Preclinical Assessment of IgY Antibodies Against Recombinant SARS-CoV-2 RBD Protein for Prophylaxis and Post-Infection Treatment of COVID-19

Abstract

Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

Keywords: COVID-19; IgY; SARS-CoV-2; egg-yolk antibodies; passive immunization; receptor binding domain.

Figure 1

SDS-PAGE patterns of IgY-R at the 2 stages of purification (4 µg per line). M stands for molecular weight marker, with size indicated on the left; Lanes 1-4: WSF of yolks belonging to hens immunized with 5, 12.5, 25, and 50 µg of rRBD; lane 5: WSF of control yolks; lanes 6-9: purified IgY of yolks belonging to hens immunized with 5, 12.5, 25, and 50 µg of rRBD; lane 10: purified control IgY. HC, Heavy chain; LC, Light chain.

Figure 2

In vitro immunoreactivity of IgY-R. (A) IgY-R raising pattern according to ELISA against the SARS-CoV-2 rRBD antigen both in serum and egg yolks of immunized hens. (B) Western Blot using IgY-R as primary antibody. Lane M: Ladder; lane 1: reduced RBD; lane 2: non reduced RBD; lane 3: PBS as control. (C) IgY-R ability to block RBD binding to Vero E6 cells. Results are presented as the mean fluorescence intensity (MFI) given by the flow cytometer (D) Neutralization percent of IgY-R using the cPass SARS-CoV-2 Neutralization Antibody Detection Kit. Cutoff value is settled as 30% following manufacturer guidelines. Mean ± SD are presented.

Figure 3

Safety of IgY-R through histopathological assessment of liver, gut, trachea, lung, and kidney of mice. No visible lesions were found on any of the slides analyzed (six slides per tissue sample). Representative images are shown. Image amplitude: 20x Scalebars: 200 µm.

Figure 4

Assessment of intranasally delivered IgY-R in hamster recovered from serum and nasal swabs. Cutoff value was set to 0.10 (CI = 95%). Mean ± SD (error bars) is presented (n = 4 per group).

Figure 5

Physical outcomes evaluation of hamsters infected with SARS-Cov-2 and intranasally administered with IgY-R. (A) Percentage variation of the weight of individuals over the experimental time-course. (B) Average speed variation (C) Average acceleration variation (D) Average displacement variation. DPI = Days Post Infection. Mean ± SD (error bars) is presented (n = 4 per group). The Mann-Whitney and Kruskal Wallis tests were performed to determine whether differences between Control and IgY-administered groups were significant (*) or non-significant (n.s.).

Figure 6

Histopathological findings in the lungs of hamsters challenged with SARS-CoV-2. (A) Areas of necrosis with hemorrhage, thickened alveolar wall, and infiltration mononucleated inflammatory cells (B) Invasion of intraalveolar mononuclear inflammatory cells (C) No visible lesions (D) The pulmonary parenchyma appears thickened in some areas with atelectasis, the alveoli are clean, there is no infiltration of inflammatory cells (E) Slightly thickened pulmonary parenchyma, the alveoli slightly dilated, but without infiltration of inflammatory cells (F) Thickening of the alveolar wall, the alveolar lumen does not present cellular infiltration. Image amplitude: 20x. Scalebars: 200 µm.