key: cord-0976941-i6q8hthr authors: León, Guillermo; Herrera, María; Vargas, Mariángela; Arguedas, Mauricio; Sánchez, Andrés; Segura, Álvaro; Gómez, Aarón; Solano, Gabriela; Corrales-Aguilar, Eugenia; Risner, Kenneth; Narayanan, Aarthi; Bailey, Charles; Villalta, Mauren; Hernández, Andrés; Sánchez, Adriana; Cordero, Daniel; Solano, Daniela; Durán, Gina; Segura, Eduardo; Cerdas, Maykel; Umaña, Deibid; Moscoso, Edwin; Estrada, Ricardo; Gutiérrez, Jairo; Méndez, Marcos; Castillo, Ana Cecilia; Sánchez, Laura; Gutiérrez, José María; Díaz, Cecilia; Alape, Alberto title: Development and pre-clinical characterization of two therapeutic equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19 date: 2020-10-19 journal: bioRxiv DOI: 10.1101/2020.10.17.343863 sha: bfd03865b44f8e8cfe28a3481edbf7ed11faeebd doc_id: 976941 cord_uid: i6q8hthr 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. COVID-19 (Coronavirus disease 2019) is a recent pandemic disease caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . It has generated millions of infections and hundreds of thousands of deaths (https://covid19.who.int/), and has become a serious threat to global public health and economies worldwide. General symptoms of COVID-19 are fever, severe respiratory illness, dyspnea, and pneumonia, with additional possible complications such as multiple-organ dysfunction or failure, that compromises the gastrointestinal, cardiovascular, renal, and central nervous systems, and can lead to septic shock [2] [3] [4] . Like other coronaviruses, the SARS-CoV-2 virus is an enveloped single-stranded RNA virus, with a virion composed of at least four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) 5 . Protein S is implicated in cellular recognition, fusion, and entry 5, 6 , making it the most attractive target for SARS-CoV-2 therapeutic development. Although contagion by this emerging virus continues to increase across the globe, more time is needed to develop and validate vaccines, drugs, and therapies to counteract the disease. Immunoglobulin-based therapies, such as treatment with human convalescent plasma, formulations of immunoglobulins purified from plasma of convalescent patients or hyperimmunized animals, or recombinant monoclonal antibodies, arise as a feasible option that may be achievable on a shorter time scale [7] [8] [9] [10] . Treatment of COVID-19 with convalescent plasma seems to be well-tolerated, reduces mortality, and has the potential to improve clinical outcomes, based on several case series studies, matched-control studies, and small randomized clinical trials [11] [12] [13] [14] [15] . However, these results must be confirmed by large and ongoing controlled clinical trials. Formulations of immunoglobulins purified from plasma of convalescent patients have also been used to treat severe respiratory illnesses with viral etiology, but to a lesser extent 7, 16, 17 . These formulations are advantageous over unpurified convalescent plasma because they are safer and have higher activity, polyvalency, and product consistency 7,9,18 . However, this strategy is donor-dependent, requires strict donor screening for both human pathogens and high levels of neutralizing anti-SARS-CoV-2 antibodies, and relies on wellestablished blood bank systems that may be scarce in developing countries 8 . On the other hand, formulations of animal-derived immunoglobulins, such as anti-SARS-CoV F(ab´)2-equine formulations, have shown neutralizing efficacy in cell culture and in in vivo murine and hamsters models, as well as in both prophylactic and therapeutic experimental settings [19] [20] [21] [22] [23] . Similarly, Zhao et al. 24 More recently, two independent groups obtained F(ab')2 preparations through immunization of horses with recombinant SARS-CoV-2 RBD (receptor binding domain; located at S1 subunit), and demonstrated preclinical efficacy as a potential therapy for COVID-19 both in vitro 25, 26 and in vivo in a murine model 25 . Also, a F(ab´)2 formulation with high in vitro neutralizing potency was developed by immunizing horses with recombinant pre-fusion trimers of SARS-CoV-2 S protein, comprising S1 and S2 subunits 27 . In light of these favorable outcomes, and harnessing our experience in manufacturing equine snake antivenom 28 , we developed two formulations of whole-IgG from plasma of horses immunized with one of two types of SARS-CoV-2 recombinant proteins: S1 (anti-S1) or a mixture of S1, SEM mosaic, and N proteins (anti-Mix). We detail the manufacturing procedure, quality and safety profiles, and in vitro preclinical efficacy of the final formulations with the aim of providing an effective, safe, and affordable potential treatment for COVID-19. All procedures involving animals in this study were approved by the Institutional Committee Two groups of three Ibero-American and mixed breed horses, ranging from 3 to 15 years old and 350 to 450 kg, were immunized with SARS-CoV-2 proteins. The first group (anti-S1) was immunized with the S1 protein alone, while the second group (anti-Mix) was immunized with a mixture of equal parts of S1, N, and Spike-E-M mosaic proteins (SEM). In brief, horses were injected subcutaneously at two weeks intervals according to the scheme summarized in Table 1 . Freund's complete adjuvant (F5881, Sigma-Aldrich; Missouri, USA), Freund's incomplete adjuvant (F5506-Sigma-Aldrich), and Emulsigen-D adjuvant (MVP adjuvants; Nebraska, USA) were included to enhance the antibody response of horses. Samples of serum were collected before and during immunization with each booster and stored at -20 °C until use. Antibody responses of horses to SARS-CoV-2 proteins was monitored via ELISA as described below. We bled horses 19 days after the last injection of viral proteins using a closed system of blood collection bags. In total, three blood collections of 6 L each were performed across three consecutive days. The first day, blood was collected and stored overnight at 2-8 °C to allow erythrocyte sedimentation. The second day, plasma from the first day was separated from erythrocytes, preserved by the addition of 0.005 % thimerosal, and stored at 2-8 °C until use. Erythrocytes were suspended in saline solution and warmed to 37 °C. Then, following the second collection of blood, tempered erythrocytes from the first day were transfused back to the same animal. The third day, we repeated the same procedure as on the second day 31 . To prepare anti-S1 and anti-Mix formulations, we purified immunoglobulins using the caprylic acid precipitation method 32 . Immunoglobulins were formulated at 65 g/L total protein, 8.5 g/L NaCl, 2.0 g/L phenol and pH 7.0. Then, purified antibodies were sterilized by 0.22 µm pore membrane filtration and dispensed in 10 mL glass vials. pre-stained molecular mass markers. Protein bands were stained with Coomassie Brilliant Blue R-250, and gels were destained with a mixture of methanol, ethanol, and acetic acid. Total protein concentration was determined using a modification of the Biuret test 34 . Fifty microliters of protein standards were mixed with 2.5 mL of Biuret reagent and incubated at room temperature for 30 min. We created a calibration curve with absorbances of the standards at 540 nm. Then, we repeated the procedure with samples and calculated protein concentration based on the equation of the calibration curve. The content of IgG monomers was assessed by FPLC gel filtration chromatography in a Superdex 200 10/300 GL column (GE Healthcare, Pharmacia; Stockholm, Sweden), using 0.15 mol/L NaCl, 20 mmol/L Tris, pH 7.5 as the mobile phase with a 0.5 mL/min flow. Protein peaks were detected by measuring absorbance at 280 nm. Turbidity was quantified using a turbidimeter (220, La Motte; Maryland, USA) calibrated with HACH Stablcal® turbidity standards prior to analysis. Turbidity was expressed in nephelometric turbidity units (NTU). We measured pH with a pH meter (Orion 4 Star, ThermoScientific; Massachusetts, USA) equipped with a glass electrode. Sodium chloride was quantified according to the Pharmacopeial methodology 35 . In brief, 5.0 mL of each sample was diluted with distilled water, mixed with acetic acid and methanol, and titrated with a standard solution of 0.1 mol/L silver nitrate. Eosin Y was used as the indicator. Osmolality was assessed by a cryoscopic technique using a micro-osmometer (3320, Advanced Instrument; Massachusetts, USA) and a reference solution. Phenol concentration was determined by a colorimetric assay 36 , using 4-aminoantipyrine and potassium ferricyanide to form a derivative compound that absorbs at 505 nm. Caprylic acid concentration was quantified by RP-HPLC following Herrera et al. 37 35 . A predetermined volume of anti-S1 and anti-Mix products was aseptically filtered through a 0.22 µm pore membrane. Membranes were cut into equal sizes, and each half was transferred to one of two types of culture media suitable for the growth of fungi as well as aerobic and anaerobic microorganisms. After inoculation, media were incubated for 14 days at either 25 °C or 35 °C depending on the media type. During and at the end of the incubation period, media were examined for macroscopic evidence of microbial growth. Sterility compliance was dependent on the absence of microbial growth. Polystyrene plates (Costar 9017, Corning Inc.; New York, USA) were coated overnight at room temperature with 0.5 g/well of S1 protein, 0.25 g/well of nucleocapsid protein, 1 g/well of mosaic protein or 0.5 g/well of RBD protein, depending on the experiment and in duplicate. After washing the plates five times with distilled water, 100 L of equine plasma or immunoglobulin formulation, diluted with 2% skim milk/PBS, were added to each well. The plates were incubated for 1 h at room temperature and washed again five times. Afterwards, 100 L of rabbit anti-equine IgG antibodies conjugated with peroxidase (A6917, Sigma-Aldrich), diluted 1:3000 or 1:5000 with 2% skim milk/PBS, were added to each well. Again, microplates were incubated for 1 h at 25 °C. After a final washing step, color was developed by the addition of H2O2 and o-phenylenediamine as a substrate (P9029, Sigma-Aldrich). Color development was stopped by the addition of 2.0 mol/L HCl. Absorbances were recorded at 492 nm. For ELISA titration of final formulations, titer was calculated as the dilution at which the absorbance was equal to five times the absorbance of a purified normal equine plasma (normal equine immunoglobulins, NEI) diluted 1:1000. For Western blot analysis, viral proteins were separated by SDS-PAGE as described above. Then, proteins were transferred to a nitrocellulose membrane and blocked with 1% skim milk/PBS for 40 min at room temperature. The membrane was incubated with a dilution 1:1000 of either anti-S1 or anti-Mix samples in 0.1% skim milk/PBS for 1 h at 25 °C. Subsequently, a second incubation was performed with a dilution 1:1000 of a rabbit antiequine IgG antibody conjugated with peroxidase. A precipitating chromogenic substrate (4-Chloro-1-Naphthol; C6788, Sigma-Aldrich) was added. Plaque Reduction Neutralization for anti-S1 and anti-Mix formulations and human Structural SARS-CoV-2 recombinant S1, N, and SEM mosaic proteins were used as immunogens for the preparation of equine formulations. S1 forms part of the transmembrane spike (S) glycoprotein homotrimer located at the viral surface. S is composed of S1 and S2 subunit domains, and is the main protein responsible for recognition of the angiotensinconverting enzyme 2 (ACE2) receptor via the receptor binding domain (RBD) at the Nterminal S1 subunit 5, 6 . Thus, S is the most suitable target for immunoglobulin-based therapy for COVID-19 1, 40 . Both S1 and S2 subunits of SARS-CoV, MERS-CoV, and SARS-CoV-2, specially conformational epitopes at S1, and particularly at RBD [41] [42] [43] [44] 53 . In contrast, the role of the envelope (E) protein is not entirely understood; this viroporin is expressed profusely during viral replication cycles, and has been implicated in viral assembly, budding, envelope formation, and pathogenesis 53, 54 . Immunization of horses with VLPs expressing MERS-CoV S, M and E proteins has successfully induced neutralizing antibodies against MERS-CoV 24 . Here, we used SARS-CoV-2 recombinant S1 protein (produced in baculovirus insect cells), N protein (expressed in E. coli), and SEM mosaic (an E. coli derived recombinant protein containing the S, E, and M immunodominant regions) as immunogens to produce two formulations of equine immunoglobulins: anti-S1 (towards S1 protein) and anti-Mix (towards a mixture of S1, N, and SEM mosaic proteins). Additionally, we used recombinant RBD (expressed in HEK293 cells) for immunoreactivity and quality control assessment. Before immunization, we verified the purity of viral protein preparations with SDS-PAGE analysis (Fig. 1) and found that S1 protein was represented by a ~85 kDa band of high purity (94%). N protein showed a band of ~57 kDa and 75% purity. SEM mosaic protein of memory T and B cells. In turn, plasma B cells produce specific antibodies, whose affinity and specificity most likely mature after each booster 55 . In this experiment, immunization of horses followed the scheme summarized in Table 1 27 . In this study, horses in anti-S1 and anti-Mix groups showed similar dynamics in their development of plasma concentrations of antibodies towards S1 (Fig. 2a) , which is not surprising because both groups received the S1 immunogen at the same dosage, schedule, and method of administration. Antibodies towards N protein were only developed by horses in the anti-Mix group (Fig. 2b) . Because RBD is a domain of S1 5,6 , we anticipated and observed that both groups would show similar responses towards RBD, even though it was not used as an immunogen (Fig. 2c ). Also, despite the fact that only the anti-Mix group was immunized against the SEM mosaic, both groups of horses developed a similar antibody response towards this immunogen (Fig. 2d) ; presumably, the S immunodominant region contained in the mosaic masked the responses to E and M proteins. Throughout the immunization schedule, animals used for hyperimmune plasma production should be under rigorous veterinary care, ensuring that the health and welfare of each animal are closely monitored, and ethical guidelines are appropriately met. During this process, the stimulation of a local inflammation is expected for the production of a cellular infiltrate, which at the beginning is mainly composed of neutrophils and at the end by antigen-presenting cells 55 . In our study, horses developed local inflammation and signs of minor pain and discomfort such as lameness or abnormal gait, which lasted in most cases two days after each booster. However, the use of an anti-inflammatory drug was necessary for pain relief in two horses in the anti-Mix group. The complete and incomplete Freund´s adjuvants produced fistulation with minor pus-like discharge between 2 and 3 weeks after each injection. In spite of their proven adjuvant efficacy and depot effect, it is recommended to limit the use of these adjuvants to the beginning of the immunization protocol because they are known to cause local injuries 56 . Therefore, for the third booster, we selected Emulsigen-D adjuvant, which only produced some ventral edema on anti-Mix horses. At the end of the immunization scheme, the immunogen dose was reduced from 1 mg to 0.5 mg of each immunogen to reduce inflammatory effects and to ration immunogen use. Overall, milder inflammation was observed in the anti-S group than in the anti-Mix group. This difference may result from protein interactions in the anti-Mix preparation and/or the fact that the anti-Mix group received 3 mg of total immunogen versus only 1 mg in the anti-S1 group. Nonetheless, further studies are necessary to confirm these hypotheses. Hematological parameters were analyzed regularly throughout the immunization scheme, and before bleeding ( In general, biochemical analysis of horse serum showed normal values, although some measures varied significantly during hyperimmune plasma production ( Table 2 ; P < 0.05). Pre-bleeding analysis revealed a significant increase in total protein and globulins and a We bled horses when the maximum anti-viral antibody level against all immunogens reached a plateau (Fig. 2) . First, horses underwent physical examination, including evaluation of body condition, auscultation of cardiorespiratory and digestive systems, and hematological tests. Strict veterinary surveillance was maintained throughout bleeding, selftransfusion, and post-bleeding. Bleeding was performed across three consecutive days. Horses had access to water and food ad libitum. By the end of the process, an average of 9.2 ± 1.0 L plasma was collected from each horse. Bleeding and plasma separation were performed by specialized technicians. Laboratories, equipment, and a closed system of blood collection bags were designed to operate with refined aseptic technique. All the plasma bags tested negatively to endotoxins, containing less than 1.5 EU/mL, and met all the specifications required to be included in the plasma pools for the immunoglobulin purification process. Bleeding resulted in no adverse acute or chronic physiological alterations. Some minor shifts were observed in serum biochemical parameters, but within accepted ranges (Table 2) as reported by Cunha et al. 27 . Horses showed an expected decrease in total protein and globulins concentration after bleeding, as a consequence of the removal of plasma proteins. After a 2-month rest period, animals recovered to normal status, and were ready to initiate a new cycle of immunization and bleeding. To prepare antisera, hyperimmune plasma was pooled by immunization group (28.9 kg anti-S1 plasma and 26.5 kg anti-Mix plasma), and subsequently fractionated by caprylic acid precipitation. This method is routinely used at the Instituto Clodomiro Picado of the University of Costa Rica for the manufacture of whole-IgG antivenoms, and produces satisfactory yield and adequate purity of preparations 32 . The antivenoms generated using this fractionation protocol have proven safe and effective in clinical trials in patients suffering snakebite envenomings [57] [58] [59] . Caprylic acid has the advantage of precipitating non-IgG proteins from plasma, while leaving the pharmacologically active ingredientthe immunoglobulinsin solution 32 . The advantage of this method is that IgG aggregation is circumvented. After precipitation, insoluble material was removed by filtration, producing a clarified solution enriched in plasma immunoglobulins. Once purified, whole-IgG preparations were properly formulated in compliance with quality control specifications (Table 3) . Solutions were dialyzed and concentrated to remove caprylic acid (Table 3 ; ≤ 250 mg/L), and to reach final protein concentration. Additionally, pH, osmolality, and ionic strength were adjusted to values compatible with parenteral administration, and phenol was added as a preservative (Table 3) . Finally, a 0.22 µm-filtration was performed prior to aseptic filling of vials as a final sterilization step to avoid contamination with potential pathogens such as bacteria, protozoa, and fungi. After downstream processing of the formulations, we filled 612 and 479 vials containing 10 mL of purified and concentrated anti-S1 and anti-Mix products, respectively. In other words, we obtained approximately 20 vials/L plasma. Because heterologous formulations are animal plasma-derived products, there is a theoretical concern regarding transmission of viral infectious agents. To prevent this possibility, viral risk assessment needs to be performed via rigorous control of the viral load of the raw material, as well as evaluation of both existing and intentionally introduced antiviral steps during production 56 . The use of caprylic acid in the production of snake antivenoms also works as an antiviral step for enveloped viruses, reducing viral loads by up to 5 log10 for several model viruses 60 . Previously, we have found that precipitation of equine plasma with 6 % caprylic acid reduces infectivity of human herpesvirus (HSV-1) in Vero cells by more than 5 log10 within 5 minutes of adding the precipitating agent (our unpublished data). Moreover, 0.25 % phenol has been reported as an efficient virucidal agent of enveloped viruses when added to final formulations of snake antivenoms 61 . Therefore, our anti-SARS-CoV-2 formulations were produced with a methodology that has been previously validated as having two viral inactivation steps. A comparison between various physicochemical parameters of both formulations is presented in Table 3 . Both preparations complied with quality control specifications. The immunoglobulin monomer content of both products indicated that they have few soluble protein aggregates, and the turbidity due to insoluble aggregates was low. This may be relevant in terms of the safety profile of the product, because the presence of aggregates of immunoglobulins in heterologous formulations has been proposed as one of the main causes of early adverse reactions 62 . Endotoxin and sterility tests were fully compliant, which also supports the microbiological safety profile of the preparations. (Table 3 ; ˃ 90%) and presented a predominant band with a molecular mass corresponding to whole IgG ~150 kDa. As a result of the efficacy of purification by caprylic acid, there are only traces of protein contaminants. The ELISA antibody response of anti-S1 and anti-Mix formulations towards the four recombinant proteins used as immunogens was significantly different (F= 797.529, df= 2;24, P< 0.0001; Fig. 4a ). Such immunoreactivity agrees with that seen during horse immunization, as the anti-S1 formulation recognized both S1 and RBD and the anti-Mix formulation recognized S1, N, and RBD. Anti-RBD immunoreactivity was higher with the anti-Mix formulation (P < 0.05), whereas the SEM mosaic was poorly recognized by both anti-S1 (ẍ= formulations. The ELISA titers of the final products towards S1 and N was calculated in reference to a normal equine immunoglobulin preparation (NEI) ( Table 3 ). The anti-S1 formulation presented a higher titer against S1 than the anti-Mix formulation, whereas only anti-Mix showed immunoreactivity towards the nucleocapsid viral protein. The Western blot results (Fig. 4b) agree with the ELISA findings, i.e. anti-S1 formulation immunoreacted with several bands of S1, particularly at 85 kDa band, and with RBD; while anti-Mix formulation immunoreacted with bands of S1, N and RBD proteins. It is worth mentioning that when compared to the electrophoretic profile of viral proteins (Fig. 1) , more immunodetected bands appeared in S1 and N, suggesting the presence of other proteins in the recombinant preparations, probably remnants of the upstream process. Because we used proteins that were expressed in non-human cells, there is no risk that immunization could have resulted in the production of equine antibodies towards human proteins that could induce adverse effects. We evaluated the ability of the final formulations to neutralize SARS-CoV-2 infection in vitro on Vero cells. In general, SARS-CoV-2 inhibition was dose-dependent (Fig. 5) . The dilution factor at which the formulations neutralized 50% of the virus (ED50) was 1:29108 (1:26885-1:31643) for anti-S1 and 1:25355 (1:22659-1:58594) for anti-Mix. In previous studies, heterologous formulations towards SARS-CoV-2 RBD were also able to neutralize the infectivity of the virus in cells 25, 26 , evidencing that the use of RBD as an immunogen can indeed trigger strong immunoreactivity and neutralization of the virus. Likewise, Cunha and colleagues 27 showed that immunization of horses with a trimeric spike protein (comprised of both S1 and S2 subunits) generated a formulation with potent in vitro neutralizing ability. Here, we demonstrated that immunization of horses with recombinant S1 elicited a strong humoral response. Such a strong neutralizing capacity is most likely due to the presence of anti-RBD antibodies and other antibodies directed towards S protein epitopes such as the Nterminal domain (NTD) 44 . During the pre-incubation phase of the PRNT assay with both anti-S1 and anti-Mix formulations, a similar potency of anti-S1 antibodies recognized and bound S1 on the viral surface, neutralizing the infection capacity of the SARS-CoV-2 virus 6 (Fig. Given that N protein is an internal antigen, its interaction with specific antibodies in a PRNT assay is unlikely. Thus, we could not assess the contribution of anti-N antibodies to viral neutralization in this study. However, vaccines that have been developed against SARS-CoV and MERS-CoV with targets other than the S protein did not successfully protect animals from infection 10, 63 . The contribution of anti-N antibodies to the control of COVID-19 requires further study. In terms of the total protein of the formulations required to neutralize 50 % of the viral infection capacity (total protein/ED50), the anti-S1 formulation required 2.3 μg/mL and the anti-Mix formulation required 2.5 μg/mL. Another formulation tested by Pan and colleagues 25 required 8.8 μg/mL, whereas a formulation reported by Zylberman et al. 26 reached titer values of 1:10240 with 3 g/dL of total protein. An anti-trimeric S-formulation resulted in a PRNT50 of 1:32000 at 9 g/dL total protein 27 . However, because different methodologies were used to assess the neutralizing ability of the above noted formulations, it would be more appropriate to compare our formulations with convalescent plasma as a control. In this study, ED50 (95% CI) of convalescent plasma was 1:339 (1:295-1:386). In other words, anti-S1 and anti-Mix formulations were 80 times more potent than the convalescent plasma pool (Fig. 5) , and a 10-mL vial of equine-derived formulation would be equivalent to 800 mL of human convalescent plasma from donors pre-selected for having a high anti-SARS-CoV-2 titer. These findings are consistent with previous reports 26, 27 . Although A surrogate assay was used to evaluate the in vitro capacity of equine formulations to trigger the activation of human FcγRIIIA (CD16) (Fig. 6 ). This assay comprised the cocultivation of anti-viral IgG, previously incubated with S1 and N proteins, with mouse BW:FcγRIIIA-ζ reporter cells expressing the extracellular portion of chimeric human FcγRIIIA. Activation of FcγRIIIA via recognition of the IgG-Fc portion was then determined by measuring IL-2 secretion as a marker 39 . When compared to normal equine IgG (Fig. 6 , bar 4), both anti-S1 and anti-Mix formulations induced the secretion of IL-2 (Fig. 6, bars 1 and 2). According to their immunoreactivity, both formulations interacted with S1 protein, and only the anti-Mix formulation reacted to N protein. A pool of anti-SARS-CoV-2 high-titer convalescent human plasma showed significantly higher IL-2 secretion than equine formulations against both S1 and N viral proteins (P < 0.005; Fig. 6 , bar 3). Interestingly, our results evidence some capacity of equine IgG to interact and activate human FcγRIIIA in vitro, suggesting the possibility of being able also to activate human suggesting that the presence of Fc fragment in the preparation is not the main culprit for these reactions. Additionally, immunoglobulin fragments have shorter half-lives than whole IgG preparations, which confers an advantage to non-digested formulations in terms of active ingredient residence time during administration 42, 71 . Clarification of the relative efficacy and safety of F(ab´)2 and IgG anti-SARS-CoV-2 formulations is an important task that must be addressed in the near future. Taking advantage of our experience with manufacturing snake antivenom, we developed two equine-IgG formulations (anti-S1 and anti-Mix) by immunizing horses with SARS-CoV-2 recombinant proteins S1, N, and SEM mosaic. Formulations were prepared with a simple, cost-effective, and scalable methodology, and showed high physicochemical and microbiological quality. We demonstrated that horses can produce large quantities of antibodies with high neutralizing potency of the virus in vitro, due to the presence of anti-S1 and anti-RBD immunoglobulins in the final products, which demonstrated to be 80 times more potent than a pool of human convalescent plasma. Both formulations have similar preclinical quality, safety, and efficacy profiles, but are yet to be validated with proper clinical trials. We suggest that the technological platform presented here could be adapted by other equine immunoglobulin producers worldwide to provide this potential treatment of COVID-19 in other regions, particularly in low-and middle-income countries. All data generated or analyzed during this study are included in this published article. ELISA antibody response of pools of plasma from the anti-S1 and anti-Mix groups of horses during the immunization schedule with SARS-CoV-2 recombinant proteins. a: anti-S1 response; b: anti-N response; c: anti-SEM mosaic response; d: anti-RBD response. Anti-S1 group is represented by a circle (•) and anti-Mix group is represented by a square (◼). Samples towards S1 and N proteins were assessed at a 1/1000 dilution, and samples towards SEM mosaic and RBD were assessed at a 1/500 dilution. Absorbances were recorded at 492 nm. Results are expressed as mean ± SD. . Immunoreactivity profile of anti-S1 and anti-Mix formulations towards SARS-CoV-2 recombinant proteins. a: ELISA antibody response of anti-S1 formulation, anti-Mix formulation, and normal equine immunoglobulin preparation (NEI) towards S1, N, SEM mosaic, and RBD. Samples against S1 and N proteins were assessed at a 1/1000 dilution, and samples against SEM mosaic and RBD were assessed at a 1/500 dilution. Absorbances were recorded at 492 nm. Results are expressed as mean ± SD. A comparison was made between groups by immunogen. Different letters indicate a statistically significant difference (P <0.05). b: Western blot analysis of anti-S1 and anti-Mix formulations towards S1, N, and RBD. Lanes 1-3: immunoreactivity of anti-S1 formulation; Lanes 4-6: immunoreactivity of anti-Mix formulation; Lanes 1 and 4: S1 protein; Lanes 2 and 5: N protein; Lanes 3 and 6: RDB. . Anti-SARS-CoV-2 equine IgG-mediated activation of human FcγRs. 1: anti-S1 formulation; 2: anti-Mix formulation; 3: anti-SARS-CoV-2 human convalescent plasma pool; 4: Normal equine immunoglobulin preparation (NEI). Plates were coated with 1 µg/well of N and S1 proteins and were incubated with 20 mg/dL of immunoglobulin preparations. BW:FcγRIIIA-ζ transfectants (BW5147 thymoma cells expressing the extracellular portion of human FcγR FcγRIIIA fused to the mouse CD3 ζ-chain) were used. Absorbance was recorded at 450 nm and corresponds to the measurement of mIL-2 by ELISA. Experiments were performed in triplicate and expressed as mean ± SD. A comparison was made between groups by immunogen. Letters indicate statistically significant differences (P <0.05). Industrial bleeding 1 anti-S1 group was immunized with S1 protein alone, and anti-Mix group with a mixture of equal parts of S1, N, and SEM mosaic proteins (1mg/protein). Table 2 . 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Anti-S1 group Anti-Mix group Preimmunization 3 21. 3 5.4 ± 0.8 3.5 ± 0.9 6.1 ± 2.6 5.6 ± 0.5 3.1 ± 0.3 6.7 ± 3.6 1 Results are expressed as mean ± SD (n = 3 horses). 2 Not determined. 3 Did not comply with the assumption of sphericity; corrected with Greenhouse-Geisser factor. GGT: Gamma-Glutamyl Transferase; AST: Aspartate Aminotransferase; CK: Creatine Kinase. *Parameter changed significantly different over time (P < 0.05). Absence of growth Absence of growth Absence of growth Anti-S1 ELISA titer 5 19062 ± 139 11172 ± 386 Pending 6 Anti-N ELISA titer 5 1:2 ± 1 3147 ± 116 Pending 6 1 Formulations were adjusted to a similar protein concentration. 2 Monomer content is expressed as the relative percent of monomeric immunoglobulin proteins as analyzed by size exclusion chromatography. 3 Turbidity is expressed in nephelometric turbidity units (NTU). 4 Endotoxin limit was calculated as the quotient K/M, where K is the threshold pyrogenic dose of endotoxin per kilogram of body weight (5 EU/kg), and M is the maximum total dose administered to a 70 kg-patient for 1 h. Based on our results, the recommended dose for anti-S1 and anti-Mix formulations is 10 mL. Therefore, the acceptable endotoxin limit for this formulation is: (5 EU/kg)/(10 mL/70 kg) = 35 EU/mL. 5 ELISA titer was calculated as the dilution at which the sample absorbance was equal to five times the absorbance of a normal equine immunoglobulin preparation (NEI) diluted 1:1000.