Applied Biotechnology Institute

Soo-Dam Kim, Ph.D., Eun Soo Park, M.Sc., Jung Hyang Park, K.M.D., Jeong Yoon Park, K.M.D., Mi-Jeong Choi, Ph.D.‖, Hwa Seung Yoo, K.M.D.. J Korean Med Rehabi 2025;35:1-12. https://doi.org/10.18325/jkmr.2025.35.3.1

Coronaviruses continue to disrupt health and economic productivity worldwide. Porcine epidemic diarrhea virus (PEDV) is a devastating swine disease and SARS-CoV-2 is the latest coronavirus to infect the human population. Both viruses display a similar spike protein on the surface that is a target of vaccine development. Despite the availability of commercial vaccines for both viruses, there is still a high occurrence of infections and a great need for enhanced efficacy and lower costs. We previously produced the PEDV spike protein (S) using transgenic maize, enabling a low-cost supply of the vaccine candidate. In this study, we (1) test orally delivered PEDV vaccine candidates in pigs to optimize the mucosal immune response; (2) generate the SARS-CoV-2 S1 protein in maize; and (3) perform structural characterization of the S1 protein for PEDV and SARS-CoV-2. We demonstrated high expression levels in maize of the S1 subunit of the SARS-CoV-2 spike protein, both with and without fusion to the heat-labile enterotoxin B (LTB) subunit. We found that the LTB fusion protein from both coronaviruses preferentially assembles into higher molecular weight multimers, consistent with the formation of trimers. For PEDV, administering the spike protein fused to LTB to young pigs elicited a higher level of mucosal IgAs compared to maize grain containing the S1 protein alone or controls. This suggests that fusing the coronavirus spike protein with LTB may provide better protection.

Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.

ABSTRACTThe Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. We determined structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramer’s voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine induces an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.

Abstract Background: Cow antibodies are very unusual in having exceptionally long CDR H3 regions. The genetic basis for this length largely derives from long heavy chain diversity (DH) regions, with a single “ultralong” DH, IGHD8-2, encoding over fifty amino acids. Many bovine IGHD regions have sequence similarity but have several nucleotide repeating units that diversify their lengths. Genomically, most DH regions exist in three clusters that appear to have formed from DNA duplication events. However, the relationship between the genomic arrangement and long CDR lengths is unclear. Results: The DH cluster containing IGHD8-2 underwent a rearrangement and deletion event in relation to the other clusters in the region corresponding to IGHD8-2, with possible fusion of two DH regions and expansion of short repeats to form the ultralong IGHD8-2 gene. Conclusions: Length heterogeneity within DH regions is a unique evolutionary genomic mechanism to create immune diversity, including formation of ultralong CDR H3 regions.

Abstract Heterologous gene expression – transferring genes from a natural cell or origin to another host cell, often across species – is a fundamental technique in biological research as well as biotechnological and pharmaceutical manufacturing. Optimization of gene sequences, therefore, is a critical factor when enhanced protein yield is needed. For any given protein, an enormous diversity of nucleotide sequences comprising the vast combinatorics of possible codon combinations could theoretically be used to encode the same amino acid sequence. The process of “codon optimization” typically replaces certain codons thought to be suboptimal with a more optimal codon encoding the same amino acid. However, such methods cannot address the enormity of possible nucleotide sequences possible to encode a given protein, or to decipher potential factors in addition to codon usage that may affect gene expression. Here we utilize the Sh ble gene encoding zeocin resistance in a system comprising bioinformatic synthetic gene production and an antibiotic selection platform. We find that supposedly codon optimized genes do not produce enhanced antibiotic resistance, suggesting that other factors are more important in synthetic gene optimization and heterologous gene expression.

The green tea polysaccharide was extracted from leaves of Camellia sinensis and hydrolyzed with enzyme. Skin moisturization effect of green tea polysaccharide was studied with 2D- PAGE analysis of epidermal hydration factors. Among up-regulated proteins extracted from Hacat cell treated with Green tea polysaccharide on 2DE gel, 5 aquaporins were observed. Among them the aquaporin 5 and 8 were increased 3.4 and 5.3 times respectively. The green tea polysaccharide was found to be effective for skin moisturization by enhanced expression of aquaporins.

Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat-stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.

Osteoarthritis is the most common form of arthritis. It is a progressive joint disease associated with aging. It may be found in the knees, hips, or other joints. It is estimated that costs associated with osteoarthritis exceed 2% of the gross national product in developed countries. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a mainstay in the treatment of inflammatory disease and are among the most widely used drugs worldwide. The main limitation in using NSAIDs consists in their side-effects, including gastrointestinal ulcerogenic activity and bronchospasm. The mechanism of action of these drugs is attributed to the inhibition of cyclooxygenase (COX), and, consequently, the conversion of arachidonic acid into prostaglandins. It is hypothesized that the undesirable side-effects of NSAIDs are due to the inhibition of COX-1 (constitutive isoform), whereas the beneficial effects are related to the inhibition of COX-2 (inducible isoform). Arachidonic acid can also be converted to leukotrienes (LTs) by the action of 5-lipoxygenase (5-LOX). Licofelone, a LOX/COX competitive inhibitor, decreases the production of proinflammatory leukotrienes and prostaglandins (which are involved in the pathophysiology of osteoarthritis and in gastrointestinal (GI) damage induced by NSAIDs) and has the potential to combine good analgesic and anti-inflammatory effects with excellent GI tolerability. Preliminary data with this drug seem promising, but further well-designed clinical trials of this agent in the elderly will be necessary before a final evaluation is possible.