Serial Llama Immunization with Various SARS-CoV-2 RBD Variants Induces Broad Spectrum Virus-Neutralizing Nanobodies

Abstract

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.

Keywords: COVID-19; SARS-CoV-2; antiviral nanobody; broadly neutralizing antibody; coronavirus variants; single-domain antibody; viral evasion.

Figure 1

(A) Outline of serial immunization of a llama by various SARS-CoV-2 RBD variants, as well as the screening results. (B) BLI analysis of the interaction between nanobodies H5, G7, and 2F4 with the RBD of SARS-CoV-2 Wuhan. (C) Competition BLI assay for nanobodies H5, G7, and 2F4 binding to RBD-His6 Wu.

Figure 2

(A) Activity of H5, G7, and 2F4 nanobodies (Nbs) and nanobody-Fc fusions in pseudovirus neutralization assay with Wuhan variant of the SARS-CoV-2 Spike protein. Data from four independent experiments are presented. (B) Activity of H5, G7, and 2F4 Nbs in pseudovirus neutralization assay using Wuhan, Delta, Lambda, and Kappa variants of the SARS-CoV-2 Spike protein. N/N—no visible neutralization.

Figure 3

(A) Scheme of using H5, G7, and 2F4 Fc-fusions in treatment and prophylaxis of SARS-CoV-2 infection in a hamster model. (B) Relative weight dynamics in the prophylaxis and treatment groups following administration of Nb-Fc or control total human IgG. Error bars represent means ± SD. (C) Impact of Nb-Fc on the levels of viral transcripts in hamster lungs (RT-qPCR with RdRp-specific primers). RT-qPCR with E gene-specific primers produced very similar results (not shown). Error bars represent means ± SEM.

Figure 4

(A) Dendrogram of similarity of nanobody amino acid sequences. Potent neutralizing nanobodies are boxed, and those devoid of neutralization activity are marked with an asterisk. Blue and green correspond to library 1 (Wuhan and Omicron), and red are from library 2. (B) Percentage of amino acid residue substitutions in the identified nanobodies compared to the germline-derived sequences.

Figure 5

(A) Activity of nanobodies (Nb) 9A4, 9A7, 9A21, 9A57, and 9A58 in pseudovirus neutralization assay using Wuhan, BA.1, BA.5, and XBB.1 SARS-CoV-2 Spike variants, as well as the activity of 9A7, 9A21, 9A57, 9A58 Fc-fusions (Nb-Fc) in pseudovirus neutralization assay using Wuhan, BA.1, BA.1.1, BA.2, BA.5, BQ.1, BQ.1.1, XBB, XBB.1, XBB.1.5, and XBB.1.16 variants. (B) Affinity values for the interaction of 9A7, 9A21, 9A57, 9A58, 9A80, and RBDs of Wuhan, BA.1, BA.5, and XBB.1.5 SARS-CoV-2 variants.

Figure 6

(A) BLI analysis of competition between 9A7-, 9A21-, 9A57-, 9A58-Fc fusions for binding the RBD-His6. (B) BLI analysis of competition between Nbs, including 9A57-SUMO fusion binding the RBD Wu.