[Construction of a chimeric cell model expressing human immunodeficiency virus-1 envelope protein based on transposon system and evaluation of chimeric antigen receptor t-cell cytotoxicity in vitro].

High-titer human packaging cells producing γ-retroviral vectors pseudotyped with engineered baboon endogenous retrovirus (BaEV) envelope proteins

This proof-of-concept study underlines the utility of the Env variant BaEV-TR for the establishment of stable pseudotype VPCLs and the utilization of HT1080 cells as an alternative to frequently used 293T host cells.

Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells

An industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs) and provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

virus-specific immune responses in C57BL/6 mice by immunization with a modified recombinant adenovirus containing the chimeric gene, GcS0.7

Presentation by Virally Infected Cells of Antigenic Proteins Utilized in Natural Killer Cell Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) 2256335

The presence of viral proteins in the plasma membranes of infected cells is essential for natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC). However, many enveloped viruses assemble inside cells rather than budding out through plasma membranes. Thus, the presence of viral proteins in plasma membranes of infected cells is uncertain, as is which of several viral structural proteins participates in ADCC. Their presence might represent leftover proteins after virion assembly. To address external viral protein display and its sufficiency for antibody-dependent recognition, we utilized the endemic, cold-causing human coronavirus OC-43, a surrogate virus for SARS-CoV-2. We used NK-92-CD16A lymphocytes as killer cells, OC-43 from BEI, and infected, 51Cr-radiolabeled lung A549 cells as ADCC ‘targets’. Viral proteins were monitored by immunofluorescent microscopy. Antibodies to OC-43 included two monoclonal anti-spike S with human Fc’s, plasma from children who had OC-43 respiratory infections, rabbit affinity purified polyclonal anti-nucleocapsid N and S, and sera from unimmunized rabbits. We found that all the antibody reagents reacted with OC-43 infected cells by immunofluorescent microscopy. Labeling of external viral proteins was punctate. However, only the immune plasma and the non-immunized rabbit sera supported ADCC. The finding with anti-S was surprising since the human mAb 1249A8 anti-S could support Fc-receptor dependent phagocytosis of beads with S protein [PMID35862475]. The finding is consistent with PMID35587364, that SARS CoV-2 S was under-expressed in plasma membranes of infected cells and that depletion of anti-S from antisera from infected persons had negligible effect on ADCC. The finding with anti-N was surprising in light of PMID41060789. Conclusions are premature but the data indicate that antibodies to the S protein alone are unlikely to support ADCC to coronavirally infected cells. University of Nevada, Reno Foundation Award. Viral Immunology (VIR)

Construction, expression, and characterization of a multi-epitope protein based on envelope glycoproteins B, C, and D of Varicellovirus bovinealpha1/5 in Escherichia coli.

Varicellovirus bovinealpha1 (BoAHV-1) and 5 (BoAHV-5) are important pathogens associated with respiratory, reproductive, and neurological disorders in cattle, remaining globally relevant since their first reports in the 1950s and 1960s. Envelope glycoproteins B, C, and D play critical roles in viral attachment and fusion, making them key targets for immune responses and promising candidates for vaccine development. This study aimed to design a multi-epitope protein using immunoinformatic approaches, incorporating highly conserved, antigenic B- and T- cell epitopes with strong predicted MHC-binding affinity from glycoproteins B, C, and D of BoAHV-1/5, followed by in silico characterization and heterologous expression in Escherichia coli. The construct was designed using bioinformatics tools, cloned into the pET-24a vector, and expressed in a prokaryotic system. The resulting protein consists of 321 amino acids, with a predicted molecular weight of 33.24kDa, high antigenicity (1.1543) and non-allergenicity. Molecular docking analyses indicated strong interactions with bovine Toll-like receptors TLR2/6 and TLR4. The protein was successfully expressed in E. coli and detected by anti-His6x antibody in Western blot, showing the expected molecular weight (~33kDa). It was also recognized by bovine serum containing neutralizing antibodies against BoAHV-1/5. Overall, these findings support the feasibility of the multi-epitope construct and provide a foundation for future investigations of its immunogenicity and protective efficacy studies in animal models.

[Construction and characterization of a stable Cas9-expressing monoclonal WSL cell line].