Convalescent human IgG, but not IgM, from COVID-19 survivors confers dose-dependent protection against SARS-CoV-2 replication and disease in hamsters.

Introduction: Antibody therapeutic strategies have served an important role during the COVID-19 pandemic, even as their effectiveness has waned with the emergence of escape variants. Here we sought to determine the concentration of convalescent immunoglobulin required to protect against disease from SARS-CoV-2 in a Syrian golden hamster model. Methods: Total IgG and IgM were isolated from plasma of SARS-CoV-2 convalescent donors. Dose titrations of IgG and IgM were infused into hamsters 1 day prior to challenge with SARS-CoV-2 Wuhan-1. Results: The IgM preparation was found to have ~25-fold greater neutralization potency than IgG. IgG infusion protected hamsters from disease in a dose-dependent manner, with detectable serum neutralizing titers correlating with protection. Despite a higher in vitro neutralizing potency, IgM failed to protect against disease when transferred into hamsters. Discussion: This study adds to the growing body of literature that demonstrates neutralizing IgG antibodies are important for protection from SARS-CoV-2 disease, and confirms that polyclonal IgG in sera can be an effective preventative strategy if the neutralizing titers are sufficiently high. In the context of new variants, against which existing vaccines or monoclonal antibodies have reduced efficacy, sera from individuals who have recovered from infection with the emerging variant may potentially remain an efficacious tool.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants.

The emergence of novel potentially pandemic pathogens necessitates the rapid manufacture and deployment of effective, stable, and locally manufacturable vaccines on a global scale. In this study, the ability of the Escherichia coli expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was evaluated. The RBD of the original Wuhan-Hu1 variant and of the Alpha and Beta variants of concern (VoC) were expressed in E. coli, and their biochemical and immunological profiles were compared to RBD produced in mammalian cells. The E. coli-produced RBD variants recapitulated the structural character of mammalian-expressed RBD and bound to human angiotensin converting enzyme (ACE2) receptor and a panel of neutralizing SARS-CoV-2 monoclonal antibodies. A pilot vaccination in mice with bacterial RBDs formulated with a novel liposomal adjuvant, Army Liposomal Formulation containing QS21 (ALFQ), induced polyclonal antibodies that inhibited RBD association to ACE2 in vitro and potently neutralized homologous and heterologous SARS-CoV-2 pseudoviruses. Although all vaccines induced neutralization of the non-vaccine Delta variant, only the Beta RBD vaccine produced in E. coli and mammalian cells effectively neutralized the Omicron BA.1 pseudovirus. These outcomes warrant further exploration of E. coli as an expression platform for non-glycosylated, soluble immunogens for future rapid response to emerging pandemic pathogens.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Evaluation of Antibody-Dependent Fc-Mediated Viral Entry, as Compared With Neutralization, in SARS-CoV-2 Infection.

Fc-mediated virus entry has been observed for many viruses, but the characterization of this activity in convalescent plasma against SARS-CoV-2 Variants of Concern (VOC) is undefined. In this study, we evaluated Fc-mediated viral entry (FVE) on FcγRIIa-expressing HEK293 cells in the presence of SARS-CoV-2 convalescent plasma and compared it with SARS-CoV-2 pseudovirus neutralization using ACE2-expressing HEK293 cells. The plasma were collected early in the pandemic from 39 individuals. We observed both neutralization and FVE against the infecting Washington SARS-CoV-2 strain for 31% of plasmas, neutralization, but not FVE for 61% of plasmas, and no neutralization or FVE for 8% of plasmas. Neutralization titer correlated significantly with the plasma dilution at which maximum FVE was observed, indicating Fc-mediated uptake peaked as neutralization potency waned. While total Spike-specific plasma IgG levels were similar between plasma that mediated FVE and those that did not, Spike-specific plasma IgM levels were significantly higher in plasma that did not mediate FVE. Plasma neutralization titers against the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOC were significantly lower than titers against the Washington strain, while plasma FVE activity against the VOC was either higher or similar. This is the first report to demonstrate a functional shift in convalescent plasma antibodies from neutralizing and FVE-mediating against the earlier Washington strain, to an activity mediating only FVE and no neutralization activity against the emerging VOC, specifically the Beta (B.1.351) and Gamma (P.1) VOC. It will be important to determine the in vivo relevance of these findings.

A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 µg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations.

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)­directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform.

Serological assessment of SARS-CoV-2 specific responses are an essential tool for determining the prevalence of past SARS-CoV-2 infections in the population especially when testing occurs after symptoms have developed and limited contact tracing is in place. The goal of our study was to test a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. We carried out the assay on samples collected from three sample groups: subjects diagnosed with COVID-19 from the U.S. Army hospital at Camp Humphreys in Pyeongtaek, South Korea; healthcare administrators from the same hospital but with no reported diagnosis of COVID-19; and pre-pandemic samples. We found that the new CoV-specific multiplex assay was highly sensitive allowing plasma samples to be diluted 1:30,000 with a robust signal. The reactivity of IgG responses to SARS-CoV-2 nucleocapsid protein and IgM responses to SARS-CoV-2 spike protein could distinguish COVID-19 samples from non-COVID-19 and pre-pandemic samples. The data from the three sample groups also revealed a unique pattern of cross-reactivity between SARS-CoV-2 and SARS-CoV-1, MERS-CoV, and seasonal coronaviruses HKU1 and OC43. Our findings show that the CoV-2 IgM response is highly specific while the CoV-2 IgG response is more cross-reactive across a range of human CoVs and also showed that IgM and IgG responses show distinct patterns of epitope specificity. In summary, this multiplex assay was able to distinguish samples by COVID-19 status and characterize distinct trends in terms of cross-reactivity and fine-specificity in antibody responses, underscoring its potential value in diagnostic or serosurveillance efforts.