ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses. Graphical The SARS-CoV-2 spike protein was engineered to recruit ESCRT proteins to its cytoplasmic tail by adding an EABR motif, which induced the assembly of enveloped virus-like particles. A COVID-19 mRNA vaccine encoding spike-EABR elicited superior antibody responses compared to a conventional mRNA vaccine in mice.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes.

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer cross-linking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb-resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models

To combat future SARS-CoV-2 variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles presenting randomly-arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against epitopes that are conserved and relatively occluded, rather than variable, immunodominant, and exposed. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD-nanoparticles in mice and macaques, observing stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants including Omicrons and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest mosaic-8 RBD-nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers. Description

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

Many anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) neutralizing antibodies target the angiotensin-converting enzyme 2 (ACE2) binding site on viral spike receptor-binding domains (RBDs). Potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly emergent zoonotic sarbecoviruses and variants, but they usually show only weak neutralization potencies. Here, we characterize two class 4 anti-RBD antibodies derived from coronavirus disease 2019 (COVID-19) donors that exhibit breadth and potent neutralization of zoonotic coronaviruses and SARS-CoV-2 variants. C118-RBD and C022-RBD structures reveal orientations that extend from the cryptic epitope to occlude ACE2 binding and CDRH3-RBD main-chain H-bond interactions that extend an RBD ß sheet, thus reducing sensitivity to RBD side-chain changes. A C118-spike trimer structure reveals rotated RBDs that allow access to the cryptic epitope and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice.

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.