Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses

Abstract

Globally, there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss after infection, decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology, and prevents viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

Fig. 1. PiN-21 protects Syrian hamsters from SARS-CoV-2 infection.

(A) Overview of the experimental design. SARS-CoV-2 (9 × 10⁴ PFU) was intratracheally (IT) inoculated followed by intranasal (IN) delivery of 100 μg of PiN-21 (shown in blue dots) or a control Nb (shown in gray circles). Animal weight changes were monitored daily. Nasal washes and throat swabs were collected on 2 and 4 d.p.i. Animals were euthanized for necropsy on 5 (n = 3) and 10 d.p.i (n = 3), with viral titers and gRNAs of lung tissues measured. (B) Protection of weight loss of infected hamsters treated with PiN-21. ***P < 0.001. (C to E) Measurement of viral titers by the plaque assay. **P < 0.01. The dashed line indicates the detection limit of the assay. The color scheme is consistent across all the panels.

Fig. 2. Assessment of Nb delivery in the hamster respiratory system.

(A) Schematic design of PiN-21 (shown in red triangles) and PiN-21_Alb (shown in blue squares) aerosolization in hamster models. (B) Nb neutralization potency before and after aerosolization measured by PRNT50 assay. (C and D) Normalized overall neutralization activity by plaque assay of PiN-21 and PiN-21_Alb of different time points after aerosolization.

Fig. 3. Treatment efficacy of aerosolized PiN-21 in the hamster model of SARS-CoV-2.

(A) Overview of the experiment design. SARS-CoV-2 (3 × 10⁴ PFU) was intranasally inoculated. PiN-21 (shown in red triangles) or a control Nb (shown in gray circles) was aerosolized to hamsters in the cage 6 h.p.i. Animal weight changes were monitored, and nasal washes and throat swabs were taken daily. Animals were euthanized 3 d.p.i. for necropsy, with viral titers and gRNA of lung tissues measured. (B) Percentage of body weight change of PiN-21 aerosol–treated animals compared to the control (n = 6). (C) Reduction of viral titers in hamster lungs (3 d.p.i.). Significant differences were observed between treated and control groups. **P < 0.01; *P < 0.05. The dashed line indicates the detection limit of the assay. (D) Lung pathology scores of treated and control groups. Significant difference was denoted by ****P < 0.0001. (E) Hematoxylin and eosin staining of necrotizing bronchointerstitial pneumonia affiliate with abundant SARS-CoV-2 S antigen in bronchiole epithelium and alveolar type 1 and 2 pneumocytes in the control group. All images were acquired at 20×; scale bar, 100 μm. Areas marked by boxes are shown at higher magnification in the rightmost panel (scale bar, 25 μm). (F) Immunostainings of bronchointerstitial compartments (3 d.p.i.). Orange, SARS-CoV-2S; magenta, CD68/macrophages; red, CD3e⁺ T cells; teal, ACE2; gray, DAPI. The bronchiole is outlined by white hash. Total magnification, ×200; scale bar, 100 μm.