Development and characterization of two equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19

Abstract

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.

Heterologous Hyperimmune Polyclonal Antibodies Against SARS-CoV-2: A Broad Coverage, Affordable, and Scalable Potential Immunotherapy for COVID-19

“…The capacity of both formulations to neutralize virus infectivity (USA-WA1/2020 isolate) in vitro is 80 times higher than that of pooled human convalescent plasma (37). Additionally, the equine IgGs in these formulations activate human FcγRIIIA in vitro, suggesting downstream mediation of effector functions via interaction with Fc receptors in immune cells (37).…”

“…SARS-CoV-2 S1 glycoprotein or its RBD were described (34)(35)(36)(37). These formulations are significantly more potent than COVID-19 convalescent plasma at neutralizing SARS-CoV-2 infectivity (35)(36)(37).…”

“…The fractionation process included two steps with robust viral inactivation/removal activity (caprylic acid precipitation and phenol addition) to ensure the viral safety of the products, as required by the WHO (21). The capacity of both formulations to neutralize virus infectivity (USA-WA1/2020 isolate) in vitro is 80 times higher than that of pooled human convalescent plasma (37). Additionally, the equine IgGs in these formulations activate human FcγRIIIA in vitro, suggesting downstream mediation of effector functions via interaction with Fc receptors in immune cells (37).…”

“…Similarly, IgG and F(ab')2 formulations from horses immunized with MERS-CoV virus-like particles exhibit in vivo neutralization ( 33 ). Recently, three F(ab')2 formulations from plasma of horses hyperimmunized with recombinant SARS-CoV-2 S1 glycoprotein or its RBD were described ( 34 – 37 ). These formulations are significantly more potent than COVID-19 convalescent plasma at neutralizing SARS-CoV-2 infectivity ( 35 – 37 ).…”

“…Recently, three F(ab')2 formulations from plasma of horses hyperimmunized with recombinant SARS-CoV-2 S1 glycoprotein or its RBD were described ( 34 – 37 ). These formulations are significantly more potent than COVID-19 convalescent plasma at neutralizing SARS-CoV-2 infectivity ( 35 – 37 ). Moreover, a multi-center, double-blind, placebo-controlled phase II/III clinical trial (NCT04494984) with RBD-specific equine F(ab')2 fragments performed in Argentina showed that this immunotherapy is well-tolerated and that clinical improvement of hospitalized severe COVID-19 patients is achieved ( 38 ).…”