Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum.

Fig 1. Schematic overview of bovine immunoglobulin preparation from colostrum.

Fig 2

(A) NAb ELISA setup and principle. Colostrum immunoglobulin preparation was added in serial dilution to the trimeric S protein-coated plates and preincubated to provide some time for the antibodies to bind. Next, conjugated ACE2, which competes with the antibodies for the same binding sites, was added. Color development becomes more intense in samples containing fewer neutralizing antibodies, and a sample without any antibodies has the most intense color since maximal binding of conjugated ACE2 could occur. (B) Binding of colostrum immunoglobulin to the different trimeric S protein variants in SARS-CoV-2. Relative optical density (OD) values are represented as the mean ± SD (n = 3). The IC₅₀ concentrations for colostrum immunoglobulin preparation were determined using 4-parameter nonlinear regression.

Fig 3. Pseudovirus neutralization assay.

Measurement of the inhibition of trimeric S protein-dependent entry of different pseudoviruses pseudotyped with wt, Alpha, Beta, Gamma, Delta or Kappa VoCs of the SARS-CoV-2 S protein into ACE2-expressing HEK293 cells in the presence of serial dilutions of colostrum immunoglobulin preparation. The rate of cell entry was measured via firefly luciferase activity and expressed as the relative infection rate (%) compared to luciferase activity in the untreated control. Data are represented as the mean ± SD (n = 3). The IC₅₀ concentrations for colostrum immunoglobulin preparation were determined using 4-parameter nonlinear regression.

Fig 4. The neutralizing activity of the colostrum immunoglobulin preparation on SARS-CoV-2 infection analyzed by cytopathic effect reduction.

Colostrum-derived immunoglobulins from a nonimmunized cow and from SARS-CoV-2 S protein-immunized cows were added to (A) Alpha and (B) Beta SARS-CoV-2 virus isolates at different concentrations and then transferred to Vero E6 cells, which were grown for 4–5 days. The neutralization activity was defined by the endpoint method, i.e., determination of colostrum immunoglobulin preparation concentrations blocking infection of Vero E6 cells. Data are represented as the mean ± SD (n = 3). The unpaired t test was performed; Alpha: ****p<0.0001, Beta: *p = 0.0164.

Fig 5. Blood serum neutralizing antibody titers over time after immunization and reimmunization of cows #6546, #3541, #3547 and #3539.

The first three pink dotted lines indicate the days of initial immunizations before the first calving. The black dotted lines denote the days of calving, and the following two pink dotted lines indicate reimmunizations. The analyses were conducted using competitive ELISA (see the materials and methods section), and the red dotted line indicates the respective OD 450 of 0.75 (y-axis), above which neutralizing antibodies are not detectable and below which their presence is measurable. The x-axis shows the days after the initial injection for immunization. The connected line shows dots on each day that blood was drawn. This figure shows 4 examples of analyses that were indeed conducted for all cows included in the study.

Fig 6. As in Fig 3, the results of the pseudovirus neutralization assay are depicted.

Here, pseudoviruses were pseudotyped with either Delta or Omicron VoCs of the SARS-CoV-2 S protein into ACE2-expressing HEK293 cells in the presence of serial dilutions of colostrum immunoglobulin preparations. As described, the rate of cell entry was measured via firefly luciferase activity and expressed as the relative infection rate (%) compared to luciferase activity in the untreated control. Data are represented as the mean ± SD (n = 2–4). The IC₅₀ concentrations for colostrum immunoglobulin preparation were determined using 4-parameter nonlinear regression.

Fig 7. The bioavailability of the final immunoglobulin formulation, which was administered intranasally to healthy volunteers at two different concentrations (0.1 mg/mL and 0.2 mg/mL), and of the residual solution 1 hour and 4 hours postadministration was measured with a cow IgG ELISA kit.

The median concentration of bovine IgG is indicated with a black line.