Efficacy, Pharmacokinetics, and Toxicity Profiles of a Broad Anti-SARS-CoV-2 Neutralizing Antibody.

We recently reported the isolation and characterization of an anti-SARS-CoV-2 antibody, called IgG-A7, that protects transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from an infection with SARS-CoV-2 Wuhan. We show here that IgG-A7 protected 100% of the transgenic mice infected with Delta (B.1.617.2) and Omicron (B.1.1.529) at doses of 0.5 and 5 mg/kg, respectively. In addition, we studied the pharmacokinetic (PK) profile and toxicology (Tox) of IgG-A7 in CD-1 mice at single doses of 100 and 200 mg/kg. The PK parameters at these high doses were proportional to the doses, with serum half-life of ~10.5 days. IgG-A7 was well tolerated with no signs of toxicity in urine and blood samples, nor in histopathology analyses. Tissue cross-reactivity (TCR) with a panel of mouse and human tissues showed no evidence of IgG-A7 interaction with the tissues of these species, supporting the PK/Tox results and suggesting that, while IgG-A7 has a broad efficacy profile, it is not toxic in humans. Thus, the information generated in the CD-1 mice as a PK/Tox model complemented with the mouse and human TCR, could be of relevance as an alternative to Non-Human Primates (NHPs) in rapidly emerging viral diseases and/or quickly evolving viruses such as SARS-CoV-2.

Discovery and Optimization of Neutralizing SARS-CoV-2 Antibodies Using ALTHEA Gold Plus Libraries™.

We recently reported the isolation and characterization of anti-SARS-CoV-2 antibodies from a phage display library built with the VH repertoire of a convalescent COVID-19 patient, paired with four naïve synthetic VL libraries. One of the antibodies, called IgG-A7, neutralized the Wuhan, Delta (B.1.617.2) and Omicron (B.1.1.529) strains in authentic neutralization tests (PRNT). It also protected 100% transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from SARS-CoV-2 infection. In this study, the four synthetic VL libraries were combined with the semi-synthetic VH repertoire of ALTHEA Gold Libraries™ to generate a set of fully naïve, general-purpose, libraries called ALTHEA Gold Plus Libraries™. Three out of 24 specific clones for the RBD isolated from the libraries, with affinity in the low nanomolar range and sub-optimal in vitro neutralization in PRNT, were affinity optimized via a method called "Rapid Affinity Maturation" (RAM). The final molecules reached sub-nanomolar neutralization potency, slightly superior to IgG-A7, while the developability profile over the parental molecules was improved. These results demonstrate that general-purpose libraries are a valuable source of potent neutralizing antibodies. Importantly, since general-purpose libraries are "ready-to-use", it could expedite isolation of antibodies for rapidly evolving viruses such as SARS-CoV-2.

Gerencia de Enfermería durante la pandemia de COVID-19 en un hospital público de Honduras / Nursing Management at a Honduran Public Hospital during the COVID-19 Pandemic

Introducción: El modelo de gestión hospitalaria es el conjunto de políticas y procesos que permiten tomar decisiones asertivas. Durante la pandemia de COVID-19, los sistemas de salud a nivel global necesitaron reorganización para responder a las necesidades presentadas. Sin embargo, no se tiene registro de dicho proceso en Honduras. Objetivo: Explorar las experiencias de profesionales de Enfermería relacionadas con la gerencia de servicios del Hospital "Mario Catarino Rivas" durante la pandemia de COVID-19. Métodos: Se realizó un estudio fenomenológico interpretativo sobre las experiencias de profesionales de Enfermería relacionadas con la gerencia de servicios del Hospital "Mario Catarino Rivas", durante la pandemia de COVID-19. La colecta de datos fue mediante entrevistas semiestructuradas a 20 profesionales de Enfermería en cargos de jefatura, como resultado de un muestreo deliberado. Los datos se analizaron con el método temático, con agrupación de las narraciones, se siguieron los siete pasos de la perspectiva de Colaizzi. Resultados: Se hallaron tres temas, siete subtemas y 30 conceptos, se encontró la calidad total el modelo gerencial utilizado para dar respuestas a las dificultades presentadas durante la pandemia COVID-19. Dentro de los principales desafíos se encontró "falta de recursos humanos" para dar respuesta a la demanda de pacientes. Conclusiones: Durante la COVID-19, se evidencia el importante rol del profesional de Enfermería en cargos gerenciales para mitigar el impacto económico en el sistema de salud hondureño, y garantizar la calidad de vida de los pacientes. Es necesario la apertura de espacios con mayor responsabilidad, con respaldo legal que fortalezca la práctica avanzada(AU)

Introduction: A hospital management model is the set of policies and processes that allow assertive decision making. During the COVID-19 pandemic, health systems worldwide required reorganization to respond to the emerging needs. However, there is no record of such process in Honduras. Objective: To explore the experiences of nursing professionals concerning the management of services at Hospital Mario Catarino Rivas during the COVID-19 pandemic. Methods: An interpretative phenomenological study was conducted on the experiences of nursing professionals concerning the management of services at Hospital Mario Catarino Rivas during the COVID-19 pandemic. The data were collected using semistructured interviews with 20 nursing professionals in head positions, as a result of a deliberate sampling. The data were analyzed using the thematic method, with clustering of narratives and following the seven steps of Colaizzi's perspective. Results: Three themes, 7 subthemes and 30 concepts were found. In addition, the overall quality of the management model used to respond to the difficulties presented during the COVID-19 pandemic was found. Among the main challenges, "lack of human resources" to respond to patient demand was found. Conclusions: During COVID-19, the important role of the nursing professional in managerial positions to mitigate the economic impact on the Honduran health system and to guarantee the quality of life of patients is evident. It is necessary to open spaces with greater responsibility, with legal support to strengthen advanced practice(AU)

In Vitro and In Vivo Characterization of a Broadly Neutralizing Anti-SARS-CoV-2 Antibody Isolated from a Semi-Immune Phage Display Library.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all the three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Taken together, our findings indicate that IgG-A7 could be a suitable candidate for development of antibody-based drugs to treat and/or prevent SARS-CoV-2 VOCs infection.

Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library.

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile.

Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing.

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14-21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

Desigualdades sociales en salud: mortalidad en menores de cinco años, Honduras, 2014 / Social inequalities in health: mortality in children under five years, Honduras, 2014

Antecedentes: Las desigualdades sociales en salud se refieren a disparidades de salud, que se consideran injustas, evitables e innecesarias y que sistemáticamente recaen sobre poblaciones vulnerables. Objetivo: Medir las desigualdades sociales en salud de la mortalidad en los niños menores de cinco años, Francisco Morazán y Yoro, Honduras, año 2014. Métodos: Estudio descriptivo transversal. Se analizó la mortalidad de menores de 5 años a través de la medición de desigualdades sociales; se estimó las métricas de desigualdad en salud, índice de Kuznets relativo y absoluto, índice de desigualdad de la pendiente, índice de concentración en salud; se describió la situación de las desigualdades sociales en salud de la mortalidad por estratificador de equidad social (alfabetismo). Resultados:En 27 municipios del departamento de Francisco Morazán se registraron 71 menores de cinco años fallecidos, y en los 11 municipios del departamento de Yoro 131. Se encontró un exceso de 18 muertes de menores de cinco años por 1000 nacidos vivos entre la población con menor alfabetismo en Francisco Morazán, y de 23 en Yoro, en comparación con los municipios de mayor alfabetismo. En ambos departamentos la curva de concentración de la salud se mantuvo sobre la línea de equidad, concluyendo que la mortalidad de menores de cinco años se concentró en la población menos alfabetizada. Discusión: Se observó que los municipios con tasas más altas de alfabetismo presentaron menos mortalidad de menores de cinco años...(AU)

Validation of a cell-based colorimetric reporter gene assay for the evaluation of Type I Interferons.

The biotherapeutic type I interferons (IFN-I) are indicated to treat several diseases. These products are regulated to guarantee safety and efficacy through critical quality attributes. For this purpose, the development of robust assays is required, followed by its validation to demonstrate their suitability for its intended purpose. Despite there are some commercial kits to evaluate IFN-I signaling, these are focused on measuring in vitro biological response instead of their validation, which is a pharmaceutical industry requirement. The aim of this work was to validate the HEK-Blue IFN-α/ß system evaluating the biological activity of IFN-α/ß under good laboratory practices, according to international standards. Our results demonstrated that HEK-Blue IFN-α/ß system comply with accuracy (r2>0.95) precision (CV < 20%) and specificity for both IFN-α/ß; confirming that this assay is robust for this biotherapeutics' evaluation. Thereby, this bioassay could be implemented as a complementary method to the classical anti-proliferative and anti-viral assays under quality control environments.

Validation of an ADCC assay using human primary natural killer cells to evaluate biotherapeutic products bearing an Fc region.

The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy. Induction of ADCC can be evaluated by employing effector cells from different sources, such as peripheral blood mononuclear cells (PBMC) and genetically modified cell lines (e.g., transfected NKs or Jurkat cells), and different approaches can be used for detection and results interpretation depending on the type of effector cells used. In this regard, validation of the assays is relevant to ensure the reliability of the results according to the intended purpose. Herein, we show the standardization and validation of ADCC assays to test the potency of three biotherapeutic proteins using primary NK cells obtained from fresh blood as effector cells and detecting cell death by flow cytometry. The advantage of using primary NKs instead of modified cells is that the response is closer to that occurring in vivo since cytotoxicity is evaluated in a direct manner. Our results indicate that in all cases, the assays exhibited a characteristic sigmoidal dose/response curve complying with accurate, precise and specific parameters. Thereby, the validated ADCC assay is an appropriate alternative to evaluate the biological activities of these type of biotherapeutics.

Development and validation of a bioassay to evaluate binding of adalimumab to cell membrane-anchored TNFα using flow cytometry detection.

Physicochemical and structural properties of proteins used as active pharmaceutical ingredients of biopharmaceuticals are determinant to carry out their biological activity. In this regard, the assays intended to evaluate functionality of biopharmaceuticals provide confirmatory evidence that they contain the appropriate physicochemical properties and structural conformation. The validation of the methodologies used for the assessment of critical quality attributes of biopharmaceuticals is a key requirement for manufacturing under GMP environments. Herein we present the development and validation of a flow cytometry-based methodology for the evaluation of adalimumab's affinity towards membrane-bound TNFα (mTNFα) on recombinant CHO cells. This in vitro methodology measures the interaction between an in-solution antibody and its target molecule onto the cell surface through a fluorescent signal. The characteristics evaluated during the validation exercise showed that this methodology is suitable for its intended purpose. The assay demonstrated to be accurate (r2 = 0.92, slope = 1.20), precise (%CV ≤ 18.31) and specific (curve fitting, r2 = 0.986-0.997) to evaluate binding of adalimumab to mTNFα. The results obtained here provide evidence that detection by flow cytometry is a viable alternative for bioassays used in the pharmaceutical industry. In addition, this methodology could be standardized for the evaluation of other biomolecules acting through the same mechanism of action.

The Macrophage Activation Induced by Bacillus thuringiensis Cry1Ac Protoxin Involves ERK1/2 and p38 Pathways and the Interaction with Cell-Surface-HSP70.

Here, we aimed to further characterize the mechanisms involved in protoxin (p) Cry1Ac-induced macrophage activation. We demonstrated that pCry1Ac induces MAPK ERK1/2, p38, and JNK phosphorylation in RAW264.7 macrophages. Because MAPK activation is mainly triggered via ligand-receptor interactions, we focused on the identification of potential pCry1Ac-receptor proteins. Flow cytometry and confocal analysis showed specific saturable pCry1Ac-binding to the macrophage surface and evidenced its internalization via the clathrin-pathway. We performed immunoprecipitation assays and identified by MALDI-TOF-TOF several possible pCry1Ac-binding proteins, such as heat shock proteins (HSPs), vimentin, α-enolase, and actin; whose interaction and presence was confirmed, respectively, by ligand blot and Western blot assays. We also detected cell-surface (cs) pCry1Ac-HSP70 colocalization, so HSP70 was chosen for further characterization. Co-immunoprecipitation with HSP70 antibodies followed by Western blot confirmed the pCry1Ac-HSP70 interaction. Furthermore, pretreatment of RAW264.7 cells with HSP70 antibodies reduced pCry1Ac-induced ERK1 phosphorylation and MCP-1 production; thus suggest the functional participation of csHSP70 in pCry1Ac-induced macrophage activation. csHSP70 also was evaluated in peritoneal-cavity (PerC) macrophages of untreated BALB/c mice, interestingly it was found that the predominant population namely large-peritoneal-macrophages (LPM) displayed csHSP70 + hi. Furthermore, the dynamics of PerC macrophage subsets, LPM, and small-peritoneal macrophages (SPM) were evaluated in response to in vivo pCry1Ac stimuli in presence or not of phenylethynesulfonamide (PES) a functional HSP70 inhibitor. It was found that pCry1Ac increased the proportion of SPM CD11b + F4/80 + lowMHCII + csHSP70 + low and markedly reduced the amount of LPM CD11b + F4/80 + hiMHCII-csHSP70 + hi; while PES, partially suppressed this pCry1Ac-induced effect, further suggesting the participation of HSP70 in macrophage activation process. J. Cell. Biochem. 119: 580-598, 2018. © 2017 Wiley Periodicals, Inc.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model.

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.

Comparative proteome analysis of Brucella abortus 2308 and its virB type IV secretion system mutant reveals new T4SS-related candidate proteins.

Brucella abortus is an alpha-2 proteobacteria with a type IV secretion system (T4SS) known as virB, which is necessary to gain virulence by building up a replicative vacuole associated with the endoplasmic reticulum of the host cell. A virB T4SS mutant of the B. abortus 2308 strain and its wild-type strain were grown in acid medium in order to obtain and analyze their proteomes, looking for putative proteins that may serve as T4SS substrates and those that may be subjected to T4SS regulation. A total of 47 overexpressed and 22 underexpressed proteins from the virB T4SS mutant strain were selected and sequenced. Some of the 69 analyzed proteins have not been described before either as over or under-expressed in relation to a virB T4SS mutation, whereas some of them have been already described by other groups as potentially important secretory proteins in other Brucella species. An important number of the proteins identified are outer membrane and periplasmic space protein, which makes them become particularly important new T4SS-related candidate proteins.