Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19

Abstract

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')₂ immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')₂ immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Figure 1

Analysis of the purified and inactivated SARS-CoV-2. (A) SDS–PAGE: 4–15% gradient SDS–PAGE gel under nonreducing conditions and silver staining. (1) LMW marker—10 to 250 kDa; (2) Purified and inactivated SARS-CoV-2. (B) Western blot: samples of the virus, which were separated on 4–15% gradient SDS-PAGE gels, were blotted onto nitrocellulose membranes and incubated with monoclonal antibodies against the SARS-CoV-2 S protein (Lane 3) or anti-SARS-CoV-2N protein (Lane 4). The membranes were incubated with specific peroxidase-conjugated anti-rabbit IgG (1:5000), and the reactions were revealed using SuperSignal West Pico chemiluminescent substrate. (C) Protein identification through tandem mass spectrometry analysis (LC–MS/MS) of the inactivated SARS-CoV-2 purified antigen. Only proteins with at least 1 unique peptide, a score − log10P > 20 and a false discovery rate < 1% were accepted for identification and are depicted in the Table. The proportion of proteins was estimated based on the mass spectra counts of each identified protein. (A) and (B) were cropped, the original results are presented in Fig. S7. (B) is composed of two panels, representing two membrane strips processed separately, and a third strip containing a molecular weight marker was used do estimate the molecular sizes of bands (Fig. S7).

Figure 2

Purified and inactivated SARS-CoV-2 virus immunogenicity in mice. (A) ELISA plates were coated with 100 µL of purified and inactivated SARS-CoV-2 (1 × 10E + 03 virus/mL), incubated with increasing dilutions of nonimmune or experimental sera obtained from BALB/c mice, and incubated with anti-mouse HRPO-conjugated IgG. The reaction was performed by adding TMB substrate, and the absorbance was detected at λ 450 nm using a spectrophotometer. The titre was established as the highest serum dilution at which the measured absorbance was twice as high as that determined for nonimmunized mouse serum (control group). (B) Sera from mice immunized with purified and inactivated SARS-CoV-2 antigen were also tested using the CPE-VNT. The neutralization titre was defined as the inverse of the highest dilution of serum that blocks viral replication. Statistical analyses were performed using Student’s t-test followed by the Mann–Whitney test (*p ≤ 0.05).

Figure 3

Challenge test in Golden Syrian hamsters. (A) Study design—scheme for infecting animals with SARS-CoV-2 followed by treatment with equine anti-SARS-CoV-2 F(ab′)₂ immunoglobulin. (B) SARS-CoV-2 RNA copies as measured using RT-qPCR per number of β-actin RNA copies per gram of tissue. Green: G1 animals (n = 6 per subgroup), i.e., serum-treated, SARS-CoV-2-infected animals; red: G2 animals (n = 6 per subgroup), i.e., nontreated, SARS-CoV-2-infected animals. p.i. = post infection; n.s.: not significant (p value > 0.05); **p value = 0.0043 (Day 3 p.i.).

Figure 4

The pneumonic pattern of animals infected and treated with equine anti-SARS-CoV-2 F(ab′)₂ immunoglobulin. Panels show the evolution of the infectious process in the course of SARS-CoV-2 hamster experimental infection and treatment with equine serum. Infected and nontreated animals showed a more marked evolution of the diffuse interstitial pneumonia pattern, mainly on Day 7 after infection, than infected and SARS-CoV-2-treated animals. Control samples show the normal histological pattern of the tissue. Bar 100 µm; haematoxylin and eosin staining (H&E).

Figure 5

Histopathological changes in hamsters infected and treated with equine anti-SARS-CoV-2 F(ab′)₂ immunoglobulin. Cellularity (a), functional lung area (b), pneumonia (c), edema (d) and bronchitis (e) scores. Infiltration kinetics of leukocytes (f), neutrophils, and (g) mononuclear cells. Evolution of lesions in the bronchial epithelium (h). Data are presented as the means ± standard deviations of samples of six animals per group for the scores and five fields/images per slide. The data were analysed statistically with one-way ANOVA, followed by Tukey’s post-test. ***p < 0.0001; ** p < 0.001. Bar 10 µm; haematoxylin and eosin staining (H&E).

Figure 6

Detection of antibodies in the Golden Syrian hamster COVID model. (a) ELISA plates were coated with 100 µL of rabbit anti-horse IgG and then incubated with a previously prepared standard curve consisting of known concentrations of anti-SARS-CoV-2 horse F(ab′)₂ parallel to hamster serum samples. Horse F(ab′)₂ in the standard curve or in the samples was then detected by adding peroxidase-conjugated anti-horse IgG followed by the TMB substrate. Absorbance values were measured at 450 nm. The standard curve was linearized by log–log transformation and then subjected to linear regression analysis. The concentration of horse F(ab′)₂ in hamster serum samples was estimated by interpolating the samples to the standard curve. (b) The cPass™ kit for SARS-CoV-2 neutralizing antibody detection was used according to the manufacturer’s instructions for qualitative direct detection of total neutralizing antibodies to SARS-CoV-2 in hamster sera. (c) Positive immunofluorescence for F(ab′)₂ in the lungs of the noninfected group. Thin lung tissue sections from the noninfected group treated with anti-SARS-CoV-2 equine serum were stained with anti-SARS-CoV-2 spike glycoprotein rabbit antibody for one hour and then washed and treated with an Alexa Fluor 647-conjugated anti-rabbit antibody for 30 min. After washing, anti-horse IgG FITC was added, incubated for one hour and washed, and the slides were mounted using Hoechst. The sections were visualized under a confocal microscope (Leica TSC SP8 DSL Hyvolution).

Figure 7

Protective effect of the anti-SARS-CoV-2 antibody on the lungs of the Golden Syrian hamster COVID model. (a‒d) Positive immunostaining for Spike protein and F(ab′)₂ in lung tissue from infected animals treated with serum. (g‒h) 3D analysis showing the detailed interaction of SARS-CoV-2 with F(ab′)₂. (e‒f) Distribution of SARS-CoV-2 in experimentally infected lung tissue and (i‒j) 3D infection kinetics. (*) Lumen of the bronchioles or alveoli; (yellow star) interstice; (blue star) vascular endothelium; (dotted arrows) the area of highest magnification to the right; (arrowhead) regions of viral adhesion and antibodies; (arrow): protective effect of F(ab′)₂ on a bronchial epithelial cell or just infected cell. Bar: 20 µm.