A phase I study to evaluate the safety, tolerance and pharmacokinetics of anti-Shiga toxin hyperimmune equine F (ab')2 fragments in healthy volunteers.

AIMS: Shiga toxin-producing Escherichia coli-haemolytic uraemic syndrome (STEC-HUS) is considered a toxaemic disorder in which early intervention with neutralizing antibodies may have therapeutic benefits. INM004, composed of F (ab')2 fragments from equine immunoglobulins, neutralizes Stx1/Stx2, potentially preventing the onset of HUS. METHODS: A single-centre, randomized, phase 1, single-blind, placebo-controlled clinical trial to evaluate INM004 safety, tolerance and pharmacokinetics (PK) in healthy adult volunteers, was conducted; in stage I, eight subjects were divided in two cohorts (n = 4) to receive a single INM004 dose of 2 or 4 mg kg-1, or placebo (INM004:placebo ratio of 3:1). In stage II, six subjects received three INM004 doses of 4 mg kg-1 repeated every 24 h, or placebo (INM004:placebo ratio of 5:1). RESULTS: Eight subjects (57.1%) experienced mild treatment-emergent adverse events (TEAEs); most frequent were rhinitis, headache and flushing, resolved within 24 h without changes in treatment or additional intervention. No serious AEs were reported. Peak concentrations of INM004 occurred within 2 h after infusion, with median Cmax values of 45.1 and 77.7 µg mL-1 for 2 and 4 mg kg-1, respectively. The serum concentration of INM004 declined in a biphasic manner (t1/2 range 30.7-52.9 h). Systemic exposures increased with each subsequent dose in a dose-proportional manner, exhibiting accumulation. Geometric median Cmax and AUC values were 149 and 10 300 µg h mL-1, respectively, in the repeated dose regimen. Additionally, samples from subjects that received INM004 at 2 mg kg-1 showed neutralizing capacity against Stx1 and Stx2 in in vitro assays. CONCLUSIONS: The results obtained in this first-in-human study support progression into the phase 2 trial in children with HUS.

Surveillance of Shiga toxin-producing Escherichia coli associated bloody diarrhea in Argentina.

In Argentina, hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC-HUS) infection is endemic, and reliable data about prevalence and risk factors have been available since 2000. However, information about STEC-associated bloody diarrhea (BD) is limited. A prospective study was performed during the period October 2018-June 2019 in seven tertiary-hospitals and 18 referral units from different regions, aiming to determine (i) the frequency of STEC-positive BD cases in 714 children aged 1-9 years of age and (ii) the rate of progression of bloody diarrhea to HUS. The number and regional distribution of STEC-HUS cases in the same hospitals and during the same period were also assessed. Twenty-nine (4.1%) of the BD patients were STEC-positive, as determined by the Shiga Toxin Quik Chek (STQC) test and/or the multiplex polymerase chain reaction (mPCR) assay. The highest frequencies were found in the Southern region (Neuquén, 8.7%; Bahía Blanca, 7.9%), in children between 12 and 23 month of age (8.8%), during summertime. Four (13.8%) cases progressed to HUS, three to nine days after diarrhea onset. Twenty-seven STEC-HUS in children under 5 years of age (77.8%) were enrolled, 51.9% were female; 44% were Stx-positive by STQC and all by mPCR. The most common serotypes were O157:H7 and O145:H28 and the prevalent genotypes, both among BD and HUS cases, were stx2a-only or -associated. Considering the endemic behavior of HUS and its high incidence, these data show that the rate of STEC-positive cases is low among BD patients. However, the early recognition of STEC-positive cases is important for patient monitoring and initiation of supportive treatment.

Safety and effectiveness of RBD-specific polyclonal equine F(ab´)2 fragments for the treatment of hospitalized patients with severe Covid-19 disease: A retrospective cohort study.

BACKGROUND: Passive immunotherapy has been evaluated as a therapeutic alternative for patients with COVID-19 disease. Equine polyclonal immunotherapy for COVID-19 (EPIC) showed adequate safety and potential efficacy in a clinical trial setting and obtained emergency use authorization in Argentina. We studied its utility in a real world setting with a larger population. METHODS: We conducted a retrospective cohort study at "Hospital de Campaña Escuela-Hogar" (HCEH) in Corrientes, Argentina, to assess safety and effectiveness of EPIC in hospitalized adults with severe COVID-19 pneumonia. Primary endpoints were 28-days all-cause mortality and safety. Mortality and improvement in modified WHO clinical scale at 14 and 21 days were secondary endpoints. Potential confounder adjustment was made by logistic regression weighted by the inverse of the probability of receiving the treatment (IPTW) and doubly robust approach. FINDINGS: Subsequent clinical records of 446 non-exposed (Controls) and 395 exposed (EPIC) patients admitted between November 2020 and April 2021 were analyzed. Median age was 58 years and 56.8% were males. Mortality at 28 days was 15.7% (EPIC) vs. 21.5% (Control). After IPTW adjustment the OR was 0.66 (95% CI: 0.46-0.96) P = 0.03. The effect was more evident in the subgroup who received two EPIC doses (complete treatment, n = 379), OR 0.58 (95% CI 0.39 to 0.85) P = 0.005. Overall and serious adverse events were not significantly different between groups. CONCLUSIONS: In this retrospective cohort study, EPIC showed adequate safety and effectiveness in the treatment of hospitalized patients with severe SARS-CoV-2 disease.

Non-clinical safety assessment and in vivo biodistribution of CoviFab, an RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required the urgent development of new therapies, among which passive immunotherapy is contemplated. CoviFab (INM005) is a RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies. We investigate their preclinical security and biodistribution by in vivo and ex vivo NIR imaging after intravenous administration of a dose of 4 mg/kg at time 0 and 48 h. Images were taken at 1, 12, 24, 36, 48, 49, 60, 72, 84, 96, 108, 120, 132 and 144 h after the first intravenous injection. At 96 and 144 h, mice were sacrificed for haematology, serum chemistry, clinical pathology, histopathology and ex vivo imaging. The biodistribution profile was similar in all organs studied, with the highest fluorescence at 1 h after each injection, gradually decreasing after that each one and until the end of the study (144 h). The toxicology study revealed no significant changes in the haematology and serum chemistry parameters. Further, there were no changes in the gross and histological examination of organs. Nonclinical data of the current study confirm that CoviFab is safe, without observable adverse effects in mice. Furthermore, we confirm that bioimaging studies are a useful approach in preclinical trials to determine biodistribution.

RBD-specific polyclonal F(ab´)2 fragments of equine antibodies in patients with moderate to severe COVID-19 disease: A randomized, multicenter, double-blind, placebo-controlled, adaptive phase 2/3 clinical trial.

BACKGROUND: passive immunotherapy is a therapeutic alternative for patients with COVID-19. Equine polyclonal antibodies (EpAbs) could represent a source of scalable neutralizing antibodies against SARS-CoV-2. METHODS: we conducted a double-blind, randomized, placebo-controlled trial to assess efficacy and safety of EpAbs (INM005) in hospitalized adult patients with moderate and severe COVID-19 pneumonia in 19 hospitals of Argentina. Primary endpoint was improvement in at least two categories in WHO ordinal clinical scale at day 28 or hospital discharge (ClinicalTrials.gov number NCT04494984). FINDINGS: between August 1st and October 26th, 2020, a total of 245 patients were enrolled. Enrolled patients were assigned to receive two blinded doses of INM005 (n = 118) or placebo (n = 123). Median age was 54 years old, 65•1% were male and 61% had moderate disease at baseline. Median time from symptoms onset to study treatment was 6 days (interquartile range 5 to 8). No statistically significant difference was noted between study groups on primary endpoint (risk difference [95% IC]: 5•28% [-3•95; 14•50]; p = 0•15). Rate of improvement in at least two categories was statistically significantly higher for INM005 at days 14 and 21 of follow-up. Time to improvement in two ordinal categories or hospital discharge was 14•2 (± 0•7) days in the INM005 group and 16•3 (± 0•7) days in the placebo group, hazard ratio 1•31 (95% CI 1•0 to 1•74). Subgroup analyses showed a beneficial effect of INM005 over severe patients and in those with negative baseline antibodies. Overall mortality was 6•9% the INM005 group and 11•4% in the placebo group (risk difference [95% IC]: 0•57 [0•24 to 1•37]). Adverse events of special interest were mild or moderate; no anaphylaxis was reported. INTERPRETATION: Albeit not having reached the primary endpoint, we found clinical improvement of hospitalized patients with SARS-CoV-2 pneumonia, particularly those with severe disease.

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.

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.

[Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome]. / Desarrollo de un producto anti-toxina Shiga para la prevención del síndrome urémico hemolítico.

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.

Desarrollo de un producto anti-toxina shiga para la prevención del síndrome urémico hemolítico / Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome

El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.

Specific recognition of a DNA immunogen by its elicited antibody.

DNA recognition by antibodies is a key feature of autoimmune diseases, yet model systems with structural information are very limited. The monoclonal antibody ED-10 recognizes one of the strands of the DNA duplex used in the immunogenic complex. Modifications of the 5' end decrease the binding affinity and short oligonucleotides retain high binding affinity. We determined crystal structures for the Fab bound to a 6-mer oligonucleotide containing the specific sequence that raised the antibody and compared it with the unliganded Fab. Only the first two bases from the 5' end (dTdC) display electron density and we observe four key hydrogen bonds at the interface. The thymine ring is stacked between TrpH50 and TrpH95, and the cytosine ring is packed against TyrL32. Upon DNA binding, TyrH97 and TrpH95 rearrange to allow subnanomolar binding affinity, five orders of magnitude higher than other reported complexes, possibly because of having gone through affinity maturation. This structure represents the first bona fide antibody DNA immunogen complex described in atomic detail.

Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domain of the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-binding activity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2 interaction has a strong electrostatic component, which correlates with the high proportion of acidic residues found in the antibody combining site. Further circular dichroism experiments in the presence of phosphate show that, in addition to electrostatic screening of key potential interactions, ionic strength affects the conformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlates with the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface. Noticeably, this finding is well correlated with the strong entropic component of the interaction. Site directed mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix of E2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociation constant of the E2-ED15 interaction is 10(-7) M and the association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower than those generally reported in the most extensively described "nonflexible" antibody-protein interactions, indicating the presence of a slow conformational rearrangement on the antigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition is discussed.

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi, which interacts with the structural flagellar protein paraflagellar rod.

Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.