Distinct in vitro and in vivo neutralization profiles of monoclonal antibodies elicited by the receptor binding domain of the ancestral SARS-CoV-2.

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains.

SARS-CoV-2 variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta) show increased transmissibility and enhanced antibody neutralization resistance. Here we demonstrate in K18-hACE2 transgenic mice that B.1.1.7 and B.1.351 are 100-fold more lethal than the original SARS-CoV-2 bearing 614D. B.1.1.7 and B.1.351 cause more severe organ lesions in K18-hACE2 mice than early SARS-CoV-2 strains bearing 614D or 614G, with B.1.1.7 and B.1.351 infection resulting in distinct tissue-specific cytokine signatures, significant D-dimer depositions in vital organs and less pulmonary hypoxia signaling before death. However, K18-hACE2 mice with prior infection of early SARS-CoV-2 strains or intramuscular immunization of viral spike or receptor binding domain are resistant to the lethal reinfection of B.1.1.7 or B.1.351, despite having reduced neutralization titers against these VOC than early strains. Our results thus distinguish pathogenic patterns in K18-hACE2 mice caused by B.1.1.7 and B.1.351 infection from those induced by early SARS-CoV-2 strains, and help inform potential medical interventions for combating COVID-19.