Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models

Abstract

The weakened protective efficacy of COVID-19 vaccines and antibodies caused by SARS-CoV-2 variants presents a global health emergency, which underscores the urgent need for universal therapeutic antibody intervention for clinical patients. Here, we screened three alpacas-derived nanobodies (Nbs) with neutralizing activity from twenty RBD-specific Nbs. The three Nbs were fused with the Fc domain of human IgG, namely aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc, which could specifically bind RBD protein and competitively inhibit the binding of ACE2 receptor to RBD. They effectively neutralized SARS-CoV-2 pseudoviruses D614G, Alpha, Beta, Gamma, Delta, and Omicron sub-lineages BA.1, BA.2, BA.4, and BA.5 and authentic SARS-CoV-2 prototype, Delta, and Omicron BA.1, BA.2 strains. In mice-adapted COVID-19 severe model, intranasal administration of aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc effectively protected mice from lethal challenges and reduced viral loads in both the upper and lower respiratory tracts. In the COVID-19 mild model, aVHH-13-Fc, which represents the optimal neutralizing activity among the above three Nbs, effectively protected hamsters from the challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and BA.2 by significantly reducing viral replication and pathological alterations in the lungs. In structural modeling of aVHH-13 and RBD, aVHH-13 binds to the receptor-binding motif region of RBD and interacts with some highly conserved epitopes. Taken together, our study illustrated that alpaca-derived Nbs offered a therapeutic countermeasure against SARS-CoV-2, including those Delta and Omicron variants which have evolved into global pandemic strains.

Keywords: Broad-spectrum; COVID-19; Nanobody; Rodent models; SARS-CoV-2; Therapeutic.

Fig. 1

Screening and characterization of SARS-CoV-2 nanobodies (Nbs). A Alpacas were immunized with SARS-CoV-2 S DNA vaccine and S-trimer protein for 6 doses at week 0, 3, 6, 9, 12, and 20, respectively. B Neutralizing titers of immunized alpaca serum was detected by SARS-CoV-2 D614G pseudovirus based on HIV pseudovirus system following vaccination. PBMCs were isolated from the whole blood of alpacas on week 21 for construction of phage library. C Positive rate of 192 monoclonal phage supernatants by phage-ELISA, the positive sample was defined as the ratio of OD₄₅₀ value between the experiment group and negative control is greater than 3. D Percentage of inhibition of 192 monoclonal phage supernatants to SARS-CoV-2 D614G pseudovirus. According to the capture of luciferase report gene, Nbs with inhibition rates greater than or equal to 97% were extracted for sequencing and alignment. E Sequence alignment of 20 VHH gene sequences. FR1 to FR4 and CDR1 to CDR3 were marked. F Metabolic dynamics of Nbs. Antibody level of aVHH-13 and aVHH-13-Fc were measured at different time points post tail vein injection in mice. T1/2: the half-life of terminal elimination, the time required for the terminal phase blood drug concentration to decrease by half. Tmax: peak time, the time for the drug action to reach the peak. Cmax: peak concentration, the highest value of blood drug concentration after administration. Data were presented as mean with standard deviation.

Fig. 2

Competitive inhibition with ACE2 and binding ability with RBD of nanobodies (Nbs). A The competitive inhibition between Nbs and ACE2-Fc in SARS-CoV-2 RBD-binding, determined by competitive ELISA. Briefly, RBD-his protein was coated, following blocking, ACE2-Fc adding and Nbs adding, anti-human IgG Fc antibody and HRP labeled secondary antibody was added. After color rendering, results were read at 450 ​nm absorbance. B The binding affinity between Nbs and SARS-CoV-2 RBD determined by indirect ELISA. Binding capacity was expressed in half effect concentration (EC₅₀). Data were presented as mean with standard deviation.

Fig. 3

Nanobodies (Nbs) possess broad-spectrum neutralizing capability against pseudoviral SARS-CoV-2 VOCs. A–I Measurement of the neutralizing potency of aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc against SARS-CoV-2 D614G, Alpha, Beta, Delta, Gamma, Omicron BA.1, Omicron BA.2, Omicron BA.4, Omicron BA.5 pseudoviruses, respectively. Briefly, neutralization titer was measured based on lentivirus pseudoviral system. Pseudoviruses of SARS-CoV-2 strains were prepared by co-transfection of SARS-CoV-2 S and lentivirus skeleton plasmids, harvested and tittered. Then pseudoviruses were added in serial diluted serum samples or antibodies. After incubation and cells adding, luciferase was added and neutralization titers were presented as IC₅₀. IC₅₀ was determined by the serum dilution that inhibiting 50% concentration of luciferase. Data were presented as mean with standard deviation.

Fig. 4

Broad-spectrum neutralization capability against authentic SARS-CoV-2. Neutralization antibody titers of aVHH-11-Fc, aVHH-13-Fc, and aVHH-14-Fc for SARS-CoV-2 BMA8, prototype, Delta, Omicron BA.1 and Omicron BA.2. Briefly, neutralization titer was detected by authentic SARS-CoV-2. Nbs were successively diluted dilution in a two-fold scale and 100 TCID₅₀ SARS-CoV-2 were added. Post incubation, cells were added. The maximum antibody dilution concentrations that neutralize all viral CPE were defined as the neutralizing titer. Statistical analysis was performed by two-way ANOVA, ∗P ​< ​0.05. Data were presented as mean with standard deviation.

Fig. 5

Protective efficacy of nanobodies (Nbs) against lethal infection of the mouse-adapted SARS-CoV-2 in mice. A Experimental schedule for nanobody therapeutics in mice. Groups of BALB/c mice (n ​= ​20) were challenged with 50 LD₅₀ of BMA8 via the intranasal route, followed by three doses successive intranasal administration with aVHH-11-Fc, aVHH-13-Fc or aVHH-14-Fc at 0.5, 1 and 2 ​h post infection. Nbs with a dose of 20 ​mg/kg were given to each mouse. The survival rate, weight change and body temperature of BALB/c mice were monitored daily after SARS-CoV-2 BMA8 infection. Lung and nasal turbinates were taken (n ​= ​3) on 3 days post infection (dpi), 7 dpi and 14 dpi, respectively for viral loads determination and virus tittering. B–D Survival rate, weight change and body temperature during the 14 days successive monitoring post challenge. E, G Viral loads quantified by RT-qPCR at 3 dpi, 7 dpi and 14 dpi in lung and nasal turbinates. F, H Virus titer was conducted by TCID₅₀ using Reed-Muench methods at 3 dpi, 7 dpi and 14 dpi in lung and nasal turbinates. I HE and IHC staining of lung tissue from nanobody treatment group (aVHH-13-Fc). No obvious pulmonary lesions or SARS-CoV-2 antigens were recorded. Scale bar ​= ​100 ​μm. J HE and IHC staining of lung tissue from PBS control group. Pulmonary lesions including mucosal epithelial cell degeneration, necrosis and sloughing, inflammatory necrosis in some lumens (blue arrows), and inflammatory cell infiltration (yellow arrows) were observed. Abundant SARS-CoV-2 antigens were observed in IHC staining (yellow region). K–O The hematological values of BALB/c mice were analyzed at 3 days after SARS-CoV-2 BMA8 infection. K neutrophil (Neu) percentage. L White blood cell (WBC) count. M platelet (PLT). N Monocyte (Mno). O lymphocyte (LYM) percentage. Data are presented as the mean ​± ​standard deviation (n ​= ​4). Statistical analysis was performed by two-way ANOVA. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001.

Fig. 6

Protective efficacy of nanobodies (Nbs) against SARS-CoV-2 VOCs in golden hamsters. A Experimental schedule for Nbs therapeutics in golden hamsters. Groups of golden hamsters (n ​= ​14) were challenged with 1000 TCID₅₀ of SARS-CoV-2 prototype, Delta, Omicron BA.1 or BA.2 via the intranasal route, followed by three doses successive intranasal administration of aVHH-11-Fc, aVHH-13-Fc or aVHH-14-Fc at 0.5, 1 and 2 ​h post infection. Nbs with a dose of 20 ​mg/kg were given to each hamster. The survival rate and weight change were monitored daily after challenge. Lung and nasal turbinates were taken on 3 days post infection (dpi), 7 dpi and 14 dpi, respectively for viral loads determination and virus tittering. B, C Survival rate and weight change during the 14 days monitoring post challenge. D, F Viral loads quantified by RT-qPCR at 3 dpi, 7 dpi and 14 dpi in lung and nasal turbinates. E, G Virus titers was conducted by TCID₅₀ using Reed-Muench methods at 3 dpi, 7 dpi and 14 dpi in lung and nasal turbinates. Data are presented as the mean ​± ​SD (n ​= ​3). Statistical analysis was performed by two-way ANOVA (∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001). J–M The basic structure of the lung tissues in treatment groups given purified aVHH-13-Fc post challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and Omicron BA.2, respectively; Scale bar ​= ​100 ​μm ​N–Q Abnormality of lung tissue structure were marked, represent as alveolar wall thickening (black arrow), mild bleeding (yellow arrow) and a small amount of lymphocyte and neutrophil infiltration (red arrow). R–U Viral antigens detected in the lung sections of purified aVHH-13-Fc treatment group post challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and Omicron BA.2, respectively. V–Y Abundant viral antigens (yellow regions) detected in lung sections in control group. The figure showed immunohistochemistry (IHC) labeling against SARS-CoV-2 N, scale bar ​= ​100 ​μm.

Fig. 7

Structure docking model of nanobodies bound to RBDs. Crystal structure of SARS-CoV-2 RBD proteins were applied in structural modeling with aVHH-13, energy minimization and energy minimized were then conducted. A Structural analysis of the combination of aVHH-13 and SASR-CoV-2 prototype-RBD. RBD is colored in green and aVHH-13 is colored in cyan (same color annotation below). B Structural analysis of the combination of aVHH-13 and SASR-CoV-2 Delta-RBD. C Structural analysis of the combination of aVHH-13 and SASR-CoV-2 Omicron BA.1-RBD. D Structural analysis of the combination of aVHH-13 and SASR-CoV-2 Omicron BA.2-RBD.