Persistent immune imprinting occurs after vaccination with the COVID-19 XBB.1.5 mRNA booster in humans.

Immune imprinting describes how the first exposure to a virus shapes immunological outcomes of subsequent exposures to antigenically related strains. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron breakthrough infections and bivalent COVID-19 vaccination primarily recall cross-reactive memory B cells induced by prior Wuhan-Hu-1 spike mRNA vaccination rather than priming Omicron-specific naive B cells. These findings indicate that immune imprinting occurs after repeated Wuhan-Hu-1 spike exposures, but whether it can be overcome remains unclear. To understand the persistence of immune imprinting, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID-19 mRNA vaccine booster. We showed that the XBB.1.5 booster elicited neutralizing antibody responses against current variants that were dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. Therefore, immune imprinting persists after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 booster vaccination, which will need to be considered to guide future vaccination.

Persistent immune imprinting after XBB.1.5 COVID vaccination in humans.

Immune imprinting - also known as 'original antigenic sin' - describes how the first exposure to a virus shapes the immunological outcome of subsequent exposures to antigenically related strains. SARS-CoV-2 Omicron breakthrough infections and bivalent COVID-19 vaccination were shown to primarily recall cross-reactive memory B cells and antibodies induced by prior mRNA vaccination with the Wuhan-Hu-1 spike rather than priming naive B cells that recognize Omicron-specific epitopes. These findings underscored a strong immune imprinting resulting from repeated Wuhan-Hu-1 spike exposures. To understand if immune imprinting can be overcome, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID mRNA vaccine booster. Our data show that the XBB.1.5 booster elicits neutralizing antibody responses against current variants that are dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. These results indicate that immune imprinting persists even after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 spike booster mRNA vaccination, which will need to be considered to guide the design of future vaccine boosters.

Anamnestic humoral correlates of immunity across SARS-CoV-2 variants of concern.

While immune correlates against SARS-CoV-2 are typically defined at peak immunogenicity following vaccination, immunologic responses that expand selectively during the anamnestic response following infection can provide mechanistic and detailed insights into the immune mechanisms of protection. Moreover, whether anamnestic correlates are conserved across variants of concern (VOC), including the Delta and more distant Omicron VOC, remains unclear. To define the anamnestic correlates of immunity, across VOCs, we deeply profiled the humoral immune response in individuals infected with sequence-confirmed Delta or Omicron VOC after completing the vaccination series. While limited acute N-terminal domain and receptor-binding domain (RBD)-specific immune expansion was observed following breakthrough infection, a significant immunodominant expansion of opsonophagocytic Spike-specific antibody responses focused largely on the conserved S2-domain of SARS-CoV-2 was observed. This S2-specific functional humoral response continued to evolve over 2-3 weeks following Delta or Omicron breakthrough, targeting multiple VOCs and common coronaviruses. Strong responses were observed on the fusion peptide (FP) region and the heptad repeat 1 (HR1) region adjacent to the RBD. Notably, the FP is highly conserved across SARS-related coronaviruses and even non-SARS-related betacoronavirus. Taken together, our results point to a critical role of highly conserved, functional S2-specific responses in the anamnestic antibody response to SARS-CoV-2 infection across VOCs. These humoral responses linked to virus clearance can guide next-generation vaccine-boosting approaches to confer broad protection against future SARS-related coronaviruses. IMPORTANCE The Spike protein of SARS-CoV-2 is the primary target of antibody-based recognition. Selective pressures, be it the adaption to human-to-human transmission or evasion of previously acquired immunity, have spurred the emergence of variants of the virus such as the Delta and Omicron lineages. Therefore, understanding how antibody responses are expanded in breakthrough cases of previously vaccinated individuals can provide insights into key correlates of protection against current and future variants. Here, we show that vaccinated individuals who had documented COVID-19 breakthrough showed anamnestic antibody expansions targeting the conserved S2 subdomain of Spike, particularly within the fusion peptide region. These S2-directed antibodies were highly leveraged for non-neutralizing, phagocytic functions and were similarly expanded independent of the variant. We propose that through deep profiling of anamnestic antibody responses in breakthrough cases, we can identify antigen targets susceptible to novel monoclonal antibody therapy or vaccination-boosting strategies.

Cytotoxic T Cells Targeting Spike Glycoprotein Are Associated with Hybrid Immunity to SARS-CoV-2.

mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared with vaccination in the absence of prior infection. However, the immune mechanisms by which this hybrid immunity is generated and maintained are unknown. Whereas genetic variation in spike glycoprotein effectively subverts neutralizing Abs, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled human T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive (n = 13) or -convalescent (n = 17) individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells and polyfunctional Th1 responses was similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multimodal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.

Dynamics of SARS-CoV-2 VOC Neutralization and Novel mAb Reveal Protection against Omicron.

New variants of SARS-CoV-2 continue to emerge and evade immunity. We isolated SARS-CoV-2 temporally across the pandemic starting with the first emergence of the virus in the western hemisphere and evaluated the immune escape among variants. A clinic-to-lab viral isolation and characterization pipeline was established to rapidly isolate, sequence, and characterize SARS-CoV-2 variants. A virus neutralization assay was applied to quantitate humoral immunity from infection and/or vaccination. A panel of novel monoclonal antibodies was evaluated for antiviral efficacy. We directly compared all variants, showing that convalescence greater than 5 months post-symptom onset from ancestral virus provides little protection against SARS-CoV-2 variants. Vaccination enhances immunity against viral variants, except for Omicron BA.1, while a three-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a two-dose. A novel Mab neutralizes Omicron BA.1 and BA.2 variants better than the clinically approved Mabs, although neither can neutralize Omicron BA.4 or BA.5. Thus, the need remains for continued vaccination-booster efforts, with innovation for vaccine and Mab improvement for broadly neutralizing activity. The usefulness of specific Mab applications links with the window of clinical opportunity when a cognate viral variant is present in the infected population.

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants.

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant-neutralizing antibody that is a strong candidate for clinical development.

Receptor binding domain (RBD) antibodies contribute more to SARS-CoV-2 neutralization when target cells express high levels of ACE2.

Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on details of the experimental assay. Here we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. But for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.

Receptor-Binding Domain (RBD) Antibodies Contribute More to SARS-CoV-2 Neutralization When Target Cells Express High Levels of ACE2.

Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on the details of the experimental assay. Here, we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor-binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. However, for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that the ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.

Dynamics of SARS-CoV-2 VOC neutralization and novel mAb reveal protection against Omicron.

To evaluate SARS-CoV-2 variants we isolated SARS-CoV-2 temporally during the pandemic starting with first appearance of virus in the Western hemisphere near Seattle, WA, USA, and isolated each known major variant class, revealing the dynamics of emergence and complete take-over of all new cases by current Omicron variants. We assessed virus neutralization in a first-ever full comparison across variants and evaluated a novel monoclonal antibody (Mab). We found that convalescence greater than 5-months provides little-to-no protection against SARS-CoV-2 variants, vaccination enhances immunity against variants with the exception of Omicron BA.1, and paired testing of vaccine sera against ancestral virus compared to Omicron BA.1 shows that 3-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a 2-dose regimen. We also reveal a novel Mab that effectively neutralizes Omicron BA.1 and BA.2 variants over clinically-approved Mabs. Our observations underscore the need for continued vaccination efforts, with innovation for vaccine and Mab improvement, for protection against variants of SARS-CoV-2. Summary: We isolated SARS-CoV-2 temporally starting with emergence of virus in the Western hemisphere. Neutralization analyses across all variant lineages show that vaccine-boost regimen provides protection against Omicron BA.1. We reveal a Mab that protects against Omicron BA.1 and BA.2 variants.

A multifaceted high-throughput assay for probing antigen-specific antibody-mediated primary monocyte phagocytosis and downstream functions.

Monocytes are highly versatile innate immune cells responsible for pathogen clearance, innate immune coordination, and induction of adaptive immunity. Monocytes can directly and indirectly integrate pathogen-destructive instructions and contribute to disease control via pathogen uptake, presentation, or the release of cytokines. Indirect pathogen-specific instructions are conferred via Fc-receptor signaling and triggered by antibody opsonized material. Given the tremendous variation in polyclonal humoral immunity, defining the specific antibody-responses able to arm monocytes most effectively remains incompletely understood. While monocyte cell line-based assays have been used previously, cell lines may not faithfully recapitulate the full biology of monocytes. Thus, here we describe a multifaceted antigen-specific method for probing antibody-dependent primary monocyte phagocytosis (ADMP) and secondary responses. The assay not only reliably captures phagocytic uptake of immune complexes, but also detects unique changes in surface markers and cytokine secretions profiles, poorly detected by monocytic cell lines. The assay captures divergent polyclonal-monocyte recruiting activity across subjects with varying SARS-CoV-2 disease severity and also revealed biological nuances in Fc-mutant monoclonal antibody activity related to differences in Fc-receptor binding. Thus, the ADMP assay is a flexible assay able to provide key insights into the role of humoral immunity in driving monocyte phenotypic transitions and downstream functions across many diseases.

Omicron spike function and neutralizing activity elicited by a comprehensive panel of vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern comprises several sublineages, with BA.2 and BA.2.12.1 having replaced the previously dominant BA.1 and with BA.4 and BA.5 increasing in prevalence worldwide. We show that the large number of Omicron sublineage spike mutations leads to enhanced angiotensin-converting enzyme 2 (ACE2) binding, reduced fusogenicity, and severe dampening of plasma neutralizing activity elicited by infection or seven clinical vaccines relative to the ancestral virus. Administration of a homologous or heterologous booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 across all vaccines evaluated. Our data suggest that although Omicron sublineages evade polyclonal neutralizing antibody responses elicited by primary vaccine series, vaccine boosters may provide sufficient protection against Omicron-induced severe disease.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes.

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD's class 1 and class 2 epitopes, including sites 417, 478, and 484-486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines.

The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.

The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes.

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD's class 1 and class 2 epitopes, including sites 417, 478, and 484-486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.

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.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody1. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.