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While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees. Here, in a longitudinal cohort of 1245 hospital care workers and 146 nursing home residents, the authors find that a large inter-individual variation in anti-spike antibody levels after one dose of BNT162b2mRNA vaccine is partially explained by age, sex, previous exposure, and treatments, while the 2nd dose is required to reach sero-conversion at the population level.

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.

When deadlines and resources of software projects become scarce, testing is usually in the first row to have its activities aborted or reduced; however, if defects cannot be found, product quality can be affected. In the software development process, aborted or reduced activities that can bring short-term benefits, but can be harmful to the project in the long run, are considered Technical Debt (TD) and, when the TDs impact testing activities, they are called Test Debt. Although there are several studies dealing with Test Debt, current solutions often deal with specific types of tests (e.g., exploratory and automated tests) and do not address the whole software testing process. Aiming to fill these gaps, this work then proposes a Test Debt Catalog, called TestDCat, with subtypes of Test Debts and Technical Debt management activities. This catalog is built based on the results of an empirical study, a literature review and semi-structured interviews conducted with practitioners who perform testing activities on five projects from industry. For the TestDCat evaluation, a case study is conducted in real projects in order to identify if the catalog is user-friendly and if its use helps the Test Debt management during the execution of test activities in these software development projects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at −80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.

Current lab-scale purification methods for ADDomer are labour-intensive, time-consuming and poorly scalable. The intracellular nature of ADDomer production further complicates downstream processing, requiring robust, scalable solutions for cell lysis and clarification. In the present work we focus on developing a scalable, GMP-compliant process for ADDomer purification. The workflow combines tangential microfiltration (TMF) using a 0.4 μm hollow fiber for cell retention, chemical lysis with 0.06 % Deviron ® C16, and in-line Benzonase ® treatment for DNA removal. Several capture approaches were explored, including anion and cation exchange, hydrophobic interaction and multimodal chromatography. Among these, the Sartobind ® Q anion exchange membrane demonstrated superior performance, achieving ADDomer purity of ~ 83 % and recovery yields > 85 %. Ultrafiltration membranes of different chemistries (regenerated cellulose, RC, and polyethersulfone, PES) and cut-off (300 kDa, 500 kDa, 1 MDa) were evaluated for polishing and buffer exchange. The 1 MDa RC membrane enabled a recovery of ~ 87 % and purity > 97%. Final sterile filtration with a PES membrane preserved particle integrity, purity and achieved > 80 % recovery. Overall, the purification process herein established yielded ~ 47 mg of ADDomer particles per L of culture volume while removing > 97 % and > 99 % of total protein and ds DNA, respectively. In summary, this study showcases the implementation of a scalable and GMP-compliant purification platform for ADDomer, paving the way for the development of next-generation ADDomer-based vaccines and antivenoms.

The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect ( Sf 9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 10 6 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His 6 ) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27–22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

Plasmodium falciparum cysteine-rich protective antigen ( Pf CyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield Pf CyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality Pf CyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved Pf CyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest Pf CyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, Pf CyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti- Pf CyRPA antibodies that impeded the multiplication of P. falciparum in vitro . This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant Pf CyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin–Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC’s MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.

Breast cancer poses a global healthcare challenge due to its prevalence and multifactorial etiology. Despite advances in early detection and treatment, therapeutic approaches like standardized chemotherapy often fall short, primarily due to tumor heterogeneity and genetic variations. In light of this, personalized treatments are imperative.To address diagnostic and subtype classification challenges, this study utilized Metabric datasets and applied machine learning models after pre-processing, feature selection and hiperparameters otimization. The K-Nearest Neighbors (KNN) model achieved a 94% accuracy rate in cancer detection with a high recall rate, minimizing false negatives. For subtype classification, the Deep Neural Network (DNN) model excelled with a 95% accuracy and an average macro recall value of 0.96.The study in the realm of cancer prediction, only two genes were commonly observed. In the part of predict of the subtype revealed a significant overlap of 32 genes with the PAM50 list, affirming genetic consistency in breast cancer.In conclusion, the integration of KNN and DNN models significantly enhances both diagnostic and therapeutic strategies in breast cancer management. These models not only demonstrated high accuracy but also provided insights into gene-specific roles, potentially revolutionizing personalized, data-driven treatment pathways.