A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has become a serious burden on global public health. Although therapeutic drugs against COVID-19 have been used in many countries, their efficacy is still limited. We here reported nanobody (Nb) phage display libraries derived from four camels immunized with the SARS-CoV-2 spike receptor-binding domain (RBD), from which 381 Nbs were identified to recognize SARS-CoV-2-RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2-RBD variants and two Nbs blocked the interaction of human ACE2 with bat-SL-CoV-WIV1-RBD and SARS-CoV-1-RBD. Among these candidates, Nb11-59 exhibited the highest activity against authentic SARS-CoV-2 with 50% neutralizing dose (ND₅₀) of 0.55 μg/ml. Nb11-59 can be produced on large scale in Pichia pastoris, with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11-59 might be a promising prophylactic and therapeutic molecule against COVID-19, especially through inhalation delivery.

Keywords: SARS‐CoV‐2; large‐scale production; nanobody; nebulization; neutralizing activity.

FIGURE 1

Schematic depicting the immunization and screening strategy that were used to isolate SARS‐CoV‐2 Spike RBD‐directed Nbs. PBLs, peripheral blood lymphocytes

FIGURE 2

Identification of Nbs specific to SARS‐CoV‐2‐RBD. (A) PE‐ELISA was performed to identify positive clones. The clones with binding ratio higher than 3 were considered as positive clones. (B) Phylogenetic tree of the isolated SARS‐CoV‐2‐RBD‐directed Nbs, based on the neighbor joining method

FIGURE 3

Identification and characterization of SARS‐CoV‐2‐RBD/ACE2 blocking Nbs. (A) Examples are given to illustrate the results of screening by FACS. (B) Screening for SARS‐CoV‐2‐RBD/ACE2 blocking Nbs. (C) Purification of blocking SARS‐CoV‐2‐RBD Nbs and SDS‐PAGE was used to check the purity of these Nbs. (D) Characterizations of blocking Nb candidates including EC₅₀, IC₅₀, yield, PA binding, and Tm were displayed. (E) Sequences of CDR3 region of seven selected SARS‐CoV‐2‐RBD Nbs

FIGURE 4

The affinity and blocking capacity of seven Nbs toward SARS‐CoV‐2‐RBD different mutants and other coronavirus species. (A) Affinity of selected candidates was measured by BLI. (B) The blocking activity against SARS‐CoV‐2‐RBD mutants/ACE2 interaction was determined by FACS. (C) The binding and blocking activities of selected seven candidates toward bat‐SL‐CoV‐WIV1‐RBD were detected through ELISA and FACS, respectively. (D) The binding and blocking activities of selected seven candidates toward SARS‐CoV‐1‐RBD were detected through ELISA and FACS, respectively

FIGURE 5

Evaluation of neutralizing potential of seven Nbs using plaque reduction neutralization test. (A) Plaques formed in Vero E6 cells inoculated with 100 PFU SARS‐CoV‐2 and 10‐fold diluted Nbs. Cry1B Nb was used as the negative control. (B) Inhibitory rate of seven Nbs against authentic SARS‐CoV‐2 at the concentration of 50 and 5 μg/ml. (C) Plaques formed in Vero E6 cells inoculated with 100 PFU SARS‐CoV‐2 plus threefold diluted Nb16‐68 or Nb11‐59 mixture. (D) 50% ND₅₀ of Nb16‐68 or Nb11‐59 was determined. The results shown are mean value ± SD

FIGURE 6

The large‐scale production and preliminary druggability analysis. (A) The yield of HuNb11‐59 in fermentation tank was determined at indicated times, which were detected by SDS‐PAGE assay, with 1 g/L Nb11‐59 as the standard protein. Lanes 1–8, 10 μl supernatants after 0, 24, 48, 72, 120, 159.5, 183.5, and 213 h of induction; lane 9, 10 μl 1/20 diluted culture supernatant induced for 213 h; lane 10, 10 μl of 1 g/L high‐purity target protein. (B) The purity of HuNb11‐59 was determined through SDS‐PAGE, following affinity chromatography and hydrophobic chromatography. (C) The purity of HuNb11‐59 was determined through SEC‐HPLC, AEX‐HPLC, and RP‐HPLC analysis. (D) The affinity of HuNb11‐59 was measured by BLI (left). The blocking activity of Nb11‐59 and HuNb11‐59 against SARS‐CoV‐2‐RBD/ACE interaction was determined by FACS (right). (E) The stability of HuNb11‐59 under different temperatures and freeze‐and‐thaw cycles was detected by SEC‐HPLC. (F) The purity of nebulized HuNb11‐59 formulation was detected by SEC‐HPLC