Analysis of antibodies from whole-cell immunization by a tANCHOR cell-based ELISA.

Monitoring specific antibodies derived from whole-cell immunization through cell-based ELISA methods poses challenges due to humoral responses against various cell proteins. In this report, we outline a technique involving pre-adsorption on cells to remove undesirable antibodies from immune serum. This step provides the subsequent monitoring of antibodies specific to the targeted antigen using a tANCHOR-based ELISA. Notably, this approach accelerates result acquisition, eliminating the necessity to purify the expressed antigen or obtain a customized peptide for coating assay plates.

tANCHOR fast and cost-effective cell-based immunization approach with focus on the receptor-binding domain of SARS-CoV-2.

Successful induction of antibodies in model organisms like mice depends strongly on antigen design and delivery. New antigen designs for immunization are helpful for developing future therapeutic monoclonal antibodies (mAbs). One of the gold standards to induce antibodies in mice is to express and purify the antigen for vaccination. This is especially time-consuming when mAbs are needed rapidly. We closed this gap and used the display technology tetraspanin anchor to develop a reliable immunization technique without the need to purify the antigen. This technique is able to speed up the immunization step enormously and we have demonstrated that we were able to induce antibodies against different proteins with a focus on the receptor-binding domain of SARS-CoV-2 and the extracellular loop of canine cluster of differentiation 20 displayed on the surface of human cells.

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications.

Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.

Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2.

Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified as a major autoantigenic target in Crohn's disease patients. It was reported recently that a long (GP2a) and a short (GP2b) isoform of GP2 exist and that in the outcome of inflammatory bowel diseases (IBD) GP2-specific autoantibodies probably appear as new serological markers for diagnosis and therapeutic monitoring. To investigate this further and in order to establish diagnostic tools for the discrimination of both GP2 isoforms, a set of different murine monoclonal and camelid recombinant single domain antibodies (camelid VHH) was generated and validated in various enzyme-linked immunosorbent assay (ELISA) formats, immunofluorescence on transgenic cell lines and immunohistochemistry on monkey pancreas tissue sections. Out of six binders identified, one was validated as highly specific for GP2a. This murine monoclonal antibody (mAb) was used as capture antibody in construction of a sandwich ELISA for the detection of GP2a. Camelid VHHs or a second murine mAb served as detection antibodies in this system. All antibodies were also able to stain GP2a or GP2b on transgenic cell lines as well as on pancreatic tissue in immunohistochemistry. The KD values measured for the camelid VHHs were between 7 nM and 23pM. This set of specific binders will enable the development of suitable diagnostic tools for GP2-related studies in IBD.

Neuroprotection by Caffeine in Hyperoxia-Induced Neonatal Brain Injury.

Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term "oxygen radical disease of prematurity". Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28-32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.