Nanoparticles Codelivering mRNA and SiRNA for Simultaneous Restoration and Silencing of Gene/Protein Expression In Vitro and In Vivo.

RNA-based agents (siRNA, miRNA, and mRNA) can selectively manipulate gene expression/proteins and are set to revolutionize a variety of disease treatments. Nanoparticle (NP) platforms have been developed to deliver functional mRNA or siRNA inside cells to overcome their inherent limitations. Recent studies have focused on siRNA to knock down proteins causing drug resistance or mRNA technology to introduce tumor suppressors.

Longitudinal determination of seroprevalence and immune response to SARS-CoV-2 in a population of food and retail workers through decentralized testing and transformation of ELISA datasets.

Background: Since the onset of the global COVID-19 pandemic in early 2020, numerous studies have been conducted worldwide to understand our immune response to the virus and to vaccination. This study investigates the humoral response elicited by SARS-CoV-2 infection and by vaccination in the poorly studied population of food and retail workers. These occupations were classified as essential by the Public Health Agency of Canada, potentially placing this population at greater risk of infection.

Dissecting the Enterococcal Polysaccharide Antigen (EPA) structure to explore innate immune evasion and phage specificity.

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E.

SARS-CoV-2 spike-based virus-like particles incorporate influenza H1/N1 antigens and induce dual immunity in mice.

A vaccine effective against both SARS-CoV-2 and influenza A (IAV) viruses could represent a cost-effective strategy to reduce their combined public health burden as well as potential complications arising from co-infection. Based on previous findings that full-length SARS-CoV-2 spike (S) expression can induce high-level, enveloped VLP (eVLP) production in CHO cells, we tested whether IAV H1N1 hemagglutinin (H1) and neuraminidase (N1) could also be displayed on these particles. We found that co-incorporation of the IAV surface antigens in spike VLPs (S-VLPs) was highly efficient: upon transient co-expression of S + H1 or S + H1 + N1 in CHO cells, the resulting VLPs contained similar amounts of the SARS-CoV-2 S and IAV antigens.

Histamine stimulates human microglia to alter cellular prion protein expression via the HRH2 histamine receptor.

Although the cellular prion protein (PrP) has been evolutionarily conserved, the role of this protein remains elusive. Recent evidence indicates that PrP may be involved in neuroinflammation and the immune response in the brain, and its expression may be modified via various mechanisms. Histamine is a proinflammatory mediator and neurotransmitter that stimulates numerous cells via interactions with histamine receptors 1-4 (HRH1-4).

Rapid Systematic Screening of Bispecific Antibody Surrogate Geometries for T-Cell Engagement Using DNA Nanotechnology.

Bispecific antibodies (bsAbs) are emerging immune-therapeutics, and many formats exist that differ considerably in structure. However, little systematic data exist about how the spatial organization of their components influences activity, requiring innovative approaches combining empirical and quantitative frameworks. This study presents a modular DNA nanotechnology platform to generate numerous bsAbs with surrogate geometries that span the structural features of the BiTE, IgG-like, and IgG-conjugate platforms to screen for T-cell engagement.

A Novel Antigen Design Strategy to Isolate Single-Domain Antibodies that Target Human Nav1.7 and Reduce Pain in Animal Models.

Genetic studies have identified the voltage-gated sodium channel 1.7 (Na1.7) as pain target.

Semi-Covariance Coefficient Analysis of Spike Proteins from SARS-CoV-2 and Its Variants Omicron, BA.5, EG.5, and JN.1 for Viral Infectivity, Virulence and Immune Escape.

Semi-covariance has attracted significant attention in recent years and is increasingly employed to elucidate statistical phenomena exhibiting fluctuations, such as the similarity or difference in charge patterns of spike proteins among coronaviruses. In this study, by examining values above and below the average/mean based on the positive and negative charge patterns of amino acid residues in the spike proteins of SARS-CoV-2 and its current circulating variants, the proposed methods offer profound insights into the nonlinear evolving trends in those viral spike proteins. Our study indicates that the charge span value can predict the infectivity of the virus and the charge density can estimate the virulence of the virus, and both predicated infectivity and virulence appear to be associated with the capability of viral immune escape.

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer.

Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients.

Recombinant Protein Production from Stable CHO Cell Pools.

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.

Role of N343 glycosylation on the SARS-CoV-2 S RBD structure and co-receptor binding across variants of concern.

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance.

Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized.

Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity.

Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22.

Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.

With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines.

CHO cells for virus-like particle and subunit vaccine manufacturing.

Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8 T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment.