Invest Clin 62(Suppl. 2): 3 - 17, 2021 https://doi.org/10.22209/IC.v62s2a01

Corresponding author: Alexis Rodríguez-Acosta, Facultad de Farmacia, Universidad Central de Venezuela, Caracas,

República Bolivariana de Venezuela. Tel: +58 491 7243; E-mail: rodriguezacosta1946@yahoo.es

Production of equine sera as a potential

immunotherapy against COVID-19

Mariana V. Cepeda

Juan C. Jiménez

, Flor H. Pujol

, Héctor R. Rangel

, Carlos Bello

José Cubillan

, María L. Serrano

, Tony Chacón

, Antonietta Saba

, Miguel A. López

and

Alexis Rodríguez-Acosta

1,5

Biotecfar C.A, Facultad de Farmacia, Universidad Central de Venezuela, Caracas,

República Bolivariana de Venezuela.

Instituto de Inmunología “Dr. Nicolás Bianco”, Facultad de Medicina, Universidad

Central de Venezuela, Caracas, República Bolivariana de Venezuela.

Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Miranda, República Bolivariana

de Venezuela.

Unidad de Química Medicinal, Facultad de Farmacia, Universidad Central de Venezuela,

Caracas, República Bolivariana de Venezuela.

Laboratorio

Inmunoquímica y Ultraestructura, Instituto Anatómico “Dr. José

Izquierdo”, Facultad de Medicina, Universidad Central de Venezuela, Caracas, República

Bolivariana de Venezuela.

Key words: Equine antiserum; anti-RBD; immunotherapy; pandemic; SARS-CoV-2;

equine coronavirus.

Abstract. Emerging viruses such as the COVID-19-inducing virus, SARS-

CoV-2, represent a threat to human health, unless effective vaccines, drugs

or alternative treatments, such as passive immunization, become accessible.

Animal-derived immunoglobulins, such as equine immunoglobulins might be

useful as immunoprophylaxis or immunotherapy against this viral disease.

Therapeutic antibodies (Abs) for SARS-CoV-2 were obtained from hyperimmune

equine plasma using the Spike protein receptor binding domain (RBD) as an

immunogen. The presence of anti-RBD antibodies was evaluated by ELISA and

the titres of neutralizing antibodies were determined in viral cell culture. Im-

munized horses generated high-titre of anti-RBD antibodies with antiviral neu-

tralizing activity on Vero-E6 cells of 1/1,000. To minimize potential adverse

effects, the immunoglobulins were digested with pepsin, and purified to obtain

the F(ab’)2 fragments with the protocol standardized by Biotecfar C.A  for

the production of snake antivenom. Pre-immune serum displayed an unexpect-

ed anti-RBD reactivity by ELISA (titre up to 1/900) and Western Blot, but no

4 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Producción de un suero equino como inmunoterapia potencial

contra la COVID-19

Invest Clin 2021; 62 (Suppl. 2): 3-17

Palabras clave: Antisuero equino; anti-RBD; inmunoterapia; pandemia; SARS-CoV-2;

coronavirus equino.

Resumen. Los virus emergentes, como el virus causante de la COVID-19,

el SARS-CoV-2, representan una amenaza para la salud de la humanidad, mien-

tras no estén disponibles vacunas, medicamentos o tratamientos alternativos

eficaces, como la inmunización pasiva. Las inmunoglobulinas de producción

animal, como las de los equinos, pueden ser útiles como inmunoprofilaxis o

inmunoterapia contra esta enfermedad viral. Se produjeron anticuerpos tera-

péuticos (Abs) contra el SARS-CoV-2 a partir de plasma equino hiperinmune in-

munizado con el dominio de unión al receptor (RBD) de la proteína de la espiga

viral. La presencia de anticuerpos contra RBD se evaluó mediante ELISA y de

anticuerpos neutralizantes por inhibición del crecimiento del virus en cultivos

celulares. Los caballos inmunizados generaron títulos elevados de anticuerpos

anti-RBD con actividad neutralizante antiviral en células Vero-E6 de 1/1.000.

El suero preinmune mostró una reactividad anti-RBD por ELISA (título hasta

1/900) y Western Blot pero sin actividad neutralizante. Con el propósito de

disminuir posibles efectos adversos, se realizó la digestión proteica con pepsina

de las inmunoglobulinas y posterior purificación para obtener los fragmentos

F(ab’)2 empleando el protocolo utilizado por Biotecfar C.A para la producción

de los antivenenos de serpientes. El modelado del RBD del coronavirus equino

mostró que algunos de los epítopos conocidos del RBD del SARS-CoV-2 se con-

servaban estructuralmente en la proteína del coronavirus equino. Esto podría

sugerir que parte de la reactividad del suero preinmune al RBD del SARS-CoV-2

podría deberse a una exposición previa al coronavirus equino en el animal.

Received: 28-02-2021 Accepted: 11-06-2021

INTRODUCTION

SARS-CoV-2, the new coronavirus re-

sponsible for the disease COVID-19, belongs

to the family Coronaviridae, genus Betacoro-

navirus, subgenera Sarbecovirus. The virus

genome encodes for four structural proteins

and several non-structural proteins. Among

the structural proteins, the Spike protein

(S) contains two domains: S1 that includes

the receptor-binding domain (RBD), which

interacts with the human receptor angio-

neutralizing activity. Modelling of the RBD of equine coronavirus showed that

some of the known epitopes of SARS-CoV-2 RBD were structurally conserved in

the equine coronavirus protein. This might suggest that some of the reactivity

observed in the pre-immune serum to the SARS-CoV-2 RBD might be due to a

previous exposure to equine coronavirus.

Production of equine sera as immunotherapy against COVID-19 5

Vol. 62(Suppl. 2): 3 - 17, 2021

tensin-converting enzyme 2 (hACE2), and

S2 that includes the fusion domain, which

promotes the fusion of the viral envelope

with the cellular membrane, to promote vi-

ral entry, through an early or late endosomal

pathway (1, 2)

SARS-CoV-2 RBD has been

documented to enclose immune epitopes

proficient of stimulating antibodies able to

neutralise viral invasion and impede viral

entry by competing with hACE2 binding

(3)

or targeting the receptor-binding domain

(RBD) (4). Several authors have found that

SARS-CoV-2 RBD immunogen stimulated

upper titres of neutralizing antibodies (5)

and displaying a strong antibody response

in the immunized mammals and non-human

primates (6).

Even if vaccines are becoming available

to prevent COVID-19 (7), the pandemic is

still highly active, and vaccination of all the

population will not be feasible in a short

term (8). Thus, therapeutic tools will still be

needed for preventing the severe evolution

of the disease.

Equine-derived polyclonal antibodies

against several proteins have shown good ef-

ficacy against infectious diseases caused by

highly pathogenic agents, such as Ebola (9),

West Nile virus (10), H

influenza virus

(11), respiratory syncytial syndrome virus

(12) and Middle East respiratory coronavi-

rus syndrome (13).

Production of equine anti-SARS-CoV-2

antibodies have been reported in China (14-

19) India (20) and Argentina (21).

The aim of this study was the produc-

tion of F(ab’)2 anti-RBD equine antibodies

as a potential passive immunization medica-

tion against COVID-19.

MATERIALS AND METHODS

Virus antigen, plasma pools, animals and

reagents

Protein RBD antigen, expressed in

HEK293 line was purchased from Acro Bio-

system, USA. Specific hyper-immune horse

plasma (SHHP) was obtained of experimen-

tal animals from Biotecfar. C.A. Mice (Mus

musculus) NIH strain (18-20 g) of both sex-

es, purchased from the Instituto Nacional de

Higiene “Rafael Rangel”, Caracas, Venezu-

ela, were used for toxicity assays. Bovine se-

rum albumin (BSA), Tris-base and Tween-20

were from Sigma-Aldrich (USA). Pepsin

(from porcine gastric mucosa) 0.7 FIP-U/

mg) was from Merck (Germany). Goat anti-

horse F(ab’)

2 IgG conjugated with horserad-

ish peroxidase (HRP) was from LSBio (USA).

All other chemicals for buffers and solutions

were from Sigma-Aldrich (USA), unless oth-

erwise stated.

Experimental horses

Three male horses (Equus ferus ca-

ballus) were selected between the ages of 3

and 5 years old from the Purebred Racing

Race. These specimens were examined pre-

vious to starting the trial: general physical

evaluation, equine infectious anaemia test,

cellular and biochemical haematology and

a complete health plan was implemented in

accordance with national regulations. The

specimens were evaluated weekly over and

done with a complete physical examination

and cellular and biochemical haematology

(22). The horses were housed in individual

stalls or boxes, feed with Bermuda grass hay,

commercial concentrate and ad libitum wa-

ter, respecting all national and international

animal welfare conditions.

Ethical approval

This research has been approved by the

Bioethics Committee of Hospital Universita-

rio de Caracas, in Ordinary Meeting via online

N° 06 dated December 22, 2020, following

the norms obtained from the ARRIVE guide-

lines and were carried out in accordance with

the U.K. Animals (Scientific Procedures) Act,

1986 and associated guidelines, EU Directive

2010/63/EU for animal experiments. Trained

staff organised all the experimental methods

relating to the use of live animals.

6 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Selection of immunogen

In order to guarantee a safe handling

of the material with which the horses were

immunised, it was decided to work with the

SARS-CoV-2 S protein RBD (N354D, D364Y),

which was expressed from human 293 cells

(HEK293) with a molecular mass of 27.0

kDa purchased from (Acro Biosystem, USA).

The protein migrates as 33-35 kDa under re-

ducing condition (SDS-PAGE) due to glyco-

sylation (data not shown).

Horse immunization

The selected horses received a variable

amount (100 to 600 µg/mL) of the RBD anti-

gen in 1 mL of PBS: first injection of 100 µg of

antigen in Complete Freund adjuvant at week

0; injection of 200 µg of antigen in Incomplete

Freund adjuvant at week 1; injection of 300 µg

of antigen in PBS buffer at week 2; injection

of 600 µg of antigen in PBS buffer at week 3;

injection of 600 µg of antigen in PBS buffer at

week 5. The antigen injection was subdermal

performed in the lateral region of the neck.

The sampling was carried out after elapsed

times and disinfection and asepsis of the jugu-

lar vein to recover the plasma was performed.

Preliminary sample collection (negative

control)

The previous sample extraction was car-

ried out from normal horses after disinfec-

tion and asepsis of the jugular vein to recov-

er the blood.

Preparation of equine IgG immunoglobu-

lins and F (ab’)

fragments

Plasma samples were taken every 7 days

(from day 14), storing them at 4°C prior to

processing and analysis. Each sample was

placed in a thermal bath at 56°C for one

hour, and at that time were centrifuged at

2000 rpm for 15 min and then pass through

a 0.45 µm filter. After the initial immuniza-

tion, horse’s plasmas were collected on the

days indicated in the previous table, to be

stored at 4°C for further processing and

analysis.

Obtaining purified immunoglobulins

A pool of horse anti-RBD sera from the

three horses was diluted with equivalent vol-

umes of saline solution, and 1/2 volume of a

saturated ammonium sulphate solution was

included at that time.

The mixture was stired together softly

at room temperature for 30 min and after-

ward centrifuged at 5000 rpm for 20 min.

The precipitates were running in saline solu-

tion, previously, 1/3 volume of ammonium

sulphate was slowly added. Subsequently,

incubation of the sample was kept at room

temperature for 30 min. Then, the solution

was centrifuged at 5000 rpm for 20 min. The

precipitates were dissolved in saline and dia-

lysed overnight at 4 °C to remove the ammo-

nium sulphate.

Preparation of F(ab′)2 Fragments

For the preparation of F(ab’)2, the pro-

tocol standardised by Biotecfar C.A was

used, Pepsin was activated in order to elimi-

nate the Fc fragment from total IgG. Briefly,

the pH of the horse anti-RBD immunoglob-

ulins was upheld to 3.3 with 1 mol/L HCl.

The total IgG was diluted in a 1: 2 ratios and

then the pepsin was added at a concentra-

tion of 1.25 g/L. The enzymatic digestion

reaction was carried out at 30°C for 30 min.

Then was stopped to adjust the pH and carry

out the first precipitation of contaminating

proteins. It was filtered and the pH of the

filtrate was adjusted for a second precipita-

tion with ammonium sulphate, to recover

the hyper-immune proteins. This prepara-

tion was diafiltered with distilled water, be-

fore evaluation, and the protein was stored

at 4°C until use.

Analysis of antibodies against SARS CoV-2

by SDS-PAGE

The F(ab′)

purity (5µg) was analysed

by non-reducing sodium dodecyl sulphate

polyacrylamide gel electrophoresis (10%

SDS-PAGE). After running, the SDS-PAGE

were stained with Coomassie brilliant blue.

Gel images after decolouration were cap-

Production of equine sera as immunotherapy against COVID-19 7

Vol. 62(Suppl. 2): 3 - 17, 2021

tured with a ChemiDoc MP imaging system

(Bio-Rad).

Determination of equine antibody titres

by Enzyme Linked Immunosorbent Assay

(ELISA)

RBD was diluted to 1 µg/mL in PBS pH

7.4, and added to 96-well polystyrene plates

(100 µL/well). The microtitre plates were

incubated at 4°C overnight. The plates were

then washed three times with PBS-Tween 20

(0.05%) and blocked with 5% skimmed milk

for 1 h at 37°C. Subsequently, the wells were

washed three times and the samples (serum

or diluted antibodies, 100 µL volume) were

added to each well; as negative controls PBS

or nonspecific antibodies were used. The

plates were placed at 37°C for one hour and

at that point incubated with horseradish per-

oxidase (HRP), and conjugated to anti-horse

IgG antibodies at room temperature for an-

other hour. After washing, 3,3’,5, 5’-tetra-

methyl-benzidine was added. The plates were

placed in the dark room for approximately 15

minutes with the chromogens and the reac-

tion was stopped by adding 50 µL well of 2M

. Finally, the absorbance was measured

at 450 nm with a microplate reader (BioTek,

USA), and values twice times greater than

controls were considered positive.

Immunoblotting assays

The equine IgG immunoglobulins and F

(ab’)

fragments reactivity versus the SARS-

CoV-2 RBD protein were estimated using

western immunoblotting (23).

With this purpose, the gel was incu-

bated for 10 min in transfer solution (50

mM Tris-HCl at pH 8.0, containing 380 mM

glycine, 0.1% SDS and 20% methanol). After-

ward, the gel was located in a transfer cham-

ber, permitting the proteins to pass from the

polyacrylamide matrix to a nitrocellulose

membrane. This process was carried out at

180 milliamps (mA) for 2 h.

Next, the transfer, the nitrocellulose

membrane was blocked for 2 h at room tem-

perature with a 0.2 M PBS solution at pH

7.0, with 5% (w/v) skimmed milk and 0.1%

(w/v) Tween 20. Following, three washes were

carried out for 5 min, each with a solution

of 0.05% (w/v) Tween 20 and 0.2 M PBS at

pH 7.0. The membrane was incubated over

at room temperature for another 90 min,

with equine IgG immunoglobulins and F

(ab’)

fragments diluted to 1:10 in blocking

solution. Next, after the incubation inter-

val, the membrane was washed three times.

Straightaway after the washes, the secondary

antibody anti-equine IgG (coupled to horse-

radish peroxidase) diluted 1:10 in blocking

solution was added. Then, the membrane

was incubated at room temperature for an-

other 90 min and washed, as specified. The

electrophoretic bands recognised by equine

IgG immunoglobulins and F (ab’)

fragments

were visualised and the image was analysed.

Anti-SARS-CoV-2 serum toxicity test

The toxicity assay of anti COVID-19 se-

rum was carried out by intraperitoneal injec-

tion of 0.5 mL of the serum in NIH mice. The

clinical toxicological manifestations were re-

corded during a period of 7 days. None of the

mice showed signs of clinical toxicity after

seven days of anti-SARS-CoV-2 serum injec-

tion. All animals were considered healthy

with a normal behaviour.

Cell line

Vero-E6 cells was maintained in RPMI-

1640 medium (Gibco, NY, USA) supplement-

ed with 8% foetal bovine serum (FBS, Gibco,

NY, USA) were cultured in an incubator at

37 °C with 5% CO

Neutralization assay

Vero-E6 cells were seeded in 96-well

plates with 2 × 10

cells/well overnight in

8% FBS-RPMI medium supplemented with

1600 U/mL of penicillin, 800 µg/mL of

streptomycin and 10 µg/mL of amphotericin

B. Horse sera were diluted in culture medi-

um and incubated with 10 TCID50 (10 times

the tissue culture infective dose, which pro-

duce cytopathic effect in half of the plates)

8 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

of SARS-CoV-2 at 37°C for 1 h. The mixture

was then added to the cells and incubated

at 37 °C for 1 h. The cells were washed with

PBS and incubated in the culture medium.

The cytopathic effect (CPE) was examined

with an inverted microscope after three days

post-infection. The neutralization titre was

defined as the dilution of sample where 50%

of the wells exhibited no CPE (24).

Phylogenetic analysis

Coronavirus RBD were aligned using

DNAman version 5.2.2 (Lynnon Biosoft, Can-

ada) and BLOSUM matrix. Phylogenetic tree

constructed with Poisson correction and 100

bootstrap replicas. RBD alignment was also

analysed for protein conservation using PRA-

LINE sequence alignment (https://www.ibi.

vu.nl/programs).

Protein modelling

The crystal structure of the RBD of

SARS-CoV-2 bound to neutralizing antibody

CR3022 (PDB code 6W41) was retrieved and

selected for the comparative analyses. Homol-

ogy structural model of the RBD of Equine

coronavirus, was generated using the crystal

structures of the RBD domain of the human

coronavirus OC43(PDB code 6nzk) and the

beta-coronavirus HKU1 (PDB code 5kwb) as

templates. The model was obtained with the

Phyre2 modelling server using the intensive

modelling mode (25). The quality of the mod-

els was established via ProSA (26) and PRO-

CHECK programs (27). The Deep-View/Swiss-

PdbViewer 4.01 and Biovia Discovery Studio

Visualizer 17.2.0 were used to superposition

and visualisation of protein structures.

B-cell epitope prediction

EIDB (Immune-Epitope-Database and

Analysis-Resource) were used to predict lin-

ear and discontinuous B-cell epitopes using

Bepipred and ElliPro with default parameter

settings (28). Biovia Discovery Studio Visu-

alizer 17.2.0 software was used to examine

the positions of discontinuous epitopes on

the 3D structures (29).

Protein-protein docking

The crystal structure of the RBD of

SARS-CoV-2 bound to neutralizing antibody

CR3022 (PDB code 6W41) was downloaded

from Protein Data Bank. In addition, the ho-

mology model for the RBD of Equine corona-

virus was assayed. The viral spike RBD and the

structural model of the ECoV RBD were eval-

uated against the CR3022 antibody through

molecular docking. Then, the obtained bind-

ing patterns and affinity estimations were

analysed and compared. This process was

performed through two steps; first, ProABC-2

(30) was used to identify the paratope resi-

dues in CR3022 and the information was

utilized to drive the modelling of antibody–

antigen complexes using HADDOCK2.4 web

server (31) and the best model was further

refined with HADDOCK Refinement inter-

face. Then, the resulting docking data were

processed and analysed by using the tools of

PRODIGY software (32). Finally, results were

evaluated considering binding energies and

main interacting residues in each complex.

RESULTS

Horse immunization

Three male horses were immunized with

the Spike RBD viral recombinant protein.

No major adverse effects or chronic clini-

cal alterations were observed for any of the

horses. The evaluation of immunized horse’s

sera binding to the SARS-CoV-2 RBD as as-

sessed by ELISA, further validated the quali-

ties of RBD antigens tested in this study and

is shown in Fig. 1. Antibody titers were devel-

oped since the first week of immunization,

increasing with the boosts of weeks 1, 2, 3

and 5, reaching a titer of 1/24,300 main-

tained after 50 days. Some antibody reactiv-

ity was observed for the pre-immune equine

serum at 1/100 (not shown in the Fig.1: OD

1.1 in pre-immune serum vs 3.4 in the im-

munised horse), 1/300 and even 1/900 (Fig.

1). This reactivity was not observed when the

ELISA plates were not coated with the RBD

antigen (OD 0.1 in the pre-immune serum).

Production of equine sera as immunotherapy against COVID-19 9

Vol. 62(Suppl. 2): 3 - 17, 2021

Anti-SARS-CoV-2 serum toxicity test

None of the mice showed signs of clini-

cal toxicity after seven days of anti-SARS-

CoV-2 serum injection. All animals were con-

sidered healthy with a normal behaviour.

Determination of equine antibody titres

by Enzyme Linked Immunosorbent Assay

(ELISA)

The evaluation of immunised horse’s

sera binding to the SARS-CoV-2 RBD as mea-

sured by ELISA binding to RBD, further vali-

dated the qualities of RBD antigens tested in

this study is showed in Fig. 1.

Purification and characterisation of the

F(ab’)2 preparation

The purity of the F(ab’)2 anti-RBD S

protein fragments was evaluated by 10%

SDS-PAGE under reduced conditions. Three

conspicuous bands between ∼ 90 and 150

kDa bands were observed (data not shown).

Specificity of anti SARS-CoV-2 to RBD S

protein via Western blot

Western Blot analysis of the antibody

reactivity showed the specific recognition of

the RBD antigen by the immune serum, to-

gether with another band of higher molecu-

lar weight, also recognised by other immune

equine sera, probably a contaminant of the

antigen preparation. Some recognition of

the RBD band was observed in the pre-im-

mune serum, together with the other upper

band (Fig. 2, band 5).

Determination of the neutralizing activity

of the F(ab’)2 preparations

Two F(ab’)2 preparations (1 and 2, at

the middle and the end of immunization,

respectively) were evaluated for the pres-

ence of total and neutralizing antibodies

against SARS-CoV-2 RBD (Fig. 3). As ex-

pected, the titre of both anti-RBD antibod-

ies and neutralizing antibodies increased

with time and immunization boosts. The

titre of neutralizing antibodies was ap-

proximatively 1/550 for the first F(ab’)2

preparation and 1/2,300 for the second

one. Pre-immune and F(ab’)2 preparations

against other antigen (anti-venom against

snake or scorpion) did not produce any vi-

ral neutralization, even at 1/30 dilution

(data not shown).

Fig. 1. Evaluation of horse sera binding to the SARS-CoV-2 RBD as measured by ELISA. One (1.0) µg/mL

of RBD protein was coated and 7-fold serially diluted serum was supplemented after blocking. Expe-

riments were carried out in triplicate and the error bars denote ± SE, n = 2. Statistical significance

was defined as * P < 0.05. O.D: optical density.

10 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Analysis of the possible cross-reactivity

of antibodies against equine coronavirus

RBD and SARS-CoV-2 RBD

Since the equine pre-immune serum

exhibited some degree of reactivity, both

by ELISA and Western Blot, the presence

of epitopes shared between the RBD of the

Equine Coronavirus (ECoV) and SARS-CoV-2

(33) was evaluated. ECoV and SARS-CoV-2

RBD shares only 25% protein homology (Fig.

Fig. 2. Western blotting. (1 and 2) Polyvalent anti-ophidic sera. (3) Anti-scorpion serum. (4) anti-RBD S

protein [oval]. (5) Normal equine serum. 6) Membrane stained with red Ponceau. (7) Molecular mass

markers. RBD band is shown with a circle.

Fig. 3: Anti-RBD and neutralizing titres in F(ab’)2 preparations produced at different immunization

times. F(ab’)2 preparation 1 was obtained after 3 immunizations and preparation 2 after the com-

plete scheme. The neutralization titre is the highest dilution of serum that prevents infection of 50%

of replicate inoculations. Neutralization tests were run in triplicate for Preparation 1 and duplicate

for Preparation 2, with 3 replicas each time.

Production of equine sera as immunotherapy against COVID-19 11

Vol. 62(Suppl. 2): 3 - 17, 2021

4A). Three domains known as main epitope

regions in the SARS-CoV-2 RBD did not ex-

hibit neither more conservation. Only one

of them (epitope region 2) shared 30 % of

sequence identity among them (Fig. 5B).

However, structural modelling of the ECoV

RBD showed that epitope domains 1,2 and 3

share structural homology (Fig. 4B).

ECoV and SARS-CoV-2 RBDs were also

analysed in order to evaluate not only identi-

cal amino acids but also conservative substi-

tutions, particularly in the structurally con-

served regions (Fig. 5).

Additionally, by docking studies, the

binding free energy of a monoclonal anti-

body against SARS-CoV-2 RBD (CR3022), to

the EqCoV RBD was -10.5 Kcal/mol, similar

to the one obtained for SARS-CoV-2 RBD

(-14.9 Kcal/mol). CR3022 interacts with

EqCoV RBD and recognizes a discontinuous

epitope that includes several residues on the

same region (epitope 2) than with SARS-

CoV-2 (Fig. 6). Although the SARSCoV2

and ECoV RBD epitopes do not completely

overlap and different binding modes were

determined, the interaction energy suggests

that CR3022 might show some degree of

ECoV recognition. Interestingly, the epitope

of CR3022 does not overlap with the ACE2-

binding site of the RBD SARS-CoV-2 (34).

Fig. 4. Protein and structural homology between ECov and SARS-CoV-2 RBD. (A) Phylogenetic analysis

of RBD proteins from different coronavirus. Percent identity between ECoV and SARS-CoV-2 RBDs is

shown. The sequences are entitled with their accession or GISAID number. (B) Structural modelling

of EqCoV RBD and comparison with SARS-CoV-2 structure. Epitope domains are shown in colours.

12 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Fig. 5. Analysis of conservative substitutions between SARS-CoV.2 and ECoV RBD. All epitope domains are

underline in colours.

Fig. 6. A. Interaction of monoclonal antibody CR30221 with Coronavirus RBDs. Binding free energies cal-

culated for the interaction of CR3022 with SARS-CoV-2 RBD or ECoV RBD are shown. B. Aminoacids

of the respective RBDs involved in the interaction with CR30221 are observed.

Production of equine sera as immunotherapy against COVID-19 13

Vol. 62(Suppl. 2): 3 - 17, 2021

DISCUSSION

COVID-19 is one of the most important

highly pathogenic viral disease at present.

The COVID-19 pandemic has keep on for al-

most a year since the beginning of 2021, and

it is expected that this severe viral pathology

will pursue as an endemic disease for many

years (33). The response to the international

health crisis necessitates a fast answer from

the medical organizations in the form of a

prompt treatment, while vaccines become

available for all individuals. Passive immuno-

therapy with equine antibodies may help to

improve the medical treatment of the SARS-

CoV-2.

In this study, we report the prepara-

tion of hyper-immune equine sera to dem-

onstrate their protective efficacy against

SARS-CoV-2 virus, using a virus neutraliza-

tion assay. The production of equine anti-

bodies was obtained by immunising horses

with the recombinant RBD antigen. High

titres of anti-RBD antibodies were obtained

in horses after a five-boost immunization

scheme. Western Blot analysis confirmed

the specific recognition of these antibodies

of the RBD protein. To minimise potential

adverse effects, the immunoglobulins were

digested with pepsin, and purified to obtain

the F(ab’)2 fragments. The F(ab’)2 prepa-

ration unambiguously recognize the RBD

antigen and exhibited anti-SARS-CoV-2

neutralizing activity. Moreover, the ratio of

neutralizing to binding antibodies obtained

in the present work and the seroconversion

rate observed in horses after 49 days of im-

munization indicates the high quality of

the antibodies generated by using the RBD

spike protein as immunogen. Other studies

have described slightly higher titres of an-

tibodies against RBD than the one found in

this study (15,21). These differences might

be due to the immunization schemes used

in each study. In our study, the RBD dose

was lower than the one used in these stud-

ies, but with an additional booster dose.

The titres of neutralizing antibodies found

in the current report were comparable to

the ones previously described (15,21). The

hyper-immune equine sera was purified to

obtain the F(ab’)2 fragments with the pro-

tocol standardised by Biotecfar C.A  for

the production of snake and scorpion an-

tivenom, and produces a satisfactory yield

and adequate purity of preparations. The

antivenoms generated using ammonium

sulphate fractionation protocol have prov-

en safe and effective in patients suffering

snakebite and scorpion envenoming.

Pre-immune serum exhibited some de-

gree of cross reactivity with the SARS-CoV-2

RBD by ELISA and Western Blot, but not in

the neutralization test. In contrast to the

F(ab’)2 preparations against ophidic or scor-

pionic venoms, the pre-immune serum of the

horses used in this study exhibit a light spe-

cific recognition of the SARS-CoV-2 RBD by

Western Blot.

ECoV belongs to Betacoronavirus spe-

cies, subgenus Embecovirus and was first

isolated from a diarrheic foal in the USA in

1999 (35). Since then, several cases of ECoV

infections have also been reported in adult

horses from the USA, Europe and Japan.

This coronavirus has been detected in fae-

cal samples from horses with clinical signs

such as anorexia, lethargy, fever and, less

frequently, diarrhoea, colic and neurologic

deficits. The morbidity rate varies from 10%

to 83% during outbreaks, with low mortality

(35, 36).

Seroprevalence of equine coronavirus in

the U.S.A is relatively high: 9.3% (37). Even

if SARS-CoV-2 and ECoV belong to different

subgenera, the cross-reactivity observed in

the pre-immune serum prompt us to evalu-

ate if these two coronaviral RBDs might

share some epitopes. Structural modelling

of ECoV and SARS-CoV-2 RBD S showed that

these two proteins shared the structural mo-

tifs of three of the main epitope domains rec-

ognised in the SARS-CoV-2 RBD. Moreover,

docking analysis showed that one monoclo-

nal antibody produced against SARS-CoV-2

RBD exhibited a similar binding energy to

14 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

the ECoV RBD. It is worth to mention that

the epitope of CR3022 does not overlap with

the ACE2-binding site of the RBD SARS-

CoV-2 (34).

Poly-specific interaction of an antibody

with two epitopes unrelated in terms of se-

quence homology has been described (37).

In this study, no neutralizing activity was

found associated to this pre-immune cross-

reactivity. Nevertheless, (15) observed a

partial neutralizing activity in pre-immune

horse serum at low dilution. This observa-

tion is in agreement with the cross-reactivity

found in this study.

Cross reactivity with other coronavirus-

es in immunoassays, detecting antibodies

against SARS-CoV-2 in humans has also been

suggested (38). It has been also proposed

that previous exposure to other human

coronaviruses might bring some protection

for COVID-19 (39). Moreover, some cross-

neutralization activity against SARS-CoV-2

has been described in some intravenous im-

munoglobulin preparations (40). Ladner et

al. (41) proposed that the SARS-CoV-2 pro-

teome reveals regions of conservation with

endemic human coronaviruses (CoVs), but

it keep unidentified to what degree of these

may be cross-recognized by the antibody re-

sponse. The SARS-CoV-2 response seems to

be modulated by previous CoV exposures and

which have the capacity to increase roughly

neutralizing responses

To conclude, in the current work, we

prepared horse anti-RBD S protein serum by

immunizing equines with the SARS-CoV-2

RBD. In our program, the horses could be

immunized numerous times, and satisfacto-

ry titres of antibodies are predictable to be

achieved from hyper-immune horse’s plasma

for the SARS-CoV-2 therapeutic (42). Work-

ing with a scheme comprising increase im-

munogen injections, high-titre of horse an-

tisera were achieved, and F(ab’)2 fragments

have been mass-produced using good man-

ufacturing practices (GMP), in order to be

used in human clinical studies.

ACKNOWLEDGEMENTS

We wish to thank the helpful commen-

taries from Dr. Guillermo León, Instituto

“Clodomiro Picado” República de Costa Rica

and Dr. Christian Dokmetjian A, Director of

the Instituto Nacional de Producción de Bi-

ológicos de la ANLIS, Secretaria de Gobierno

de Salud de la Nación, República Argentina.

Funding for the research was provided by

Biotecfar C.A and Ministerio del Poder Popu-

lar para Ciencia y Tecnología de la República

Bolivariana de Venezuela.

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