An antidote approach to reduce risk and broaden utility of antibody-based therapeutics.

Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.

Ubiquibodies, synthetic E3 ubiquitin ligases endowed with unnatural substrate specificity for targeted protein silencing.

The ubiquitin-proteasome pathway (UPP) is the main route of protein degradation in eukaryotic cells and is a common mechanism through which numerous cellular pathways are regulated. To date, several reverse genetics techniques have been reported that harness the power of the UPP for selectively reducing the levels of otherwise stable proteins. However, each of these approaches has been narrowly developed for a single substrate and cannot be easily extended to other protein substrates of interest. To address this shortcoming, we created a generalizable protein knock-out method by engineering protein chimeras called "ubiquibodies" that combine the activity of E3 ubiquitin ligases with designer binding proteins to steer virtually any protein to the UPP for degradation. Specifically, we reprogrammed the substrate specificity of a modular human E3 ubiquitin ligase called CHIP (carboxyl terminus of Hsc70-interacting protein) by replacing its natural substrate-binding domain with a single-chain Fv (scFv) intrabody or a fibronectin type III domain monobody that target their respective antigens with high specificity and affinity. Engineered ubiquibodies reliably transferred ubiquitin to surface exposed lysines on target proteins and even catalyzed the formation of biologically relevant polyubiquitin chains. Following ectopic expression of ubiquibodies in mammalian cells, specific and systematic depletion of desired target proteins was achieved, whereas the levels of a natural substrate of CHIP were unaffected. Taken together, engineered ubiquibodies offer a simple, reproducible, and customizable means for directly removing specific cellular proteins through accelerated proteolysis.

Immunogenicity and protection of a variant nanoparticle vaccine that confers broad neutralization against SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with elevated transmission and a higher risk of infection for vaccinated individuals. We demonstrate that a recombinant prefusion-stabilized spike (rS) protein vaccine based on Beta/B.1.351 (rS-Beta) produces a robust anamnestic response in baboons against SARS-CoV-2 variants when given as a booster one year after immunization with NVX-CoV2373. Additionally, rS-Beta is highly immunogenic in mice and produces neutralizing antibodies against WA1/2020, Beta/B.1.351, and Omicron/BA.1. Mice vaccinated with two doses of Novavax prototype NVX-CoV2373 (rS-WU1) or rS-Beta alone, in combination, or heterologous prime-boost, are protected from challenge. Virus titer is undetectable in lungs in all vaccinated mice, and Th1-skewed cellular responses are observed. We tested sera from a panel of variant spike protein vaccines and find broad neutralization and inhibition of spike:ACE2 binding from the rS-Beta and rS-Delta vaccines against a variety of variants including Omicron. This study demonstrates that rS-Beta vaccine alone or in combination with rS-WU1 induces antibody-and cell-mediated responses that are protective against challenge with SARS-CoV-2 variants and offers broader neutralizing capacity than a rS-WU1 prime/boost regimen alone. Together, these nonhuman primate and murine data suggest a Beta variant booster dose could elicit a broad immune response to fight new and future SARS-CoV-2 variants.

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice.

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination.

Recently approved vaccines have shown remarkable efficacy in limiting SARS-CoV-2-associated disease. However, with the variety of vaccines, immunization strategies, and waning antibody titers, defining the correlates of immunity across a spectrum of antibody titers is urgently required. Thus, we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike glycoprotein (NVX-CoV2373) at two doses, administered as a single- or two-dose regimen. Both antigen dose and boosting significantly altered neutralization titers and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were associated with distinct levels of protection in the upper and lower respiratory tract. Moreover, NVX-CoV2373 elicited antibodies that functionally targeted emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease via combined Fc/Fab functions but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 in nonhuman primates following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination.

Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants. HIGHLIGHTS: NVX-CoV2373 subunit vaccine elicits receptor blocking, virus neutralizing antibodies, and Fc-effector functional antibodies.The vaccine protects against respiratory tract infection and virus shedding in non-human primates (NHPs).Both neutralizing and Fc-effector functions contribute to protection, potentially through different mechanisms in the upper and lower respiratory tract.Both macaque and human vaccine-induced antibodies exhibit altered Fc-receptor binding to emerging mutants.

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge.

There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M™ adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988).

Influenza Hemagglutinin Nanoparticle Vaccine Elicits Broadly Neutralizing Antibodies against Structurally Distinct Domains of H3N2 HA.

Influenza vaccine effectiveness varies annually due to the fast evolving seasonal influenza A(H3N2) strain and egg-derived mutations-both of which can cause a mismatch between the vaccine and circulating strains. To address these limitations, we have developed a hemagglutinin (HA)-based protein-detergent nanoparticle influenza vaccine (NIV) with a saponin-based Matrix-M™ adjuvant. In a phase 1 clinical trial of older adults, the vaccine demonstrated broadly cross-reactive A(H3N2) HA antibody responses. Two broadly neutralizing monoclonal antibodies derived from NIV-immunized mice were characterized by transmission electron microscopy (TEM), antibody competition assays, fluorescence-activated cell sorting (FACS) analysis, and protein-protein docking. These antibodies recognize two conserved regions of the head domain, namely the receptor binding site and the vestigial esterase subdomain, thus demonstrating the potential for an HA subunit vaccine to elicit antibodies targeting structurally and antigenically distinct but conserved sites. Antibody competition studies with sera from the phase 1 trial in older adults confirmed that humans also make antibodies to these two head domains and against the highly conserved stem domain. This data supports the potential of an adjuvanted recombinant HA nanoparticle vaccine to induce broadly protective immunity and improved vaccine efficacy.

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo-election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.

The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of α-synuclein was isolated that has ~8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen.

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 ß-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed ß-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.