Diagnostic TR-FRET assays for detection of antibodies in patient samples.

Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immune response post vaccination, and managing spread of the infectious agent among the population. Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate an alternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneous assays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performance of the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S) protein; and SARS-CoV-2 S and nucleocapsid (N) proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers.

Humoral responses to the SARS-CoV-2 spike and receptor binding domain in context of pre-existing immunity confer broad sarbecovirus neutralization.

Since the emergence of SARS-CoV-2 (SARS-2), multiple vaccine candidates were developed and studied both preclinically and clinically. Nearly all are based on the SARS-2 spike glycoprotein or its receptor binding domain (RBD). Studies of these vaccine candidates have largely been in a SARS-2 naïve context. However, pre-existing immunity to SARS-2 acquired through infection or vaccination continues to increase. Evaluating future vaccine candidates in context of this pre-existing immunity is necessary to understand how immune responses are subsequently influenced. Here, we evaluated the serum and IgG+ B cell responses to the SARS-2 RBD in context of pre-existing immunity elicited by the full SARS-2 spike, and we compared this to boosting with the full SARS-2 spike. Boosting with the SARS-2 RBD resulted in increased reactivity to RBD epitopes, but both immunization regimens resulted in similarly broad neutralization across diverse sarbecoviruses. These findings may inform comparison among SARS-2 RBD-based vaccine candidates to currently approved spike-based candidates.

SARS-CoV-2 mRNA vaccination elicits robust antibody responses in children.

Although children have been largely spared from coronavirus disease 2019 (COVID-19), the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with increased transmissibility, combined with fluctuating mask mandates and school reopenings, has led to increased infections and disease among children. Thus, there is an urgent need to roll out COVID-19 vaccines to children of all ages. However, whether children respond equivalently to adults to mRNA vaccines and whether dosing will elicit optimal immunity remain unclear. Here, we aimed to deeply profile the vaccine-induced humoral immune response in 6- to 11-year-old children receiving either a pediatric (50 µg) or adult (100 µg) dose of the mRNA-1273 vaccine and to compare these responses to vaccinated adults, infected children, and children who experienced multisystem inflammatory syndrome in children (MIS-C). Children elicited an IgG-dominant vaccine-induced immune response, surpassing adults at a matched 100-µg dose but more variable immunity at a 50-µg dose. Irrespective of titer, children generated antibodies with enhanced Fc receptor binding capacity. Moreover, like adults, children generated cross-VOC humoral immunity, marked by a decline of omicron-specific receptor binding domain, but robustly preserved omicron spike protein binding. Fc receptor binding capabilities were also preserved in a dose-dependent manner. These data indicate that both the 50- and 100-µg doses of mRNA vaccination in children elicit robust cross-VOC antibody responses and that 100-µg doses in children result in highly preserved omicron-specific functional humoral immunity.

Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting.

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes.

Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting

Hauser et al. use structure-guided design to engineer SARS-2 immunogens that direct immune responses to conserved viral sites in context of preexisting immunity. In mice, these immunogens elicit antibodies that potently neutralize related coronaviruses, including those of potential pandemic concern. Structural characterization of selected antibodies explains this observation.

Immunogenicity and Reactogenicity of SARS-CoV-2 Vaccines in Patients With Cancer: The CANVAX Cohort Study.

PURPOSE: The immunogenicity and reactogenicity of SARS-CoV-2 vaccines in patients with cancer are poorly understood. METHODS: We performed a prospective cohort study of adults with solid-organ or hematologic cancers to evaluate anti-SARS-CoV-2 immunoglobulin A/M/G spike antibodies, neutralization, and reactogenicity ≥ 7 days following two doses of mRNA-1273, BNT162b2, or one dose of Ad26.COV2.S. We analyzed responses by multivariate regression and included data from 1,638 healthy controls, previously reported, for comparison. RESULTS: Between April and July 2021, we enrolled 1,001 patients; 762 were eligible for analysis (656 had neutralization measured). mRNA-1273 was the most immunogenic (log10 geometric mean concentration [GMC] 2.9, log10 geometric mean neutralization titer [GMT] 2.3), followed by BNT162b2 (GMC 2.4; GMT 1.9) and Ad26.COV2.S (GMC 1.5; GMT 1.4; P < .001). The proportion of low neutralization (< 20% of convalescent titers) among Ad26.COV2.S recipients was 69.9%. Prior COVID-19 infection (in 7.1% of the cohort) was associated with higher responses (P < .001). Antibody titers and neutralization were quantitatively lower in patients with cancer than in comparable healthy controls, regardless of vaccine type (P < .001). Receipt of chemotherapy in the prior year or current steroids were associated with lower antibody levels and immune checkpoint blockade with higher neutralization. Systemic reactogenicity varied by vaccine and correlated with immune responses (P = .002 for concentration, P = .016 for neutralization). In 32 patients who received an additional vaccine dose, side effects were similar to prior doses, and 30 of 32 demonstrated increased antibody titers (GMC 1.05 before additional dose, 3.17 after dose). CONCLUSION: Immune responses to SARS-CoV-2 vaccines are modestly impaired in patients with cancer. These data suggest utility of antibody testing to identify patients for whom additional vaccine doses may be effective and appropriate, although larger prospective studies are needed.

T cell reactivity to the SARS-CoV-2 Omicron variant is preserved in most but not all individuals.

The SARS-CoV-2 Omicron variant (B.1.1.529) contains mutations that mediate escape from antibody responses, although the extent to which these substitutions in spike and non-spike proteins affect T cell recognition is unknown. In this study, we show that T cell responses in individuals with prior infection, vaccination, both prior infection and vaccination, and boosted vaccination are largely preserved to Omicron spike and non-spike proteins. However, we also identify a subset of individuals (∼21%) with a >50% reduction in T cell reactivity to the Omicron spike. Evaluation of functional CD4+ and CD8+ memory T cell responses confirmed these findings and revealed that reduced recognition to Omicron spike is primarily observed within the CD8+ T cell compartment potentially due to escape from HLA binding. Booster vaccination enhanced T cell responses to Omicron spike. In contrast to neutralizing immunity, these findings suggest preservation of T cell responses to the Omicron variant, although with reduced reactivity in some individuals.

Significant Reduction in Vaccine-Induced Antibody Levels and Neutralization Activity Among Healthcare Workers and Nursing Home Residents 6 Months Following Coronavirus Disease 2019 BNT162b2 mRNA Vaccination.

Antibody decline occurred from 2 weeks to 6 months post-BNT162b2 mRNA vaccination in nursing home (NH) residents and healthcare workers. Antispike, receptor-binding domain, and neutralization levels dropped >81% irrespective of prior infection. Notably, 69% of infection-naive NH residents had neutralizing antibodies at or below the assay's limit of detection.

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant.

Recent surveillance has revealed the emergence of the SARS-CoV-2 Omicron variant (BA.1/B.1.1.529) harboring up to 36 mutations in spike protein, the target of neutralizing antibodies. Given its potential to escape vaccine-induced humoral immunity, we measured the neutralization potency of sera from 88 mRNA-1273, 111 BNT162b, and 40 Ad26.COV2.S vaccine recipients against wild-type, Delta, and Omicron SARS-CoV-2 pseudoviruses. We included individuals that received their primary series recently (<3 months), distantly (6-12 months), or an additional "booster" dose, while accounting for prior SARS-CoV-2 infection. Remarkably, neutralization of Omicron was undetectable in most vaccinees. However, individuals boosted with mRNA vaccines exhibited potent neutralization of Omicron, only 4-6-fold lower than wild type, suggesting enhanced cross-reactivity of neutralizing antibody responses. In addition, we find that Omicron pseudovirus infects more efficiently than other variants tested. Overall, this study highlights the importance of additional mRNA doses to broaden neutralizing antibody responses against highly divergent SARS-CoV-2 variants.

Neutralization breadth of SARS-CoV-2 viral variants following primary series and booster SARS-CoV-2 vaccines in patients with cancer.

Patients with cancer are more likely to have impaired immune responses to SARS-CoV-2 vaccines. We study the breadth of responses against SARS-CoV-2 variants after primary vaccination in 178 patients with a variety of tumor types and after booster doses in a subset. Neutralization of alpha, beta, gamma, and delta SARS-CoV-2 variants is impaired relative to wildtype, regardless of vaccine type. Regardless of viral variant, mRNA1273 is the most immunogenic, followed by BNT162b2, and then Ad26.COV2.S. Neutralization of more variants (breadth) is associated with a greater magnitude of wildtype neutralization, and increases with time since vaccination; advancing age associates with a lower breadth. The concentrations of anti-spike protein antibody are a good surrogate for breadth (positive predictive value of =90% at >1,000 U/mL). Booster SARS-CoV-2 vaccines confer enhanced breadth. These data suggest that achieving a high antibody titer is desirable to achieve broad neutralization; a single booster dose with the current vaccines increases the breadth of responses against variants.

Comparative Immunogenicity and Effectiveness of mRNA-1273, BNT162b2, and Ad26.COV2.S COVID-19 Vaccines.

BACKGROUND: Understanding immunogenicity and effectiveness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is critical to guide rational use. METHODS: We compared the immunogenicity of mRNA-1273, BNT-162b2, and Ad26.COV2.S in healthy ambulatory adults. We performed an inverse-variance meta-analysis of population-level effectiveness from public health reports in > 40 million individuals. RESULTS: A single dose of either mRNA vaccine yielded comparable antibody and neutralization titers to convalescent individuals. Ad26.COV2.S yielded lower antibody concentrations and frequently undetectable neutralization titers. Bulk and cytotoxic T-cell responses were higher in mRNA1273 and BNT162b2 than Ad26.COV2.S recipients. Regardless of vaccine, <50% of vaccinees demonstrated CD8+ T-cell responses. Antibody concentrations and neutralization titers increased comparably after the first dose of either vaccine, and further in recipients of a second dose. Prior infection was associated with high antibody concentrations and neutralization even after a single dose and regardless of vaccine. Neutralization of Beta, Gamma, and Delta strains were poorer regardless of vaccine. In meta-analysis, relative to mRNA1273 the effectiveness of BNT162b2 was lower against infection and hospitalization, and Ad26COV2.S was lower against infection, hospitalization, and death. CONCLUSIONS: Variation in the immunogenicity correlates with variable effectiveness of the 3 vaccines deployed in the United States.

Reduced BNT162b2 Messenger RNA Vaccine Response in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Naive Nursing Home Residents.

After BNT162b2 messenger RNA vaccination, antibody levels to spike, receptor-binding domain, and virus neutralization were examined in 149 nursing home residents and 110 healthcare worker controls. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naive nursing home residents' median post-second vaccine dose antibody neutralization titers are one-quarter that of SARS-CoV-2-naive healthcare workers.

Heterogeneous immunogenicity of SARS-CoV-2 vaccines in cancer patients receiving radiotherapy.

The immunogenicity of SARS-CoV-2 vaccines in cancer patients receiving radiotherapy is unknown. This prospective cohort study demonstrates that anti-SARS-CoV-2 spike antibody and neutralization titers are reduced in a subset of thoracic radiotherapy patients, possibly due to immunosuppressive conditions. Antibody testing may be useful to identify candidates for additional vaccine doses.

Does a lack of vaccine side effects correlate with reduced BNT162b2 mRNA vaccine response among healthcare workers and nursing home residents?

BACKGROUND: The BNT162b2 SARS-CoV-2 mRNA vaccination has mitigated the burden of COVID-19 among residents of long-term care facilities considerably, despite being excluded from the vaccine trials. Data on reactogenicity (vaccine side effects) in this population are limited. AIMS: To assess reactogenicity among nursing home (NH) residents. To provide a plausible proxy for predicting vaccine response among this population. METHODS: We enrolled and sampled NH residents and community-dwelling healthcare workers who received the BNT162b2 mRNA vaccine, to assess local or systemic reactogenicity and antibody levels (immunogenicity). RESULTS: NH residents reported reactions at a much lower frequency and lesser severity than the community-dwelling healthcare workers. These reactions were mild and transient with all subjects experiencing more local than systemic reactions. Based on our reactogenicity and immunogenicity data, we developed a linear regression model predicting log-transformed anti-spike, anti-receptor-binding domain (RBD), and neutralizing titers, with a dichotomous variable indicating the presence or absence of reported reactions which revealed a statistically significant effect, with estimated shifts in log-transformed titers ranging from 0.32 to 0.37 (all p < 0.01) indicating greater immunogenicity in subjects with one or more reported reactions of varying severity. DISCUSSION: With a significantly lower incidence of post-vaccination reactions among NH residents as reported in this study, the BNT162b2 mRNA vaccine appears to be well-tolerated among this vulnerable population. If validated in larger populations, absence of reactogenicity could help guide clinicians in prioritizing vaccine boosters. CONCLUSIONS: Reactogenicity is significantly mild among nursing home residents and overall, subjects who reported post-vaccination reactions developed higher antibody titers.

Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses.

Initial exposure to a pathogen elicits an adaptive immune response to control and eradicate the threat. Interrogating the abundance and specificity of the naive B cell repertoire drives understanding of how to mount protective responses. Here, we isolated naive B cells from eight seronegative human donors targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD). Single-cell B cell receptor (BCR) sequencing identified diverse gene usage and no restriction on complementarity determining region length. A subset of recombinant antibodies produced by naive B cell precursors bound to SARS-CoV-2 RBD and engaged circulating variants including B.1.1.7, B.1.351, and B.1.617.2, as well as preemergent bat-derived coronaviruses RaTG13, SHC104, and WIV1. By structural characterization of a naive antibody in complex with SARS-CoV-2 spike, we identified a conserved mode of recognition shared with infection-induced antibodies. We found that representative naive antibodies could signal in a B cell activation assay, and by using directed evolution, we could select for a higher-affinity RBD interaction, conferred by a single amino acid change. The minimally mutated, affinity-matured antibodies also potently neutralized SARS-CoV-2. Understanding the SARS-CoV-2 RBD­specific naive repertoire may inform potential responses capable of recognizing future SARS-CoV-2 variants or emerging coronaviruses, enabling the development of pan-coronavirus vaccines aimed at engaging protective germline responses.

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity.

Vaccination elicits immune responses capable of potently neutralizing SARS-CoV-2. However, ongoing surveillance has revealed the emergence of variants harboring mutations in spike, the main target of neutralizing antibodies. To understand the impact of these variants, we evaluated the neutralization potency of 99 individuals that received one or two doses of either BNT162b2 or mRNA-1273 vaccines against pseudoviruses representing 10 globally circulating strains of SARS-CoV-2. Five of the 10 pseudoviruses, harboring receptor-binding domain mutations, including K417N/T, E484K, and N501Y, were highly resistant to neutralization. Cross-neutralization of B.1.351 variants was comparable to SARS-CoV and bat-derived WIV1-CoV, suggesting that a relatively small number of mutations can mediate potent escape from vaccine responses. While the clinical impact of neutralization resistance remains uncertain, these results highlight the potential for variants to escape from neutralizing humoral immunity and emphasize the need to develop broadly protective interventions against the evolving pandemic.

COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells.

The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.