Use of stable CHO pools and CHO TGE to accelerate SARS-CoV-2 vaccine development

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Immunological study of COVID-19 vaccine candidate based on recombinant spike trimer protein from different SARS-CoV-2 variants of concern

The emergency of new SARS-CoV-2 variants that feature increased immune escape marks an urgent demand for better vaccines that will provide broader immunogenicity. Here, we evaluated the immunogenic capacity of vaccine candidates based on the recombinant trimeric spike protein (S) of different SARS-CoV-2 variants of concern (VOC), including the ancestral Wuhan, Beta and Delta viruses. In particular, we assessed formulations containing either single or combined S protein variants. Our study shows that the formulation containing the single S protein from the ancestral Wuhan virus at a concentration of 2µg (SW2-Vac 2µg) displayed in the mouse model the highest IgG antibody levels against all the three (Wuhan, Beta, and Delta) SARS-CoV-2 S protein variants tested. In addition, this formulation induced significantly higher neutralizing antibody titers against the three viral variants when compared with authorized Gam-COVID-Vac-rAd26/rAd5 (Sputnik V) or ChAdOx1 (AstraZeneca) vaccines. SW2-Vac 2µg was also able to induce IFN-gamma and IL-17, memory CD4 populations and follicular T cells. Used as a booster dose for schedules performed with different authorized vaccines, SW2-Vac 2µg vaccine candidate also induced higher levels of total IgG and IgG isotypes against S protein from different SARS-CoV-2 variants in comparison with those observed with homologous 3-dose schedule of Sputnik V or AstraZeneca. Moreover, SW2-Vac 2µg booster induced broadly strong neutralizing antibody levels against the three tested SARS-CoV-2 variants. SW2-Vac 2µg booster also induced CD4+ central memory, CD4+ effector and CD8+ populations. Overall, the results demonstrate that SW2-Vac 2 µg is a promising formulation for the development of a next generation COVID-19 vaccine.Fil: Rudi, Erika. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Martin Aispuro, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Zurita, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: González López Ledesma, María Mora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bottero, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Malito, Juan Pablo Alfonso. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. Grupo Vinculado Incuinta al IVIT | Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas. Grupo Vinculado Incuinta al IVIT; ArgentinaFil: Gabrielli, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Gaillard, María Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Stuible, Matthew. National Research Council Canada, Montreal; CanadáFil: Durocher, Yves. National Research Council Canada, Montreal; CanadáFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit.; ArgentinaFil: Hozbor, Daniela Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Heterologous booster with a novel formulation containing glycosylated trimeric S protein is effective against Omicron

In this study, we evaluated the efficacy of a heterologous three-dose vaccination schedule against the Omicron BA.1 SARS-CoV-2 variant infection using a mouse intranasal challenge model. The vaccination schedules tested in this study consisted of a primary series of 2 doses covered by two commercial vaccines: an mRNA-based vaccine (mRNA1273) or a non-replicative vector-based vaccine (AZD1222/ChAdOx1, hereafter referred to as AZD1222). These were followed by a heterologous booster dose using one of the two vaccine candidates previously designed by us: one containing the glycosylated and trimeric spike protein (S) from the ancestral virus (SW-Vac 2µg), and the other from the Delta variant of SARS-CoV-2 (SD-Vac 2µg), both formulated with Alhydrogel as an adjuvant. For comparison purposes, homologous three-dose schedules of the commercial vaccines were used. The mRNA-based vaccine, whether used in heterologous or homologous schedules, demonstrated the best performance, significantly increasing both humoral and cellular immune responses. In contrast, for the schedules that included the AZD1222 vaccine as the primary series, the heterologous schemes showed superior immunological outcomes compared to the homologous 3-dose AZD1222 regimen. For these schemes no differences were observed in the immune response obtained when SW-Vac 2µg or SD-Vac 2µg were used as a booster dose. Neutralizing antibody levels against Omicron BA.1 were low, especially for the schedules using AZD1222. However, a robust Th1 profile, known to be crucial for protection, was observed, particularly for the heterologous schemes that included AZD1222. All the tested schedules were capable of inducing populations of CD4 T effector, memory, and follicular helper T lymphocytes. It is important to highlight that all the evaluated schedules demonstrated a satisfactory safety profile and induced multiple immunological markers of protection. Although the levels of these markers were different among the tested schedules, they appear to complement each other in conferring protection against intranasal challenge with Omicron BA.1 in K18-hACE2 mice. In summary, the results highlight the potential of using the S protein (either ancestral Wuhan or Delta variant)-based vaccine formulation as heterologous boosters in the management of COVID-19, particularly for certain commercial vaccines currently in use

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific to the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS)

Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation

Assessment of the longitudinal humoral response in non-hospitalized SARS-CoV-2-positive individuals at decentralized sites: Outcomes and concordance

IntroductionEarly in the COVID-19 pandemic, reagent availability was not uniform, and infrastructure had to be urgently adapted to undertake COVID-19 surveillance.MethodsBefore the validation of centralized testing, two enzyme-linked immunosorbent assays (ELISA) were established independently at two decentralized sites using different reagents and instrumentation. We compared the results of these assays to assess the longitudinal humoral response of SARS-CoV-2-positive (i.e., PCR-confirmed), non-hospitalized individuals with mild to moderate symptoms, who had contracted SARSCoV-2 prior to the appearance of variants of concern in Québec, Canada.ResultsThe two assays exhibited a high degree of concordance to identify seropositive individuals, thus validating the robustness of the methods. The results also confirmed that serum immunoglobulins persist ≥ 6 months post-infection among non-hospitalized adults and that the antibodies elicited by infection cross-reacted with the antigens from P.1 (Gamma) and B.1.617.2 (Delta) variants of concern.DiscussionTogether, these results demonstrate that immune surveillance assays can be rapidly and reliably established when centralized testing is not available or not yet validated, allowing for robust immune surveillance

Characterization of novel substrates and inhibitors of protein tyrosine phosphatase 1B

Protein tyrosine phosphatase 1B (PTP1B) has been studied extensively as a modulator of metabolic signaling. In addition to the insulin receptor, which is a key physiological target of PTP1B, a number of other receptor and receptor-associated tyrosine kinases have also been reported as PTP1B substrates. Although hyperactivation of several of these kinases is associated with cancer, mice lacking PTP1B are not prone to tumorigenesis. In fact, PTP1B deficiency confers resistance to particular types of cancer; however, the mechanisms responsible for this and other cellular effects of PTP1B remain poorly characterized.With the aim of better understanding its functions at a molecular level, two phosphoproteins, cortactin and STAM2, were identified as substrates of PTP1B. Cortactin regulates actin cytoskeletal dynamics in various contexts while STAM2 is involved in sorting ubiquitinated receptor tyrosine kinases (RTKs) to the lysosomal degradative pathway following their activation. In both cases, the phosphorylation of specific tyrosine residues was regulated by PTP1B, and mutation of these sites affected the cellular response to different stimuli. These novel targets could each contribute to the known effects of PTP1B deficiency on processes including RTK signaling, cell migration, and tumorigenesis.PTP1B and other members of the PTP enzyme family are considered promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, limiting cellular permeability. As an alternative approach that could aid our functional studies, a novel, uncharged, competitive inhibitor of PTP1B was characterized. This compound effectively inhibited PTP1B in cell-based assays as well as a subset of other PTPs in vitro. A series of derivatives were tested and molecular docking studies were performed in order to understand its important functional groups and identify its likely site of action.Through the identification of cortactin and STAM2 as PTP1B targets, this thesis research has contributed significantly to our understanding of the molecular events regulated by PTP1B. Furthermore, the characterization of a small-molecule inhibitor provides a biochemical tool for probing PTP function, which could also be used as a scaffold for future drug development.La protéine tyrosine phosphatase 1B (PTP1B) a été longtemps étudiée en tant que modulateur de signalisation métabolique. En plus du récepteur de l'insuline, qui est une cible physiologique clée de PTP1B, un grand nombre de recepteurs et de tyrosine-kinases associées aux recepteurs ont été rapportés comme substrats de PTP1B. Bien que l'hyperactivation de plusieurs de ces kinases est associée au cancer, les souris déficientes en PTP1B ne sont pas prédisposées au développement de tumeurs. En fait, la déficience en PTP1B confère une résistance à certains types de cancers, mais les mécanismes responsables de ceci ainsi que d'autres effets cellulaires de PTP1B ne sont pas bien caractérisés.En étudiant les fonctions de PTP1B au niveau moléculaire, deux phosphoprotéines ont été identifiées comme substrats : la cortactine et STAM2. La cortactine régule la dynamique du cytosquelette d'actine alors que STAM2 est impliqué dans le tri des récepteurs tyrosine kinases (RTKs) ubiquitinés vers la voie de dégradation lysosomale. Pour les deux substrats, la phosphorylation de certains résidus tyrosines était régulée par PTP1B et la mutation de ces sites affectait la réponse cellulaire à différents stimuli. Ces nouveaux substrats de PTP1B peuvent contribuer aux effets connus de déficience en PTP1B notamment la signalisation des RTKs, la migration cellulaire et le développement de tumeurs.PTP1B ainsi que les autres membres de la famille d'enzymes PTP sont considérés comme des cibles prometteuses pour le développement de médicaments. Par contre, les inhibiteurs de PTP les plus efficaces à ce jour sont fortement chargés ce qui limite la perméabilité cellulaire. Comme approche alternative qui pourrait aider nos études de fonction, un nouvel inhibiteur compétitif et non-chargé de PTP1B a été identifié. Ce composé inhibe efficacement PTP1B dans des essais cellulaires ainsi qu'un sous-ensemble de PTPs in vitro. Une série de dérivés ont été testés et des études de docking moléculaire ont été faites afin de mieux comprendre les groupes fonctionnels importants de cet inhibiteur et d'identifier son site d'action probable.En identifiant la cortactine et STAM2 comme substrats de PTP1B, cette thèse de recherche a contribuée significativement à notre compréhension d'événements moléculaires régulés par PTP1B. En outre, la caractérisation d'un inhibiteur fourni un outil biochimique pour étudier la fonction des PTP et peut servir d'échafaudage pour le développement de médicaments

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections

Impact of the temperature on the interactions between common variants of the SARS-CoV-2 receptor binding domain and the human ACE2

Several key mutations in the Spike protein receptor binding domain (RBD) have been identified to influence its affinity for the human Angiotensin-Converting Enzyme 2 (ACE2). Here, we perform a comparative study of the ACE2 binding to the wild type (Wuhan) RBD and some of its variants: Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Kappa B.1.617.1, B.1.1.7 + L452R and Omicron B.1.1.529. Using a coiled-coil mediated tethering approach of ACE2 in a novel surface plasmon resonance (SPR)-based assay, we measured interactions at different temperatures. Binding experiments at 10 °C enhanced the kinetic dissimilarities between the RBD variants and allowed a proper fit to a Langmuir 1:1 model with high accuracy and reproducibility, thus unraveling subtle differences within RBD mutants and ACE2 glycovariants. Our study emphasizes the importance of SPR-based assay parameters in the acquisition of biologically relevant data and offers a powerful tool to deepen our understanding of the role of the various RBD mutations in ACE2 interaction binding parameters

PTP1B Regulates Cortactin Tyrosine Phosphorylation by Targeting Tyr446*S⃞

The emergence of protein-tyrosine phosphatase 1B (PTP1B) as a potential drug target for treatment of diabetes, obesity, and cancer underlies the importance of understanding its full range of cellular functions. Here, we have identified cortactin, a central regulator of actin cytoskeletal dynamics, as a substrate of PTP1B. A trapping mutant of PTP1B binds cortactin at the phosphorylation site Tyr446, the regulation and function of which have not previously been characterized. We show that phosphorylation of cortactin Tyr446 is induced by hyperosmolarity and potentiates apoptotic signaling during prolonged hyperosmotic stress. This study advances the importance of Tyr446 in the regulation of cortactin and provides a potential mechanism to explain the effects of PTP1B on processes including cell adhesion, migration, and tumorigenesis