Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants

Abstract

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two Nb-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

Development of Nanobody-Displayed Whole-Cell Biosensors for the Colorimetric Detection of SARS-CoV-2

“…As shown in Figure c, the yeast-Nb33 achieved the highest signal response among these bioreceptors. According to the ELISA results from our collaborator, the Nb-33 achieved the lowest half-maximal effective concentration (EC 50 ) toward spike protein among the three nanobodies . This result indicated that Nb-33 has a higher capture efficiency and could be regarded as an ideal candidate for SARS-CoV-2 detection.…”

“…According to the ELISA results from our collaborator, the Nb-33 achieved the lowest half-maximal effective concentration (EC 50 ) toward spike protein among the three nanobodies. 21 This result indicated that Nb-33 has a higher capture efficiency and could be regarded as an ideal candidate for SARS-CoV-2 detection. During culturing, yeast-Nb33 grew at the slowest rate, which might indicate there are more nanobodies expressed on the yeast surface.…”

“…Nb-33 gives a positive band in Western Blot (WB) with spike protein under native conditions and by peptide array analysis seems to be directed to an epitope located at the S2 domain and does not possess neutralizing activity, Nb-45 also recognized the spike protein in a native gel, possesses low neutralizing activity, and according to biliverdin blocking assays can be directed to the NTD region. Finally, Nb-46 is not neutralizing, directed to a conformational epitope in Spike (negative by WB) …”

“…Finally, Nb-46 is not neutralizing, directed to a conformational epitope in Spike (negative by WB). 21 2.3. Construction of Nanobody-Display Vectors.…”

“…Our whole-cell biosensor takes advantage of yeast cells and AuNPs, offering a low-cost, highly sensitive, and user-friendly detection of SARS-CoV-2. 21 The Nb selected for the development of the present diagnosis assay were obtained after panning an immune library with the full-length, locked, prefused spike protein of the Wuhan-Hu-1 strain SARS-CoV-2. The three selected clones (Nb-33, Nb-45, and Nb-46) recognized the spike protein but no receptor binding domain (RBD) by enzyme-linked immunoassay (ELISA).…”