune responses. The ability of sera to neutralize the SARS-CoV-2 spike protein (S) from Beta, Gamma, Delta, and Epsilon variants of concern (VOCs) relative to the ancestral Wuhan-Hu-1 strain was compared in Comirnaty COVID-19-vaccinated elderly nursing home residents, either SARS-CoV-2 naïve (n = 22) or experienced (n = 8), or SARS-CoV-2 naïve younger individuals (n = 18) and non-vaccinated individuals who recovered from severe COVID-19 (n = 19). In all groups, except that including SARS-CoV-2-experienced nursing home residents, some participants lacked NtAb against one or more VOCs, mainly the Beta variant (15-20%). Serum NtAb titers were lowest against the Beta variant followed by Gamma, Delta and Epsilon variants. Overall, fold change reduction in NtAb titers relative to the ancestral strain was greatest for the Beta variant (6.7-19.4) followed by Gamma (4.8-16.0), Epsilon (2.9-13.4), and Delta (3.5-6.5) variants, although subtle differences were observed for Beta, Epsilon and Delta variants across comparison groups. In summary, older age, frailty, and concurrence of co-morbidities had no major impact on the serum NtAb activity profile against SARS-CoV-2 VOCs.

Severe Acute Respiratory Syndrome Coronavirus 2 Adaptive Immunity in Nursing Home Residents Following a Third Dose of the Comirnaty Coronavirus Disease 2019 Vaccine.

A third Comirnaty vaccine dose increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain antibody levels (median, 93-fold) and neutralizing antibody titers against Wuhan-Hu-1 (median, 57-fold), Beta (me 22-fold), Delta, (median, 43-fold), and Omicron (median, 8-fold) variants, but had less impact on S-reactive T-cell immunity in nursing home residents.

SARS-CoV-2 Omicron BA.1 variant breakthrough infections in nursing home residents after an homologous third dose of the Comirnaty® COVID-19 vaccine: Looking for correlates of protection.

We investigated whether peripheral blood levels of SARS-CoV-2 Spike (S) receptor binding domain antibodies (anti-RBD), neutralizing antibodies (NtAb) targeting Omicron S, and S-reactive-interferon (IFN)-γ-producing CD4+ and CD8+ T cells measured after a homologous booster dose (3D) with the Comirnaty® vaccine was associated with the likelihood of subsequent breakthrough infections due to the Omicron variant. An observational study including 146 nursing home residents (median age, 80 years; range, 66-99; 109 female) evaluated for an immunological response after 3D (at a median of 16 days). Anti-RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells were enumerated by whole-blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti-RBD antibody levels were comparable in infected and uninfected participants (21 123 vs. 24 723 BAU/ml; p = 0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p = 0.32) and frequency of virus-reactive CD4+ (p = 0.82) and CD8+ (p = 0.91) T cells were similar across participants in both groups. anti-RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445 vs. 4345 BAU/ml; p = 0.59 and 188.5 vs. 88.9; p = 0.70, respectively). Having detectable NtAb against Omicron or SARS-CoV-2-S-reactive-IFNγ-producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p = 0.54; OR, 0.0; p = 0.99 and OR 3.70; p = 0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID-19 due to the Omicron variant in nursing home residents.

Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

Preservation of anti-SARS-CoV-2 neutralising antibodies in convalescent plasma after pathogen reduction with methylene blue and visible light.

BACKGROUND: COVID-19 convalescent plasma (CCP) is an experimental treatment against SARS-CoV-2. Although there has so far been no evidence of transmission through transfusion, pathogen reduction technologies (PRT) have been applied to CCP to mitigate risk of infectious disease. This study aims to assess the impact of methylene blue (MB) plus visible light PRT on the virus-neutralising activity of the specific antibodies against SARS-CoV-2. MATERIAL AND METHODS: Thirty-five plasma doses collected by plasmapheresis from COVID-19 convalescent donors were subjected to MB plus visible light PRT. Anti-SARS-CoV-2 RBD S1 epitope IgGs antibodies were quantified by ELISA. Titres of SARS-CoV-2 neutralising antibodies (NtAbs) were measured before and after the PRT process. A Spearman's correlation was run to determine the relationship between antibody neutralisation ability and SARS-CoV-2 IgG ELISA ratio. Pre- and post-inactivation neutralising antibody titres were evaluated using a Wilcoxon test. RESULTS: The plasma pathogen reduction procedure did not diminish NtAbS titres and so did not cause a change in the viral neutralisation capacity of CCP. There was a strong correlation between pre-and post-PRT NtAbs and anti-SARS-CoV-2 IgGs titres. DISCUSSION: Our results showed PRT with MB did not impair the CCP passive immunity preserving its potential therapeutic potency. Therefore, PRT of CCP should be recommended to mitigate the risk for transmission of transfusion-associated infectious disease. There is a good correlation between SARS-CoV-2 IgG titres determined by ELISA and the neutralising capacity. This allows blood centres to select CCP donors based on IgG ELISA titres avoiding the much more labour-intensive laboratory processes for determining neutralising antibodies.

Suitability of two rapid lateral flow immunochromatographic assays for predicting SARS-CoV-2 neutralizing activity of sera.

Assessment of commercial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially available lateral flow immunochromatographic assays (LFIC; Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for coronavirus disease 2019 (COVID-19) diagnosis in hospitalized patients and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). The overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM, and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%, 93.9%, and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers (≥1/160). Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.

Adoptive transfer of ex vivo expanded SARS-CoV-2-specific cytotoxic lymphocytes: A viable strategy for COVID-19 immunosuppressed patients?

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients.

Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays.

Whether antibody levels measured by commercially available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. We evaluated the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Ninety sera from 51 hospitalized COVID-19 patients were tested by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG, and the COVID-19 ELISA IgG assays. Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (rho = 0.73) and moderate for the remaining assays (rho = 0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. The suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

Potential Influence of Helminth Molecules on COVID-19 Pathology.

In recent months, the parasitology research community has been tasked with investigation of the influence of parasite coinfection on coronavirus disease 2019 (COVID-19) outcomes. Herein, we share our approach to analyze the effect of the trematode Fasciola hepatica as a modulator of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and of COVID-19 pathology.

SARS-CoV-2 antibodies, serum inflammatory biomarkers and clinical severity of hospitalized COVID-19 patients.

BACKGROUND: The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. PATIENTS AND METHODS: This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporterbased pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. RESULTS: The overall correlation between levels of both antibody measurements was good (Rho = 0.82; P = 0 < 0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). Four ICU patients died; two of these achieved NtAb50 titers ≥1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. CONCLUSIONS: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity.