Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

Abstract

The emergence of SARS-CoV-2 variants stresses the continued need for broad-spectrum therapeutic antibodies. Several therapeutic monoclonal antibodies or cocktails have been introduced for clinical use. However, unremitting emerging SARS-CoV-2 variants showed reduced neutralizing efficacy by vaccine induced polyclonal antibodies or therapeutic monoclonal antibodies. In our study, polyclonal antibodies and F(ab')₂ fragments with strong affinity produced after equine immunization with RBD proteins produced strong affinity. Notably, specific equine IgG and F(ab')₂ have broad and high neutralizing activity against parental virus, all SARS-CoV-2 variants of concern (VOCs), including B.1.1,7, B.1.351, B.1.617.2, P.1, B.1.1.529 and BA.2, and all variants of interest (VOIs) including B.1.429, P.2, B.1.525, P.3, B.1.526, B.1.617.1, C.37 and B.1.621. Although some variants weaken the neutralizing ability of equine IgG and F(ab')₂ fragments, they still exhibited superior neutralization ability against mutants compared to some reported monoclonal antibodies. Furthermore, we tested the pre-exposure and post-exposure protective efficacy of the equine immunoglobulin IgG and F(ab')₂ fragments in lethal mouse and susceptible golden hamster models. Equine immunoglobulin IgG and F(ab')₂ fragments effectively neutralized SARS-CoV-2 in vitro, fully protected BALB/c mice from the lethal challenge, and reduced golden hamster's lung pathological change. Therefore, equine pAbs are an adequate, broad coverage, affordable and scalable potential clinical immunotherapy for COVID-19, particularly for SARS-CoV-2 VOCs or VOIs.

Keywords: SARS-CoV-2; broad-spectrum neutralizing activity; equine antibody; variants of concern; variants of interest.

Figure 1

In vitro characterization of purified equine immunoglobulin against SARS-CoV-2. (A) The neutralizing titers of hyperimmune serum, purified IgG, and F(ab’)₂ derived from equine No. 15 and No. 16 were tested with wild type SARS-CoV-2 Wuhan 01. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (B) The titers of purified SARS-CoV-2-specific IgG in equine sera were examined via RBD-capture ELISA. Two repeated tests were performed on each sample.

Figure 2

Broad-spectrum neutralizing activity test against SARS-CoV-2 VOC and VOI. The neutralizing antibody titers were calculated as the highest dilution of sera that completely inhibited virus-caused CPE. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (A) Neutralizing antibody titers of purified IgG and F(ab’)₂ of equine No.15 against SARS-CoV-2 VOC; (B) Neutralizing antibody titers of purified IgG and F(ab’)₂ of equine No.16 against SARS-CoV-2 VOC; (C) Neutralizing antibody titers of purified equine immunoglobulin of equine No.15 against SARS-CoV-2 VOI; (D) Neutralizing antibody titers of purified equine immunoglobulin of equine No.16 against SARS-CoV-2 VOI. Comparison to the wild type SARS-CoV-2 Wuhan01, the number above the column represented the fold by which the neutralizing titer of the IgG or F(ab’)₂ was weakened by the SARS-CoV-2 VOC and VOI. Samples were processed in triplicate, and error bars indicate standard error. Data are presented as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3

Evaluation of the protective efficacy of purified equine immunoglobulin in a mouse model. Groups of 13 BALB/c mice were administered with IgG or F(ab’)₂ at 1 day before mouse-adapted SARS-CoV-2 (BMA8) infection or 1 dpi with BMA8. Each mouse was given 250 µg of antibody at a dose of 10 mg/kg. BALB/c mice were challenged intranasally with a lethal dose 50 LD₅₀ of BMA8 before treatment or after administration. The survival rate, weight change, body temperature and clinical scores of BALB/c mice were monitored daily after SARS-CoV-2 BMA8 infection. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure; (B) Survival rate. (C) Percent weight change. (D) Body temperature change. Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (****P < 0.0001).

Figure 4

Blood counts in SARS-CoV-2-infected mice. The hematological values of BALB/c mice were analysed, including lymphocyte (LYM), neutrophil percentage (Neu%), monocytes (Mon), platelet count (PLT) and white blood cell count (WBC), at 3 dpi after SARS-CoV-2 BMA8 infection. Four infected mice were sacrificed at 3 dpi to collect the whole blood for blood counts test. (A) White blood cell (WBC) count; (B) neutrophil (Neu) percentage; (C) lymphocyte (LYM) percentage; (D) platelet (PLT) (E) Monocyte(Mno). Data are presented as the mean ± SEM (n=4). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5

Viral loads in the upper and lower respiratory tracts of mice. Four infected mice were sacrificed at 3 dpi and 5 dpi, respectively, and the turbinate and lungs were harvested to analyze the viral RNA loads by RT‒qPCR and TCID₅₀. (A) The viral load quantified by RT‒qPCR at 3 dpi in the prevention group. (B) The viral load quantified by RT‒qPCR at 5 dpi in the prevention group. (C) The viral load quantified by RT‒qPCR at 3 dpi in the treatment group. (D) The viral load quantified by RT‒qPCR of the turbinate and lungs at 5 dpi in the treatment group. (E) The viral load confirmed by TCID50 at 3 dpi in the prevention group; (F) The viral load confirmed by TCID₅₀ at 5 dpi in the prevention group; (G) The viral load confirmed by TCID₅₀ at 3 dpi in the treatment group; (H) The viral load confirmed by TCID₅₀ at 5 dpi in the treatment group. Data are presented as the mean ± SEM (n=4). (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6

Histopathological and immunohistochemistry findings in SARS-CoV-2-infected mice. The lungs and spleens were collected from the control mice infected with SARS-CoV-2 without equine immunoglobulin drug injection at 3dpi, and the lungs, spleens, livers and kidneys were harvested from recovered mice. After each tissue was embedded in paraffin, the sections were sectioned for HE staining. (A, B, E, F) Lung tissue changes of control mice were characterized by more necrotic epithelial cells (blue arrow), a small amount of neutrophil infiltration, and perivascular edema with a small amount of inflammatory cell infiltration in the local alveolar cavity (yellow arrow). (C, D, G, H) Spleen tissue changes of control mice were characterized with spotted apoptosis of lymphocytes, nuclear pyknosis and deep staining or fragmentation in the spleen nodules (black arrows), and the expansion of germinal centers (yellow arrow), scattered neutrophils mostly seen in the red pulp granulocyte infiltration (red arrow), and more brown‒yellow particles in the red pulp (blue arrow). (I-L) The basically normal structure of the lung, spleen liver, and kidney tissues were found in administration groups given equine IgG or F(ab’)₂. The figure showed immunohistochemistry (IHC) labeling against SARS-CoV-2 N. (M) Viral antigen was not detectable in prevention group given purified IgG; (N) Viral antigen was not detectable in prevention group given purified F(ab’)₂; (O) Viral antigen was detected for positive in prevention control group; (P) Viral antigen was not detectable in treatment group given purified IgG; (Q) Viral antigen was not detectable in treatment group given purified IgG F(ab’)₂; (R) Viral antigen was detected for positive in treatment control group. (scale bar = 100 μm).

Figure 7

Evaluation of the protective efficacy of purified equine immunoglobulin in the golden hamster model. Each golden hamster was given 500 µg of antibody at a dose of 10 mg/kg. Groups of golden hamsters were infected intranasally with 1,000 TCID₅₀ of wild-type SARS-CoV-2 Wuhan 01 before treatment and or after administration. The survival rate and weight change of BALB/c mice were monitored daily after SARS-CoV-2 Wuhan01 infection. Four infected golden hamsters in each group were sacrificed at 3 dpi, and the turbinate and lung samples were collected to analyze the viral RNA loads by RT‒qPCR and TCID₅₀, respectively. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure. (B) Survival rate. (C) Percent weight change; Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (D) The viral loads of turbinate were quantified by RT‒qPCR at 3 dpi in each group; (E) The viral loads of lung were quantified by RT‒qPCR at 3 dpi in each group; (F) The viral loads of turbinate were determined by TCID₅₀ at 3 dpi in each group; (G) The viral loads of lung were determined by TCID₅₀ at 3 dpi in each group. Data are presented as the mean ± SEM (n=5). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).