Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif.

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the ß-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to ß-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the ß-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to ß-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with ß-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA ß-lactam antibiotic. The ability of an anti-MRSA ß-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second ß-lactam molecule, opens an unprecedented realm for ß-lactam antibiotic structure-based design.

Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one.

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ß-strands constituting the central ß-sheet of the αßα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.

Structural basis for the broad specificity of a new family of amino-acid racemases.

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

Disruption of allosteric response as an unprecedented mechanism of resistance to antibiotics.

Ceftaroline, a recently approved ß-lactam antibiotic for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.

A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

Cross-protection and cross-neutralization capacity of ancestral and VOC-matched SARS-CoV-2 adenoviral vector-based vaccines.

COVID-19 vaccines were originally designed based on the ancestral Spike protein, but immune escape of emergent Variants of Concern (VOC) jeopardized their efficacy, warranting variant-proof vaccines. Here, we used preclinical rodent models to establish the cross-protective and cross-neutralizing capacity of adenoviral-vectored vaccines expressing VOC-matched Spike. CoroVaxG.3-D.FR, matched to Delta Plus Spike, displayed the highest levels of nAb to the matched VOC and mismatched variants. Cross-protection against viral infection in aged K18-hACE2 mice showed dramatic differences among the different vaccines. While Delta-targeted vaccines fully protected mice from a challenge with Gamma, a Gamma-based vaccine offered only partial protection to Delta challenge. Administration of CorovaxG.3-D.FR in a prime/boost regimen showed that a booster was able to increase the neutralizing capacity of the sera against all variants and fully protect aged K18-hACE2 mice against Omicron BA.1, as a BA.1-targeted vaccine did. The neutralizing capacity of the sera diminished in all cases against Omicron BA.2 and BA.5. Altogether, the data demonstrate that a booster with a vaccine based on an antigenically distant variant, such as Delta or BA.1, has the potential to protect from a wider range of SARS-CoV-2 lineages, although careful surveillance of breakthrough infections will help to evaluate combination vaccines targeting antigenically divergent variants yet to emerge.

Site-directed mutations and kinetic studies show key residues involved in alkylammonium interactions and reveal two sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho) to produce choline and inorganic phosphate. PchP belongs to the haloacid dehalogenase superfamily (HAD) and possesses the three characteristic motifs of this family: motif I ((31)D and (33)D), motif II ((166)S), and motif III ((242)K, (261)G, (262)D and (267)D), which fold to form the catalytic site that binds the metal ion and the phosphate moiety of Pcho. Based on comparisons to the PHOSPHO1 and PHOSPHO2 human enzymes and the choline-binding proteins of Gram-(+) bacteria, we selected residues (42)E and (43)E and the aromatic triplet (82)YYY(84) for site-directed mutagenesis to study the interactions with Pcho and p-nitrophenylphosphate as substrates of PchP. Because mutations in (42)E, (43)E and the three tyrosine residues affect both the substrate affinity and the inhibitory effect produced by high Pcho concentrations, we postulate that two sites, one catalytic and one inhibitory, are present in PchP and that they are adjacent and share residues.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Prevention of tubulin/aldose reductase association delays the development of pathological complications in diabetic rats.

In recent studies, we found that compounds derived from phenolic acids (CAFs) prevent the formation of the tubulin/aldose reductase complex and, consequently, may decrease the occurrence or delay the development of secondary pathologies associated with aldose reductase activation in diabetes mellitus. To verify this hypothesis, we determined the effect of CAFs on Na+,K+-ATPase tubulin-dependent activity in COS cells, ex vivo cataract formation in rat lenses and finally, to evaluate the antidiabetic effect of CAFs, diabetes mellitus was induced in Wistar rats, they were treated with different CAFs and four parameters were determinates: cataract formation, erythrocyte deformability, nephropathy and blood pressure. After confirming that CAFs are able to prevent the association between aldose reductase and tubulin, we found that treatment of diabetic rats with these compounds decreased membrane-associated acetylated tubulin, increased NKA activity, and thus reversed the development of four AR-activated complications of diabetes mellitus determined in this work. Based on these results, the existence of a new physiological mechanism is proposed, in which tubulin is a key regulator of aldose reductase activity. This mechanism can explain the incorrect functioning of aldose reductase and Na+,K+-ATPase, two key enzymes in the pathogenesis of diabetes mellitus. Moreover, we found that such alterations can be prevented by CAFs, which are able to dissociate tubulin/aldose reductase complex.

Dimer Asymmetry and Light Activation Mechanism in Brucella Blue-Light Sensor Histidine Kinase.

The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level.

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light­absorbing) and Pfr (far-red light­absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/ß-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.

Crystallization and preliminary X-ray diffraction analysis of Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine to produce choline and inorganic phosphate. Phosphorylcholine is released by the action of haemolytic phospholipase C (PlcH) on phosphatidylcholine or sphingomyelin. PchP belongs to the HAD superfamily and its activity is dependent on Mg2+, Zn2+ or Cu2+. The possible importance of PchP in the pathogenesis of P. aeruginosa, the lack of information about its structure and its low identity to other members of this family led us to attempt its crystallization in order to solve its three-dimensional structure. Crystals of the protein have been grown and diffraction data have been obtained to 2.7 A resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=137.16, b=159.15, c=73.31 A, beta=117.89 degrees. Statistical analysis of the unit-cell contents and the self-rotation function suggest a tetrameric state of the molecule with 222 point-group symmetry.

Transition from octahedral to tetrahedral geometry causes the activation or inhibition by Znf2+ of Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine, which is produced by the action of hemolytic phospholipase C on phosphatidylcholine or sphyngomielin, to generate choline and inorganic phosphate. Among divalent cations, its activity is dependent on Mg(2+) or Zn(2+). Mg(2+) produced identical activation at pH 5.0 and 7.4, but Zn(2+) was an activator at pH 5.0 and became an inhibitor at pH 7.4. At this higher pH, very low concentrations of Zn(2+) inhibited enzymatic activity even in the presence of saturating Mg(2+) concentrations. Considering experimental and theoretical physicochemical calculations performed by different authors, we conclude that at pH 5.0, Mg(2+) and Zn(2+) are hexacoordinated in an octahedral arrangement in the PchP active site. At pH 7.4, Mg(2+) conserves the octahedral coordination maintaining enzymatic activity. The inhibition produced by Zn(2+) at 7.4 is interpreted as a change from octahedral to tetrahedral coordination geometry which is produced by hydrolysis of the [Zn(2+)L(2)(-1)L(2)(0) (H(2)O)(2)] complex.

Crystal structures of peanut lectin in the presence of synthetic ß-N- and ß-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.