A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

SARS-CoV-2 breakthrough infections elicit potent, broad, and durable neutralizing antibody responses.

Although infections among vaccinated individuals lead to milder COVID-19 symptoms relative to those in unvaccinated subjects, the specificity and durability of antibody responses elicited by breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum-binding and -neutralizing antibody responses that are markedly more potent, durable, and resilient to spike mutations observed in variants than those in subjects who received only 2 doses of vaccine. However, we show that breakthrough cases, subjects who were vaccinated after infection, and individuals vaccinated three times have serum-neutralizing activity of comparable magnitude and breadth, indicating that an increased number of exposures to SARS-CoV-2 antigen(s) enhance the quality of antibody responses. Neutralization of SARS-CoV was moderate, however, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

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.

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.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron.

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373) cross-recognize early SARS-CoV-2 variants. T cell responses to early variants were preserved across vaccine platforms. By contrast, significant overall decreases were observed for memory B cells and neutralizing antibodies. In subjects ∼6 months post-vaccination, 90% (CD4+) and 87% (CD8+) of memory T cell responses were preserved against variants on average by AIM assay, and 84% (CD4+) and 85% (CD8+) preserved against Omicron. Omicron RBD memory B cell recognition was substantially reduced to 42% compared with other variants. T cell epitope repertoire analysis revealed a median of 11 and 10 spike epitopes recognized by CD4+ and CD8+ T cells, with average preservation > 80% for Omicron. Functional preservation of the majority of T cell responses may play an important role as a second-level defense against diverse variants.

Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2.

Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants.

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone progressive change, with variants conferring advantage rapidly becoming dominant lineages, e.g., B.1.617. With apparent increased transmissibility, variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the United Kingdom. Here we study the ability of monoclonal antibodies and convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2, complement this with structural analyses of Fab/receptor binding domain (RBD) complexes, and map the antigenic space of current variants. Neutralization of both viruses is reduced compared with ancestral Wuhan-related strains, but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2, suggesting that individuals infected previously by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insights for immunization policy with future variant vaccines in non-immune populations.

Competing Roles of Slow Oscillations and Delta Waves in Memory Consolidation versus Forgetting.

Sleep has been implicated in both memory consolidation and forgetting of experiences. However, it is unclear what governs the balance between consolidation and forgetting. Here, we tested how activity-dependent processing during sleep might differentially regulate these two processes. We specifically examined how neural reactivations during non-rapid eye movement (NREM) sleep were causally linked to consolidation versus weakening of the neural correlates of neuroprosthetic skill. Strikingly, we found that slow oscillations (SOs) and delta (δ) waves have dissociable and competing roles in consolidation versus forgetting. By modulating cortical spiking linked to SOs or δ waves using closed-loop optogenetic methods, we could, respectively, weaken or strengthen consolidation and thereby bidirectionally modulate sleep-dependent performance gains. We further found that changes in the temporal coupling of spindles to SOs relative to δ waves could account for such effects. Thus, our results indicate that neural activity driven by SOs and δ waves have competing roles in sleep-dependent memory consolidation.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.

The magnitude and timing of recalled immunity after breakthrough infection is shaped by SARS-CoV-2 variants.

Vaccination against SARS-CoV-2 protects from infection and improves clinical outcomes in breakthrough infections, likely reflecting residual vaccine-elicited immunity and recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and cellular immunity after vaccination of seropositive individuals and after Delta or Omicron breakthrough infection in vaccinated individuals. Early longitudinal sampling revealed the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titers. While vaccination of seropositive individuals resulted in robust recall of humoral and T cell immunity, recall of vaccine-elicited responses was delayed and variable in magnitude during breakthrough infections and depended on the infecting variant of concern. While the delayed kinetics of immune recall provides a potential mechanism for the lack of early control of viral replication, the recall of antibodies coincided with viral clearance and likely underpins the protective effects of vaccination against severe COVID-19.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.