Antigenic cartography using hamster sera identifies SARS-CoV-2 JN.1 evasion seen in human XBB.1.5 booster sera (preprint)

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection (preprint)

Background: We sought to determine immune and behavioral pre-infection correlates of protection against SARS-CoV-2 post-vaccine infections in a joint analysis of epidemiological and immunological cohort data. Methods: Serum and saliva samples from 176 BNT162b2-vaccinated adults in the Prospective Assessment of SARS-CoV-2 Seroconversion study were collected between October and December 2021 and assessed for serum and saliva levels of Wuhan-1 wild-type (WT) SARS-CoV-2 Spike (S)-specific IgG and IgA binding antibodies (bAb) using a multiplex microsphere-based immunoassay (MMIA). Serum samples were also assessed for WT receptor binding domain (RBD)-specific bAb by two commercial assays, BA.1 S-specific IgG bAb by MMIA, and neutralization activity against D614G, Delta (B.1.617.2), and Omicron BA.1 and BA.1.1 variants using a lentiviral pseudovirus neutralization assay. After the Fall 2021 visit, participants reported all positive PCR and/or antigen tests for SARS-CoV-2. Duration, severity, and type of symptoms, as well as risk exposures and adherence to precautionary measures, were assessed by questionnaires during the Spring 2022 visit. Results: Thirty-two participants (18.2%) developed symptomatic post-vaccination SARS-CoV-2 infections (PVI) between December 7, 2021 and April 1, 2022. Pre-infection WT (geometric mean (GM) of 3,863 vs 2,736 binding antibody unit [BAU]/ml, uninfected vs PVI, p=0.0098) and BA.1 (GM of 276.9 vs 179.9 arbitrary bAb unit [AU]/ml, uninfected vs PVI, p=0.04) anti-S IgG bAb levels measured by MMIA and neutralizing titers (NT) against BA.1 (GM titer [GMT] of 493.6 vs 286.2, uninfected vs PVI, p=0.0313) and BA.1.1 (GMT of 552.0 vs 302.5, uninfected vs PVI, p=0.021) were significantly higher in individuals that did not develop PVIs. WT anti-S bAb levels greater than 5,000 BAU/ml were associated with > 90% protection against symptomatic PVI. In individuals that developed PVI, WT anti-S IgG bAb levels correlated with lower disease severity scores ({rho}= -0.3859, p=0.032) and shorter duration of clinical disease ({rho}= -0.5273, p=0.0023). WT anti-RBD bAb levels measured by commercial assays correlated strongly with bAb levels measured by MMIA ({rho}=0.8239, p<0.0001 and {rho}=0.6929, p<0.0001, Roche and Siemens assays, respectively), but did not reach statistical significance for correlation with protection against PVI. Home risk score, but neither work nor home precautionary measures, correlated strongly with risk of PVI (mean score of 20.77 vs 47.33, uninfected vs PVI respectively, p<0.0001). Conclusions: Anti-S IgG bAb levels (directed against either WT or Omicron BA.1 subvariant) and NTs served as correlates of protection against symptomatic SARS-CoV-2 infection. Anti-S (WT) IgG bAb levels remained a significant correlate of protection against PVIs when adjusting for demography and risk behavior. Results of this study also suggest that commercial assays for anti-S bAb may need to be reformatted to enable detection of higher maximum values for use as predictors of increased susceptibility to SARS-CoV-2 infection.

Comparative Cohort Study of Post-Acute Covid-19 Infection with a Nested, Randomized Controlled Trial of Ivabradine for Those With Postural Orthostatic Tachycardia Syndrome (The COVIVA Study) (preprint)

Background: Significant clinical similarities have been observed between the recently described Long-Haul COVID-19 (LHC) syndrome, Postural Orthostatic Tachycardia Syndrome (POTS) and Inappropriate Sinus Tachycardia (IST). Shared symptoms include light-headedness, palpitations, tremulousness, generalized weakness, blurred vision, chest pain, dyspnea, brain-fog, and fatigue. Ivabradine is a selective sinoatrial node blocker FDA-approved for management of tachycardia associated with stable angina and heart failure not fully managed by beta blockers. In our study we aim to identify risk factors underlying LHC, as well as the effectiveness of ivabradine in controlling heart rate dysregulations and POTS/IST related symptoms. Methods/Design: A detailed prospective phenotypic evaluation combined with multi-omic analysis of 200 LHC volunteers will be conducted to identify risk factors for autonomic dysfunction. A comparator group of 50 volunteers with documented COVID-19 but without LHC will be enrolled to better understand the risk factors for LHC and autonomic dysfunction. Those in the cohort who meet diagnostic criteria for POTS or IST will be included in a nested prospective, randomized, placebo-controlled trial to assess the impact of ivabradine on symptoms and heart rate, assessed non-invasively based on physiologic response and ambulatory electrocardiogram. Additionally, studies on catecholamine production, mast cell and basophil degranulation, inflammatory biomarkers, and indicators of metabolic dysfunction will be measured to potentially provide molecular classification and mechanistic insights. Discussion: Optimal therapies for dysautonomia, particularly associated with LHC, have yet to be defined. In the present study, ivabradine, one of numerous proposed interventions, will be systematically evaluated for therapeutic potential in LHC-associated POTS and IST. Additionally, this study will further refine the characteristics of the LHC-associated POTS/IST phenotype, genotype and transcriptional profile, including immunologic and multi-omic analysis of persistent immune activation and dysregulation. The study will also explore and identify potential endotheliopathy and abnormalities of the clotting cascade.

Comparative Cohort Study of Post-Acute Covid-19 Infection with a Nested, Randomized Controlled Trial of Ivabradine for Those With Postural Orthostatic Tachycardia Syndrome (The COVIVA Study) (preprint)

Background:  Significant clinical similarities have been observed between the recently described ‘Long-Haul’ COVID-19 (LHC) syndrome, Postural Orthostatic Tachycardia Syndrome (POTS) and Inappropriate Sinus Tachycardia (IST). Shared symptoms include light-headedness, palpitations, tremulousness, generalized weakness, blurred vision, chest pain, dyspnea, “brain-fog”, and fatigue. Ivabradine is a selective sinoatrial node blocker FDA-approved for management of tachycardia associated with stable angina and heart failure not fully managed by beta blockers. In our study we aim to identify risk factors underlying LHC, as well as the effectiveness of ivabradine in controlling heart rate dysregulations and POTS/IST related symptoms. Methods/Design:  A detailed prospective phenotypic evaluation combined with multi-omic analysis of 200 LHC volunteers will be conducted to identify risk factors for autonomic dysfunction.  A comparator group of 50 volunteers with documented COVID-19 but without LHC will be enrolled to better understand the risk factors for LHC and autonomic dysfunction.  Those in the cohort who meet diagnostic criteria for POTS or IST will be included in a nested prospective, randomized, placebo-controlled trial to assess the impact of ivabradine on symptoms and heart rate, assessed non-invasively based on physiologic response and ambulatory electrocardiogram. Additionally, studies on catecholamine production, mast cell and basophil degranulation, inflammatory biomarkers, and indicators of metabolic dysfunction will be measured to potentially provide molecular classification and mechanistic insights. Discussion: Optimal therapies for dysautonomia, particularly associated with LHC, have yet to be defined. In the present study, ivabradine, one of numerous proposed interventions, will be systematically evaluated for therapeutic potential in LHC-associated POTS and IST. Additionally, this study will further refine the characteristics of the LHC-associated POTS/IST phenotype, genotype and transcriptional profile, including immunologic and multi-omic analysis of persistent immune activation and dysregulation. The study will also explore and identify potential endotheliopathy and abnormalities of the clotting cascade. Trial registration:ClinicalTrials.gov, ID:NCT05481177 Registered on 29 July 2022.

Bivalent COVID-19 vaccine antibody responses to Omicron variants suggest that responses to divergent variants would be improved with matched vaccine antigens (preprint)

We compared neutralizing antibody responses to BA.4/5, BQ.1.1, XBB, and XBB.1.5 Omicron SARS-CoV-2 variants after a bivalent or ancestral COVID-19 mRNA booster vaccine or post-vaccination infection. We found that the bivalent booster elicited moderately high antibody titers against BA.4/5 that were approximately two-fold higher against all Omicron variants than titers elicited by the monovalent booster. The bivalent booster elicited low but similar titers against both XBB and XBB.1.5 variants. These findings inform risk assessments for future COVID-19 vaccine recommendations and suggest that updated COVID-19 vaccines containing matched vaccine antigens to circulating divergent variants may be needed.

Post-vaccination Omicron infections induce broader immunity across antigenic space than prototype mRNA COVID-19 booster vaccination or primary infection (preprint)

The rapid emergence of new SARS-CoV-2 variants challenges vaccination strategies. Here, we measured antigenic diversity among variants and interpreted neutralizing antibody responses following single and multiple exposures in longitudinal infection and vaccine cohorts. Antigenic cartography using primary infection antisera showed that BA.2, BA.4/BA.5, and BA.2.12.1 are distinct from BA.1 and closer to the Beta cluster. Three doses of an mRNA COVID-19 vaccine increased breadth to BA.1 more than to BA.4/BA.5 or BA.2.12.1. Omicron BA.1 post-vaccination infection elicited antibody landscapes characterized by broader immunity across antigenic space than three doses alone, although with less breadth than expected to BA.2.12.1 and BA.4/BA.5. Those with Omicron BA.1 infection after two or three vaccinations had similar neutralizing titer magnitude and antigenic breadth. Accounting for antigenic differences among variants of concern when interpreting neutralizing antibody titers aids understanding of complex patterns in humoral immunity and informs selection of future COVID-19 vaccine strains.

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster (preprint)

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants. One Sentence Summary Third dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.

Durability of antibody responses and frequency of clinical and subclinical SARS-CoV-2 infection six months after BNT162b2 COVID-19 vaccination in healthcare workers (preprint)

Antibodies against SARS-CoV-2 decay but persist six months post-vaccination, with lower levels of neutralizing titers against Delta than wild-type. Only 2 of 227 vaccinated healthcare workers experienced outpatient symptomatic breakthrough infections despite 59 of 227 exhibiting serological evidence of exposure to SARS-CoV-2 as defined by development of anti-nucleocapsid protein antibodies.

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers (preprint)

Background: mRNA COVID-19 vaccines are playing a key role in controlling the COVID-19 pandemic. The relationship between post-vaccination symptoms and strength of antibody responses is unclear. Objective: To determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. Design: Single center, prospective, observational cohort study. Setting: Participants worked at Walter Reed National Military Medical Center and were seen monthly at the Naval Medical Research Center Clinical Trials Center. Participants: Generally healthy adults that were not severely immunocompromised, had no history of COVID-19, and were seronegative for SARS-CoV-2 spike protein prior to vaccination. Measures: Severity of vaccine-associated symptoms was obtained through participant completed questionnaires. Testing for IgG antibodies against SARS-CoV-2 spike protein and receptor binding domain was conducted using microsphere-based multiplex immunoassays. Results: 206 participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers one month after vaccination. We also observed that 1) post-vaccination symptoms were inversely correlated with age and weight and more common in women, 2) systemic symptoms were more frequent after the second vaccination, 3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and 4) older age was associated with lower titers. Limitations: Study only observes antibody responses and consists of healthy participants. Conclusions: Lack of post-vaccination symptoms following receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies one month after vaccination. This study also suggests that it may be possible to design future mRNA vaccines that confer robust antibody responses with lower frequencies of vaccine-associated symptoms.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions (preprint)

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we developed and applied a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human betacoronaviruses, HCoV-HKU1 and HCoV-OC43, that enables measurement of off-target pre-existing cross-reactive antibodies. The MMIA performances characteristics are: 98% sensitive and 100% specific for human subject samples collected as early as 10 days from symptom onset. The MMIA permitted the simultaneous identification of SARS-CoV-2 seroconversion and the induction of SARS-CoV-2 IgG antibody cross reactions to SARS-CoV-1 and MERS-CoV. Further, synchronous increases of HCoV-OC43 IgG antibody levels was detected with SARS-CoV-2 seroconversion in a subset of subjects for whom early infection sera were available prior to their SARS-CoV-2 seroconversion, suggestive of an HCoV-OC43 memory response triggered by SARS-CoV-2 infection.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity (preprint)

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 uL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

Additional analyses exploring the hypothesized transdifferentiation of plasmablasts to developing neutrophils in severe COVID-19 (preprint)

We thank Alquicira-Hernandez et al. for their reanalysis of our single-cell transcriptomic dataset profiling peripheral immune responses to severe COVID-19. We agree that careful analysis of single-cell sequencing data is important for generating cogent hypotheses but find several aspects of their criticism of our analysis to be problematic. Here we respond briefly to misunderstandings and inaccuracies in their commentary that may have led to misinformed interpretation of our results.

Abnormal Antibodies to Self-Carbohydrates in SARS-CoV-2 Infected Patients (preprint)

SARS-CoV-2 is a deadly virus that is causing the global pandemic coronavirus disease 2019 (COVID-19). Our immune system plays a critical role in preventing, clearing, and treating the virus, but aberrant immune responses can contribute to deleterious symptoms and mortality. Many aspects of immune responses to SARS-CoV-2 are being investigated, but little is known about immune responses to carbohydrates. Since the surface of the virus is heavily glycosylated, pre-existing antibodies to glycans could potentially recognize the virus and influence disease progression. Furthermore, antibody responses to carbohydrates could be induced, affecting disease severity and clinical outcome. In this study, we used a carbohydrate antigen microarray with over 800 individual components to profile serum anti-glycan antibodies in COVID-19 patients and healthy control subjects. In COVID-19 patients, we observed abnormally high IgG and IgM antibodies to numerous self-glycans, including gangliosides, N-linked glycans, LacNAc-containing glycans, blood group H, and sialyl Lewis X. Some of these anti-glycan antibodies are known to play roles in autoimmune diseases and neurological disorders, which may help explain some of the unusual and prolonged symptoms observed in COVID-19 patients. The detection of antibodies to self-glycans has important implications for using convalescent serum to treat patients, developing safe and effective SARS-CoV-2 vaccines, and understanding the risks of infection. In addition, this study provides new insight into the immune responses to SARS-CoV-2 and illustrates the importance of including host and viral carbohydrate antigens when studying immune responses to viruses.

Antibody Response to a Nucleocapsid Epitope as a Marker for COVID-19 Disease Severity (preprint)

Characterization of antibody response to SARS-CoV-2 is urgently needed to predict COVID-19 disease trajectories. Ineffective antibodies or antibody-dependent enhancement (ADE) could derail patient immune responses, for example. ELISA and coronavirus antigen microarray (COVAM) analysis epitope-mapped plasma from 86 COVID-19 patients. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including ICU stay, requirement for ventilators, and death. Furthermore, anti-Ep9 antibodies correlate both with various comorbidities and ADE hallmarks, including increased IL-6 levels and early IgG response. Importantly, anti-Ep9 antibodies can be detected within five days post-symptom onset and sometimes within one day. The results lay the groundwork for a new type of COVID-19 diagnostic for the early prediction of disease severity to guide more effective therapeutic interventions.

Early induction of SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients (preprint)

Virus-specific humoral and cellular immunity act synergistically to protect the host from viral infection. We interrogated the dynamic changes of virological and immunological parameters in 12 patients with symptomatic acute SARS-CoV-2 infection from disease onset to convalescence or death. We quantified SARS-CoV-2 viral RNA in the respiratory tract in parallel with antibodies and circulating T cells specific for various structural (NP, M, ORF3a and spike) and non-structural proteins (ORF7/8, NSP7 and NSP13). We observed that while rapid induction and quantity of humoral responses were associated with increased disease severity, an early induction of SARS-CoV-2 specific T cells was present in patients with mild disease and accelerated viral clearance. These findings provide further support for a protective role of SARS-CoV-2 specific T cells over antibodies during SARS-CoV-2 infection with important implications in vaccine design and immune-monitoring.