Molecular Design of Encapsulin Protein Nanoparticles to Display Rotavirus Antigens for Enhancing Immunogenicity.

Rotavirus considerably threatens global health, particularly for children <5 years. Current, licensed oral attenuated vaccine formulations have limitations including insufficient efficacy in children in low- and middle-income countries, warranting urgent development of novel vaccines with improved efficacy and safety profiles. Herein, we present a novel approach utilizing an encapsulin (ENC) nanoparticle (NP)-based non-replicating rotavirus vaccine. ENC, originating from bacteria, offers a self-assembling scaffold that displays rotavirus VP8* antigens on its surface. To enhance the correct folding and soluble expression of monomeric antigens and their subsequent assembly into NP, we adopted an RNA-interacting domain (RID) of mammalian transfer RNA synthetase as an expression tag fused to the N-terminus of the ENC-VP8* fusion protein. Using the RID-ENC-VP8* tripartite modular design, insertion of linkers of appropriate length and sequence and the universal T cell epitope P2 remarkably improved the production yield and immunogenicity. Cleavage of the RID rendered a homogenous assembly of ENC-P2-VP8* into protein NPs. Immunization with ENC-P2-VP8* induced markedly higher levels of VP8*-specific antibodies and virus neutralization titers in mice than those induced by P2-VP8* without ENC. Altogether, these results highlight the potential of the designed ENC NP-based rotavirus vaccine as an effective strategy against rotavirus disease to address global health challenges.

Chemoembolization as first-line treatment for hepatocellular carcinoma invading segmental portal vein with tumour burden limited to a monosegmental level.

OBJECTIVES: To evaluate the safety and effectiveness of chemoembolization for hepatocellular carcinoma (HCC) with portal vein tumour thrombosis (PVTT) confined to a monosegment of the liver. METHODS: A total of 192 treatment-naive patients who received chemoembolization between March 2008 and January 2023 as a first-line treatment for locally advanced HCC with PVTT limited to a monosegment were retrospectively analysed. Overall survival (OS) and the identification of pretreatment risk factors related to OS were investigated using Cox regression analysis. Complications, radiologic tumour response, and progression-free survival (PFS) following chemoembolization were investigated. RESULTS: After chemoembolization, the 1-, 3-, and 5-year OS rates were 86%, 48%, and 39%, respectively, and the median OS was 33 months. Multivariable analyses revealed four significant pretreatment risk factors: infiltrative HCC (P = .02; HR, 1.60), beyond the up-to-11 criteria (P = .002; HR, 2.26), Child-Pugh class B (P = .01; HR, 2.35), and serum AFP ≥400 ng/mL (P = .01; HR, 1.69). The major complication rate was 5%. Of the 192 patients, 1 month after chemoembolization, 35% achieved a complete response, 47% achieved a partial response, 11% had stable disease, and 7% showed progressive disease. The median PFS after chemoembolization was 12 months. CONCLUSIONS: Chemoembolization shows high safety and efficiency, and contributes to improved survival in patients with HCC with PVTT confined to a monosegment. Four risk factors were found to be significantly associated with improved survival rates after chemoembolization in patients with HCC with PVTT confined to a monosegment. ADVANCES IN KNOWLEDGE: (1) Although systemic therapy with a combination of atezolizumab and bevacizumab (Atezo-Bev) is recommended as the first-line treatment when HCC invades the portal vein, chemoembolization is not infrequently performed in HCC cases in which tumour burden is limited. (2) Our study cohort (n=192) had a median OS of 33 months and a 5% major complication rate following chemoembolization, findings in the range of candidates typically accepted as ideal for chemoembolization. Thus, patients with HCC with PVTT confined to a monosegment may be good candidates for first-line chemoembolization.

L-Pampo™, a Novel TLR2/3 Agonist, Acts as a Potent Cancer Vaccine Adjuvant by Activating Draining Lymph Node Dendritic Cells.

TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy.

Expression of SLC22A18 regulates oxaliplatin resistance by modulating the ERK pathway in colorectal cancer.

Although oxaliplatin-based chemotherapy is the current standard adjuvant therapy for colorectal cancer (CRC), the molecular mechanisms underlying oxaliplatin resistance remain unclear. Here, we examined the molecular mechanisms underlying SLC22A18-associated oxaliplatin resistance and strategies for overcoming oxaliplatin resistance. We evaluated the association between SLC22A18 and prognosis in 337 patients with CRC and its functional significance and studied the mechanisms through which SLC22A18 affects oxaliplatin resistance development in CRC cells, using CRC cell lines and patient-derived cells (PDCs). SLC22A18 downregulation was positively correlated with worse survival in patients with CRC. Low SLC22A18-expressing cells showed relatively lower sensitivity to oxaliplatin than high SLC22A18-expressing cells. In addition, ERK activation was found to be involved in the mechanisms underlying SLC22A18-related oxaliplatin resistance. To confirm ERK pathway dependence, we used an ERK inhibitor and found that combined treatment with oxaliplatin and the ERK inhibitor overcame oxaliplatin resistance in the low SLC22A18-expressing cells. Ex vivo approaches using PDC confirmed the correlation between SLC22A18 expression and oxaliplatin resistance. Results of the in vivo study showed that SLC22A18 expression regulated oxaliplatin efficacy, and that combined treatment with an ERK inhibitor could be a useful therapeutic strategy when SLC22A18 is downregulated. Together, our findings indicate that SLC22A18 could serve as a biomarker for the prediction of oxaliplatin resistance. In cases of oxaliplatin resistance due to low SLC22A18 expression, resistance can be overcome by combined treatment with an ERK inhibitor.

Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.

BACKGROUND: Statins preferentially promote tumor-specific apoptosis by depleting isoprenoid such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. However, statins have not yet been approved for clinical cancer treatment due, in part, to poor understanding of molecular determinants on statin sensitivity. Here, we investigated the potential of statins to elicit enhanced immunogenicity of KRAS-mutant (KRASmut) tumors. METHODS: The immunogenicity of treated cancer cells was determined by western blot, flow cytometry and confocal microscopy. The immunotherapeutic efficacy of mono or combination therapy using statin was assessed in KRASmut tumor models, including syngeneic colorectal cancer and genetically engineered lung and pancreatic tumors. Using NanoString analysis, we analyzed how statin influenced the gene signatures associated with the antigen presentation of dendritic cells in vivo and evaluated whether statin could induce CD8+ T-cell immunity. Multiplex immunohistochemistry was performed to better understand the complicated tumor-immune microenvironment. RESULTS: Statin-mediated inhibition of KRAS prenylation provoked severe endoplasmic reticulum (ER) stress by attenuating the anti-ER stress effect of KRAS mutation, thereby resulting in the immunogenic cell death (ICD) of KRASmut cancer cells. Moreover, statin-mediated ICD enhanced the cross-priming ability of dendritic cells, thereby provoking CD8+ T-cell immune responses against KRASmut tumors. Combination therapy using statin and oxaliplatin, an ICD inducer, significantly enhanced the immunogenicity of KRASmut tumors and promoted tumor-specific immunity in syngeneic and genetically engineered KRASmut tumor models. Along with immune-checkpoint inhibitors, the abovementioned combination therapy overcame resistance to PD-1 blockade therapies, improving the survival rate of KRASmut tumor models. CONCLUSIONS: Our findings suggest that KRAS mutation could be a molecular target for statins to elicit potent tumor-specific immunity.

Immune Responses to Irradiated Pneumococcal Whole Cell Vaccine.

Streptococcus pneumoniae (pneumococcus) can cause respiratory and systemic diseases. Recently, γ-irradiation-inactivated, non-encapsulated, intranasal S. pneumoniae (r-SP) vaccine has been introduced as a novel serotype-independent and cost-effective vaccine. However, the immunogenic mechanism of r-SP is poorly understood. Here, we comparatively investigated the protective immunity and immunogenicity of r-SP to the heat-(h-SP) or formalin-inactivated vaccine (f-SP) without adjuvants. Mice were intranasally immunized with each vaccine three times and then challenged with a lethal dose of S. pneumoniae TIGR4 strain and then subsequently evaluated for their immune responses. Immunization with r-SP elicited modestly higher protection against S. pneumoniae than h-SP or f-SP. Immunization with r-SP enhanced pneumococcal-specific IgA in the nasal wash and IgG in bronchoalveolar lavage fluid. Immunization with r-SP enhanced S. pneumoniae-specific IgG, IgG1, and IgG2b in the serum. r-SP more potently induced the maturation of dendritic cells in the cervical lymph nodes than h-SP or f-SP. Interestingly, populations of follicular helper T cells and IL-4-producing cells were potently increased in cervical lymph nodes of r-SP-immunized mice. Collectively, r-SP could be an effective intranasal, inactivated whole-cell vaccine in that it elicits S. pneumoniae-specific antibody production and follicular helper T cell activation leading to protective immune responses against S. pneumoniae infection.

Structures and Free Energies of Cerium Ions in Acidic Electrolytes.

The Ce3+/Ce4+ redox potential changes with the electrolyte, which could be due to unequal anion complexation free energies between Ce3+ and Ce4+ or a change in the solvent electrostatic screening. Ce complexation with anions and solvent screening also affect the solubility of Ce and charge transfer kinetics for electrochemical reactions involving waste remediation and energy storage. We report the structures and free energies of cerium complexes in seven acidic electrolytes based on Extended X-ray Absorption Fine Structure, UV-vis, and Density Functional Theory calculations. Ce3+ coordinates with nine water molecules as [Ce(H2O)9]3+ in all studied electrolytes. However, Ce4+ complexes with anions in all electrolytes except HClO4. Thus, our results suggest that Ce4+-anion complexation leads to the large shifts in standard redox potential. Long range screening effects are smaller than the anion complexation energies but could be responsible for changes in the Ce solubility with acid.

Size-controllable and uniform gold bumpy nanocubes for single-particle-level surface-enhanced Raman scattering sensitivity.

Gold nanocubes modified to form roughened structures, namely, gold bumpy nanocubes (Au BNCs), with very strong and uniform single-particle surface-enhanced Raman scattering (SERS) intensity were developed. The Au BNCs were synthesized by controlled regrowth, competing with 4-aminothiophenol during gold nanocube growth. Under controlled conditions, Au BNCs of various sizes were successfully generated while maintaining a cubic outline. As the bumpy surfaces of the Au BNCs increased the number of hot spots on a single cubic nanoparticle, these nanoparticles exhibited 15-times stronger SERS than normal cubic nanoparticles. We expect that this unique nanostructure will be applicable in versatile fields as an ultrasensitive SERS nanoprobe or nanoantenna owing to its cubic outline and high uniformity, as well as the ease of particle size adjustment.

A fast and reliable readout method for quantitative analysis of surface-enhanced Raman scattering nanoprobes on chip surface.

Surface-enhanced Raman scattering techniques have been widely used for bioanalysis due to its high sensitivity and multiplex capacity. However, the point-scanning method using a micro-Raman system, which is the most common method in the literature, has a disadvantage of extremely long measurement time for on-chip immunoassay adopting a large chip area of approximately 1-mm scale and confocal beam point of ca. 1-µm size. Alternative methods such as sampled spot scan with high confocality and large-area scan method with enlarged field of view and low confocality have been utilized in order to minimize the measurement time practically. In this study, we analyzed the two methods in respect of signal-to-noise ratio and sampling-led signal fluctuations to obtain insights into a fast and reliable readout strategy. On this basis, we proposed a methodology for fast and reliable quantitative measurement of the whole chip area. The proposed method adopted a raster scan covering a full area of 100 µm × 100 µm region as a proof-of-concept experiment while accumulating signals in the CCD detector for single spectrum per frame. One single scan with 10 s over 100 µm × 100 µm area yielded much higher sensitivity compared to sampled spot scanning measurements and no signal fluctuations attributed to sampled spot scan. This readout method is able to serve as one of key technologies that will bring quantitative multiplexed detection and analysis into practice.