Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.

Introduction: Hemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research. Methods: As a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination. Results: This qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria. Discussion: These results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates.

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs.

An epitope-enriched immunogen expands responses to a conserved viral site.

Pathogens evade host humoral responses by accumulating mutations in surface antigens. While variable, there are conserved regions that cannot mutate without compromising fitness. Antibodies targeting these conserved epitopes are often broadly protective but remain minor components of the repertoire. Rational immunogen design leverages a structural understanding of viral antigens to modulate humoral responses to favor these responses. Here, we report an epitope-enriched immunogen presenting a higher copy number of the influenza hemagglutinin (HA) receptor-binding site (RBS) epitope relative to other B cell epitopes. Immunization in a partially humanized murine model imprinted with an H1 influenza shows H1-specific serum and >99% H1-specific B cells being RBS-directed. Single B cell analyses show a genetically restricted response that structural analysis defines as RBS-directed antibodies engaging the RBS with germline-encoded contacts. These data show how epitope enrichment expands B cell responses toward conserved epitopes and advances immunogen design approaches for next-generation viral vaccines.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

Development of flow cytometry-based assays to assess the ability of antibodies to bind to SARS-CoV-2-infected and spike-transfected cells and mediate NK cell degranulation.

Since the beginning of the SARS-CoV-2 pandemic, antibody responses and antibody effector functions targeting SARS-CoV-2-infected cells have been understudied. Consequently, the role of these types of antibodies in SARS-CoV-2 disease (COVID-19) and immunity is still undetermined. To provide tools to study these responses, we used plasma from SARS-CoV-2-infected individuals (n = 50) and SARS-CoV-2 naive healthy controls (n = 20) to develop four specific and reproducible flow cytometry-based assays: (i) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2-infected cells and (ii) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2 Spike-transfected cells. All four assays demonstrated the ability to detect the presence of these functional antibody responses in a specific and reproducible manner. Interestingly, we found weak to moderate correlations between the four assays (Spearman rho ranged from 0.50 to 0.74), suggesting limited overlap in the responses captured by the individual assays. Lastly, while we initially developed each assay with multiple dilutions in an effort to capture the full relationship between antibody titers and assay outcome, we explored the relationship between fewer antibody dilutions and the full dilution series for each assay to reduce assay costs and improve assay efficiency. We found high correlations between the full dilution series and fewer or single dilutions of plasma. Use of single or fewer sample dilutions to accurately determine the response rates and magnitudes of the responses allows for high-throughput use of these assays platforms to facilitate assessment of antibody responses elicited by SARS-CoV-2 infection and vaccination in large clinical studies.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine.

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

Rapid test to assess the escape of SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are concerning in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a rapid test, termed CoVariant-SCAN, that detects neutralizing antibodies (nAbs) capable of blocking interactions between the angiotensin-converting enzyme 2 receptor and the spike protein of wild-type (WT) SARS-CoV-2 and three other variants: B.1.1.7, B.1.351, and P.1. Using CoVariant-SCAN, we assessed neutralization/blocking of monoclonal antibodies and plasma from COVID-19­positive and vaccinated individuals. For several monoclonal antibodies and most plasma samples, neutralization against B.1.351 and P.1 variants is diminished relative to WT, while B.1.1.7 is largely cross-neutralized. We also showed that we can rapidly adapt the platform to detect nAbs against an additional variant­B.1.617.2 (Delta)­without reengineering or reoptimizing the assay. Results using CoVariant-SCAN are consistent with live virus neutralization assays and demonstrate that this easy-to-deploy test could be used to rapidly assess nAb response against multiple SARS-CoV-2 variants.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA "breathing" on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Multiplexed, quantitative serological profiling of COVID-19 from blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens-spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)-simultaneously from 60 µl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ-induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.

Differential immune imprinting by influenza virus vaccination and infection in nonhuman primates.

Immune memory of a first infection with influenza virus establishes a lasting imprint. Recall of that memory dominates the response to later infections or vaccinations by antigenically drifted strains. Early childhood immunization before infection may leave an imprint with different characteristics. We report here a comparison of imprinting by vaccination and infection in a small cohort of nonhuman primates (NHPs). We assayed serum antibody responses for binding with hemaglutinnins (HAs) both from the infecting or immunizing strain (H3 A/Aichi 02/1968) and from strains representing later H3 antigenic clusters ("forward breadth") and examined the effects of defined HA mutations on serum titers. Initial exposure by infection elicited strong HA-binding and neutralizing serum antibody responses but with little forward breadth; initial vaccination with HA from the same strain elicited a weaker response with little neutralizing activity but considerable breadth of binding, not only for later H3 HAs but also for HA of the 2009 H1 new pandemic virus. Memory imprinted by infection, reflected in the response to two immunizing boosts, was largely restricted (as in humans) to the outward-facing HA surface, the principal region of historical variation. Memory imprinted by immunization showed exposure to more widely distributed epitopes, including sites that have not varied during evolution of the H3 HA but that yield nonneutralizing responses. The mode of initial exposure thus affects both the strength of the response and the breadth of the imprint; design of next-generation vaccines will need to take the differences into account.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys.

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

Cold sensitivity of the SARS-CoV-2 spike ectodomain.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates.

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.