SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice

Abstract

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Keywords: SARS-CoV-2; intranasal treatment; nanobodies; variants of concern; virus neutralization.

Figure 1

SARS-CoV-2 llama immunization, immune response, and Nb-library construction. (A) Immunization schedule: a llama was injected intramuscularly on days 0 and 14 with 200 μg of SARS-CoV-2 S-2P protein produced in HEK-293T, and on days 28 and 56 with 200 μg of SARS-CoV-2 S-2P produced in CHO cells and 100 μg of RBD protein emulsified in Freund’s adjuvant. Four days after the last boost, 200 mL of blood was collected, and peripheral lymphocytes were isolated to produce an immune library; (B) Total IgG titer determined by ELISA and neutralizing Ab titer determined by pVNT induced 4 and 7 days after each immunization. Four days after the third immunization (PID 32) a maximal antibody response was reached; (C) Picture illustrating neutralizing activity in llama serum determined by pVNT. The neutralization capacity increased after each immunization and correlated with a decrease in the number of fluorescent cells. A higher neutralizing titer was detected for a dilution of 1:1296 at PID 54; (D) Analysis of PCR products by agarose gel electrophoresis to confirm the number of transformants that had an insert of the proper size: each of the 48 clones that were randomly selected contained a genuine Nb fragment (~700 bp).

Figure 2

BCoV Mebus llama immunization and immune response. (A) Immunization schedule followed to produce the Nb immune library: a llama was injected intramuscularly on days 0, 14, 28, and 95 with 4.00 × 10⁹ UFF of the inactivated BCoV Mebus strain in Freund’s adjuvant. Peripheral lymphocytes were isolated from 200 mL of blood collected four days after the final boost to generate the immune library; (B) Total IgG titer determined by ELISA for BCoV and SARS-CoV-2 RBD and S-2P proteins; (C) Picture showing non-neutralizing activity of sera from a llama immunized with BCoV Mebus against SARS-CoV-2 determined by pVNT. Superimposition of the S protein structures from SARS-CoV-2 (orange) and BCoV Mebus (blue) using VMD software. Frontal (D) and upper (E) view.

Figure 3

Nanobody affinity to RBD and S-2P from SARS-CoV-2 by ELISA. The binding of the selected Nbs was analyzed in plates coated with S-2P (A) and RBD (B) proteins from the Wuhan-Hu-1 SARS-CoV-2. Different colors were assigned to each curve according to the Nbs used. Error bars represent the standard deviation (SD) of triplicates; (C) Summary table of EC₅₀ values against RBD and S domains.

Figure 4

Neutralizing activity of Nbs against SARS-CoV-2 WT strain. The neutralization potency of eight Nbs was calculated based on the pVNT results. (A) Blue, violet, and magenta symbols and lines denote Nb-104, Nb-110, and Nb-145 selected with the SARS-CoV-2 RBD protein, respectively; (B) Green, light-blue, pink, orange, and yellow symbols and lines represent Nbs selected against the S-2P protein. Black symbols and lines denote an anti-rotavirus control Nb (2KD1). Inhibition curves were performed with the selected Nbs at two-fold serial dilutions. After 48 h, the GFP signal from two images per well was quantified using ImageJ/Fiji and normalized to the number of GFP-positive cells of wells containing only pseudovirus. Inhibition curves are presented in log-transformed dilution with IC₅₀ values for each Nb. Each experiment was replicated three times. The IC₅₀ was calculated by fitting the inhibition from serially diluted Nbs to a sigmoidal dose–response curve; (C) Virus neutralization potency of each Nb was assessed by three different methodologies: pseudovirus neutralization test (pVNT), plaque reduction neutralization test (PRNT), and immunofluorescence assay (IF). Half-maximal inhibitory concentrations IC₉₀ were calculated for each Nb in each assay as described in the methods section. ND: not determined.

Figure 5

ACE2-RBD and biliverdin competition assays. (A) Competitive ELISA of ACE2 binding to RBD immobilized on plates by increasing concentrations of Nbs. The specific binding of ACE2-HRP to RBD was detected with a chromogenic reagent. The IC₅₀ was calculated by fitting the inhibition from serially diluted Nbs to a sigmoidal dose–response curve. The experiment was performed in triplicate; (B) Competitive ELISA of Nbs at different concentrations binding to S-2P protein in the presence of 5 μM of biliverdin.

Figure 6

Protection against SARS-CoV-2 challenge in k18-hACE2 mouse model. k18-hACE2 mice were challenged with 1 × 10⁵ PFU of WA-1 SARS-CoV-2 after the intranasal administration of 10 or 20 μg of Nbs. (A) The body weight changes in the animals in the control group and treatment groups were recorded for two weeks and compared; (B) Survival curves of the different treatment groups. Statistical significance was determined using One-way ANOVA with Dunnett’s post-hoc analysis. * p < 0.05, ** p < 0.01, ns: not significant.

Figure 7

Viral load in different tissues after Nb treatment and virus challenge. Virus load measured by RT qPCR in nasal turbinates (A), in the lung (B), and the brain (C). Tissues were collected at four days after challenge (n = 3 mice per tissue, 9 mice per group). Mice were treated with each Nb by the intranasal route and challenged with WA-1 SARS-CoV-2, 4 h later. Samples were prepared from infected mice for RNA isolation and RT-qPCR. Statistical significance was determined using the general mixed 2-way ANOVA model, LSD Fisher’s exact test, and Bonferroni correction. Different letters on top of bars indicate significant differences in viral load among groups for the same tissue (p < 0.001), while shared letters indicate no significant difference.

Figure 8

Enhancement of neutralization potencies against Omicron variants using Nbs cocktails. (A) Percentage of neutralization exerted by a mixture of two or three Nbs against the B.1.1.529 Omicron variant; (B) Similar experiments to A performed with the BA.2 Omicron variant. Comparison of IC₅₀ values of Nbs combinations vs. individual Nbs for the B.1.1.529 Omicron variant (C) and the BA.2 Omicron variant (D). Individual Nbs or Nb-mixtures with different letters differ significantly in IC₅₀ (One-way ANOVA, Tukey multiple comparison, p < 0.001), (E) while shared letters indicate no significant difference.

Figure 9

Analysis of Nanobody-S protein interactions. Prediction of the interaction of Nb-43 ((A), red), Nb-45 ((B), gray), and Nb-53 ((C), yellow) with the S protein (blue). Biliverdin is highlighted in green. The three panels display the S-Nb overall view and a close-up view of the interaction zone. The close-up view displays the predicted binding site, with AA in red representing those of the S protein and in black those belonging to the Nb. Amino acids enclosed in a black box and indicated with an arrow show residues that could form hydrogen bonds (dotted lines). Section B shows green circles representing possible Pi-stacking interactions.

Figure 10

Network analysis of unique nanobodies. A visual representation summarizing the RBD and S-2P binding and neutralization for the 43 isolated Nbs. Nanobodies are depicted as dots in different sizes and colors, and those with a CDR3 sequence identity greater than 70% are connected. Their neutralization potencies against pseudotyped SARS-CoV-2 WT are represented by the size of dots and the filled gradient color represents the breadth of SARS-CoV-2 variant neutralization. Dots are colored on the outer circle based on the antigen used for biopanning and whether they bind to RBD or not.