Katja Hanack

Transferring the complexity of the in vivo immune response into a controlled in vitro system represents a promising strategy for the rapid generation of antigen-specific antibodies while significantly reducing the reliance on animal experimentation, thus adhering to the 3Rs principle. In this study, we present an in vitro immunization (IVI) approach that employs conditionally immortalized dendritic cells (DCs), derived from a transgenic irtTA-GBD/T-Ag mouse model, to initiate antigen-specific immune responses in vitro . These dendritic cells can be kept in a proliferative state under tetracycline-controlled expression of the SV40 large T antigen (termed iniDCs) and maintained long-term in culture. Upon de-induction of the immortalizing transgene, the resulting deinduced iniDCs (de-iniDCs) regain physiological properties and exhibit full functional maturation in response to appropriate stimuli. When co-cultured with naïve murine T and B lymphocytes, these mature de-iniDCs are capable of initiating antigen-specific adaptive immune responses, culminating in the production of antigen-specific antibodies by B cells. By utilizing this standardized population of antigen-presenting cells, we have established a robust and reproducible IVI protocol that enables the generation of antigen-specific antibody responses in vitro . Antibody-secreting B cells were subsequently fused with myeloma cells, and antigen-specific hybridomas were rapidly identified and isolated using the novel selma ( selection of monoclonal antibody ) technology, allowing for an accelerated and efficient selection of antigen-specific monoclonal antibodies.

The Omicron variants of SARS‐CoV‐2 are characterized by their high transmissibility and immune evasion. Existing treatments using neutralizing antibodies have shown different effectiveness due to variants with mutations occurring mainly in the RBD and NTD regions. In this study, the functional neutralizing ability of a camelid full‐length antibody (hcAb‐B10) and its corresponding VHH fragment (VHH‐B10) is investigated. Experimental binding studies demonstrated clear recognition and neutralization of Wild‐type (WT) and Omicron variants, but not Delta. Epitope mapping, peptide fragment inhibition, and neutralization studies using pseudovirus expressing respective SARS‐CoV‐2 Spike variants, along with in silico molecular docking studies and AI‐directed structural design, reveal that the B10 antibody interacts effectively with the Spike trimer in a closed position of both WT and Omicron, by targeting the RBD region. This newly generated B10 antibody shows a wide coverage, including the currently dominant Omicron variants, and demonstrates its potential to efficiently neutralize SARS‐CoV‐2.

The COVID-19 pandemic had a profound impact on global health. This study focuses on an in-depth analysis of the structural proteins (Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E) protein) of SARS-CoV-2 and its variants, aiming to develop a multiepitope vaccine construct that targets the virus independently of its variants. The analysis began by examining genetic variations in viral proteins relative to the reference strain Wuhan-Hu2, particularly in the S, M, N, and E proteins. T-cell epitope predictions for MHC Class-I and Class-II binding were conducted, shedding light on potential cytotoxic and helper T lymphocyte recognition. Identification of linear B-cell epitopes laid the groundwork for antibody-based humoral immune responses. The safety and efficacy of these epitopes were assessed for antigenicity, allergenicity, toxicity, immunogenicity, and conservancy. Population coverage analysis indicated promising global effectiveness of the designed vaccine construct. By incorporating 28 epitopes, we validated that was designed vaccine construct for stability through structural analysis. Molecular dynamics simulations and docking studies revealed its robust interaction with Toll-like receptor 4 (TLR4). Immune simulation studies suggested that the vaccine construct could induce a potent immune response by enhancing antibody titers, B-cell proliferation, memory cell development, and activation of T cells and natural killer cells upon administration. This comprehensive approach offers a promising multiepitope vaccine against SARS-CoV-2, with the potential for broad global coverage and strong immunogenicity. Further experimental validation holds the prospect of introducing a novel candidate vaccine to aid in the ongoing battle against the COVID-19 pandemic.

The discovery of antibodies through phage display is significantly influenced by antigen presentation during panning, particularly for membrane-anchored proteins, which pose challenges due to their complex structures. Traditional approaches, such as whole cells expressing the target protein, often result in low antigen density and high background signals. In this study, we describe an alternative method using stably transfected cell lines that express the target antigen on their surface, regulated by an intracellular enhanced green fluorescent protein (EGFP) signal. This system enables high-throughput flow cytometry-based screening of phage display libraries to isolate human antibodies that recognize the native conformation of membrane proteins. Using human epithelial cell adhesion molecule (EpCAM) and human neuroplastin 65 (NP65) as model antigens, we established an optimized screening workflow with polyclonal phage pools. Selected EpCAM-specific single-chain variable fragments (scFvs) from a naïve library were recombinantly expressed with an IgG4 scaffold and characterized for specific binding. This approach provides an effective platform for the identification of antibodies against membrane proteins in their native state.

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.

In the original paper, Li and co-workers [ACS Appl. Mater. Interfaces 2022, 14, 17128-17141] described their approach to select specific hybridoma cells from a polyclonal hybridoma pool by using a cell surface anchor to catch the secreted antibody. The antigen-specific detection was performed with streptavidin-labeled antigen and a PE-labeled anti-F(ab & PRIME;)2 antibody. The present comment offers a clearer description of the selection system originally published by Listek et al. in 2020 and provides further information about the importance of controls and recent adaptations made by our lab.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by western blot, ELISA and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy-, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and 44 samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5 % and a specificity of 100 % for the certified IVD. There were 7 samples with an uncertain outcome. Our VLP-ELISA showed superior performance with a sensitivity of 97.5 %, a specificity of 100 %, and only 3 uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

We established an in vitro activation protocol for the generation of monoclonal antibodies. Purified murine B lymphocytes were activated in vitro with combinations of antigen and selected stimuli. Within ten days we induced specific IgM and IgG antibody responses against a viral coat protein. Permanently antibody-producing hybridomas were generated to produce monoclonal antibodies.

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.