Reduced antigenicity of Omicron lowers host serologic response (preprint)

SARS-CoV-2 Omicron variant of concern (VOC) contains fifteen mutations on the receptor binding domain (RBD), evading most neutralizing antibodies from vaccinated sera. Emerging evidence suggests that Omicron breakthrough cases are associated with substantially lower antibody titers than other VOC cases. However, the mechanism remains unclear. Here, using a novel geometric deep-learning model, we discovered that the antigenic profile of Omicron RBD is distinct from the prior VOCs, featuring reduced antigenicity in its remodeled receptor binding sites (RBS). To substantiate our deep-learning prediction, we immunized mice with different recombinant RBD variants and found that the Omicron's extensive mutations can lead to a drastically attenuated serologic response with limited neutralizing activity in vivo, while the T cell response remains potent. Analyses of serum cross-reactivity and competitive ELISA with epitope-specific nanobodies revealed that the antibody response to Omicron was reduced across RBD epitopes, including both the variable RBS and epitopes without any known VOC mutations. Moreover, computational modeling confirmed that the RBS is highly versatile with a capacity to further decrease antigenicity while retaining efficient receptor binding. Longitudinal analysis showed that this evolutionary trend of decrease in antigenicity was also found in hCoV229E, a common cold coronavirus that has been circulating in humans for decades. Thus, our study provided unprecedented insights into the reduced antibody titers associated with Omicron infection, revealed a possible trajectory of future viral evolution and may inform the vaccine development against future outbreaks.

Super-immunity by broadly protective nanobodies to sarbecoviruses (preprint)

Vaccine boosters and infection can facilitate the development of SARS-CoV-2 antibodies with improved potency and breadth. Here, we observed super-immunity in a camelid extensively immunized with the SARS-CoV-2 receptor-binding domain (RBD). We rapidly isolated a large repertoire of specific ultrahigh-affinity nanobodies that bind strongly to all known sarbecovirus clades using integrative proteomics. These pan-sarbecovirus nanobodies (psNbs) are highly effective against SARS-CoV and SARS-CoV-2 variants including the Omicron, with the best median neutralization potency at single-digit ng/ml. Structural determinations of 13 psNbs with the SARS-CoV-2 spike or RBD revealed five epitope classes, providing insights into the mechanisms and evolution of their broad activities. The highly evolved psNbs target small, flat, and flexible epitopes that contain over 75% of conserved RBD surface residues. Their potencies are strongly and negatively correlated with the distance of the epitopes to the receptor binding sites. A highly potent, inhalable and bispecific psNb (PiN-31) was developed. Our findings inform on the development of broadly protective vaccines and therapeutics.

Llamanade: an open-source computational pipeline for robust nanobody humanization (preprint)

Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite having marked physicochemical properties, Nbs are derived from camelids and may require "humanization" to improve translational potentials for clinical trials. Here we have systematically analyzed the sequence and structural properties of Nbs based on NGS (next-generation sequencing) databases and high-resolution structures. Our analysis reveals substantial framework diversities and underscores the key differences between Nbs and human Immunoglobulin G (IgG) antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen-binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade, a user-friendly, open-source to facilitate rational humanization of Nbs. Using Nb sequence as input, Llamanade provides information on the sequence features, model structures, and optimizes solutions to humanize Nbs. The full analysis for a given Nb takes less than a minute on a local computer. To demonstrate the robustness of this tool, we applied it to successfully humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server to support the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective trials. Author SummaryCamelid Nbs are characterized by small size, excellent pharmacological properties and high flexibility in bioengineering for therapeutic development. However, Nbs are "xeno" antibodies, which require "humanization" to improve their translational potential. Currently, there is a lack of systematic investigation of Nbs to rationally guide humanization. No dedicated software has been developed for this purpose. Here, we report the development of Llamanade, an open-source computational pipeline and the first dedicated software to facilitate rational humanization of Nbs. To subjectively evaluate Llamanade, we used it to humanize a cohort of structurally diverse and ultrapotent antiviral Nbs against SARS-CoV-2. Robust humanization by Llamanade significantly improved the humanness level of Nbs to closely resemble fully human IgGs. Importantly, these highly humanized antiviral Nbs remained excellent solubility and comparably high bioactivities to the non-humanized Nb precursors. We envision that Llamanade will help advance Nb research into therapeutic development.

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes (preprint)

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses (preprint)

Globally there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals weight loss post-infection and decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology and prevents viral pneumonia. Combined with the marked stability and low production cost, this novel therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

Versatile, Multivalent Nanobody Cocktails for Highly Efficient SARS-CoV-2 Neutralization (preprint)

The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered multiple elite Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization. These promising agents are readily translated into efficient, cost-effective, and convenient therapeutics to help end this once-in-a-century health crisis.