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.

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.

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.

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.

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.

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.

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.

High-Resolution Sequencing of Viral Populations during Early Simian Immunodeficiency Virus Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses.

Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (≤29 weeks postinfection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultradeep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing-based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies, leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep-sequencing-based fitness assay, we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

Heterologous Sarbecovirus Receptor Binding Domains as Scaffolds for SARS-CoV-2 Receptor Binding Motif Presentation.

Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center on targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potent neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.

Ongoing evolution of SARS-CoV-2 drives escape from mRNA vaccine-induced humoral immunity.

Since the COVID-19 pandemic began in 2020, viral sequencing has documented 131 individual mutations in the viral spike protein across 48 named variants. To determine the ability of vaccine-mediated humoral immunity to keep pace with continued SARS-CoV-2 evolution, we assessed the neutralization potency of sera from 76 vaccine recipients collected after 2 to 6 immunizations against a comprehensive panel of mutations observed during the pandemic. Remarkably, while many individual mutations that emerged between 2020 and 2022 exhibit escape from sera following primary vaccination, few escape boosted sera. However, progressive loss of neutralization was observed across newer variants, irrespective of vaccine doses. Importantly, an updated XBB.1.5 booster significantly increased titers against newer variants but not JN.1. These findings demonstrate that seasonal boosters improve titers against contemporaneous strains, but novel variants continue to evade updated mRNA vaccines, demonstrating the need for novel approaches to adequately control SARS-CoV-2 transmission.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

Heterologous sarbecovirus receptor binding domains as scaffolds for SARS-CoV-2 receptor binding motif presentation.

Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center upon targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potently neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.

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.

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.

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.