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.

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.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

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.

Physicochemical and Biological Characterization of RTXM83, a New Rituximab Biosimilar.

BACKGROUND: RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries. OBJECTIVE: The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product. METHODS: Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). RESULTS: RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent. CONCLUSION: RTXM83 is similar to its reference product in all critical quality attributes.

EcXyl43 ß-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

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.

Long-lasting immunoprotective and therapeutic effects of a hyperstable E7 oligomer based vaccine in a murine human papillomavirus tumor model.

Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high-risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV-induced cancer cells. We have previously shown that E7 can self-assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV-tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG-rich oligonucleotide (ODN) as adjuvant elicits a strong long-lasting protection against E7-expressing tumor cells, preventing tumor outgrowth after rechallenge 90-days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose-dependent manner. These proof-of-principle preclinical experiments denote the potential applicability of our E7SOx-based vaccine to the treatment of cervical cancer and other mucosal HPV-related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low-cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age.

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.