Broadening sarbecovirus neutralization with bispecific antibodies combining distinct conserved targets on the receptor binding domain.

Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs and cocktails against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2-02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2-02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.

Broad SARS-CoV-2 neutralization by monoclonal and bispecific antibodies derived from a Gamma-infected individual.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins.

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses.

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses.

Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate.

Hepatitis C virus (HCV) infection affects approximately 58 million people and causes ~300,000 deaths yearly. The only target for HCV neutralizing antibodies is the highly sequence diverse E1E2 glycoprotein. Eliciting broadly neutralizing antibodies that recognize conserved cross-neutralizing epitopes is important for an effective HCV vaccine. However, most recombinant HCV glycoprotein vaccines, which usually include only E2, induce only weak neutralizing antibody responses. Here, we describe recombinant soluble E1E2 immunogens that were generated by permutation of the E1 and E2 subunits. We displayed the E2E1 immunogens on two-component nanoparticles and these nanoparticles induce significantly more potent neutralizing antibody responses than E2. Next, we generated mosaic nanoparticles co-displaying six different E2E1 immunogens. These mosaic E2E1 nanoparticles elicit significantly improved neutralization compared to monovalent E2E1 nanoparticles. These results provide a roadmap for the generation of an HCV vaccine that induces potent and broad neutralization.