Search Results (828)

Porcine epidemic diarrhea virus (PEDV) has emerged as a major pathogen responsible for porcine diarrheal diseases, causing outbreaks of severe diarrhea and high mortality in neonatal piglets, thereby inflicting severe economic losses on the global swine industry. Current commercial PED vaccines, comprising conventional inactivated and live attenuated formulations, have exhibited progressively diminished efficacy in the face of emerging PEDV variants. The development of high-efficiency vaccine platforms is therefore critical for PED control. This study engineered a cellular membrane nanovesicle (CMN)-based vaccine, which differs from existing inactivated or subunit vaccines by presenting the PEDV spike (S) protein on the cell membranes to mimic the bilayer phospholipid structure of the viral envelope. The full-length S protein (FS, aa 19-1309) or a truncated S protein fragment (TS, aa 19-726) was expressed in Expi293F cells, followed by extraction of cell membranes to assemble antigen-displaying CMN vaccines. Compared with commercial live attenuated vaccine, administration of the CMN vaccine elicited high-titer neutralizing antibodies and elevated IFN-γ-producing CD8⁺ T cells in murine studies. Safety assessments revealed no adverse effects on body weight, hepatic/renal function indices, or histopathological parameters in vaccinated mice. Furthermore, immunization of piglets elicited notable humoral and CD8⁺ T cell immune responses. Collectively, the strategy of CMN-based vaccine described herein delivers a potential PEDV vaccine platform, thereby offering a novel avenue for next-generation veterinary vaccine development. Full article

The rapid evolution of SARS-CoV-2, particularly the emergence of Omicron subvariants, has significantly reduced the efficacy of existing vaccines and monoclonal antibodies. This study investigates the phenomenon of immune imprinting by comparing two phage display antibody libraries derived from early 2020 wild-type SARS-CoV-2 convalescents (WT-AbLib) and early 2023 Omicron convalescents (Omi-AbLib). The capacity and diversity of both antibody libraries were systematically evaluated. The libraries were screened using BF.7 and XBB.1.5 antigens. WT-AbLib showed markedly reduced diversity after Omicron antigen selection, with dominant clones shifting from IGHV3-66-class broadly neutralizing antibodies (bnAbs) targeting the receptor-binding motif to IGHV1-46-class broadly non-neutralizing antibodies targeting conserved lateral receptor-binding domain (RBD) sites. Omi-AbLib maintained higher diversity, but dominant antibodies were also non-neutralizing and targeted the same conserved lateral region. These findings suggest that immune imprinting drives the dominance of broadly non-neutralizing antibodies following Omicron breakthrough or reinfection. This phenomenon provides a mechanistic explanation for persistent viral evasion and recurrent infection, and highlights major challenges for the development of next-generation broadly neutralizing therapeutics. Full article

Tetanus is a zoonotic disease posing significant threats to both humans and animals, particularly horses, sheep, and ruminants. Current antitoxin therapies rely on animal-derived immunoglobulins, presenting challenges including animal welfare concerns, pathogen contamination risks, and manufacturing complexity. Alpaca-derived nanobodies (VHH) are promising alternatives owing to their high antigen-binding affinity, thermostability, and potential for microbial production. We developed highly active trivalent VHH antibodies (tVHH) that target multiple epitopes of tetanus neurotoxin (TeNT). Following alpaca immunization with tetanus toxoid, 41 VHH clones were isolated using phage display. Six VHH clones were selected through in vivo neutralization assays, from which three clones of VHH (8, 11, 36) were selected to construct tVHH-8/11/36 and tVHH-8/36/11. Using an improved 21-day mouse neutralization assay, tVHH-8/11/36 demonstrated exceptional neutralizing activity of approximately 1580 IU/mg against 4000 LD₅₀ of toxin, substantially exceeding current human and veterinary anti-tetanus immunoglobulin preparations. Surface plasmon resonance and ELISA confirmed that each VHH recognizes different TeNT domains, producing synergistic neutralizing effects through multimerization. Since antitoxin therapy challenges are common to both animals and humans, this tVHH technology supports One Health by providing a unified therapeutic platform applicable across species through sustainable microbial production. Full article

Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4⁺ fraction, activation, exhaustion, effector/terminal differentiation, and IFN-γ/TNF-α production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays. Full article

Dendritic cells (DCs) are essential for antiviral immunity but are also susceptible to HIV-1 infection. Although sensing and restriction pathways in DCs are well described, the mechanisms underlying latent infection and its functional consequences remain unclear. In this study, we performed transcriptomic profiling of monocyte-derived DCs harboring transcriptionally active (Active-HIV) or latent HIV-1 (Latent-HIV) proviruses using a dual-reporter virus. Gene set enrichment analysis revealed suppression of metabolic and stress-modulatory programs in Active-HIV compared to unexposed DCs. In contrast, Latent-HIV showed broad downregulation of pathways, including interferon and innate responses and metabolic programs, indicating a hyporesponsive and dampened antiviral state despite the absence of differentially expressed genes (DEGs). DEG analysis of Active-HIV versus Latent-HIV showed that active transcription associates with cellular stress, cytoskeletal remodeling, and RNA processing. Functional analyses further demonstrated the activation of RNA processes, the suppression of antigen-presentation pathways, and altered membrane and cytoskeletal signaling in Active-HIV. These pathways suggest that transcriptionally active HIV-1 is linked to cellular programs supporting replication, coinciding with a metabolically strained yet immunologically engaged state that may impair antigen presentation. Conversely, latently infected DCs display a hyporesponsive state consistent with proviral silencing. This dichotomy reveals distinct mechanisms of DC dysfunction that may facilitate HIV-1 persistence and immune evasion. Full article

Chagas disease, caused by Trypanosoma cruzi, affects a significant proportion of patients who develop digestive and cardiac complications, including megaviscera. This pathogenesis has been associated with autoimmune mechanisms mediated by molecular mimicry. In this study, an in silico evaluation of the potential structural basis of cross-reactivity of β-tubulin 1.9 of T. cruzi and the human β-4A tubulin isoform 3 was conducted. Using bioinformatics tools, homologous regions were identified and potentially immunogenic epitopes were predicted, considering their structural modeling and molecular docking. The proteins shared 87% sequence identity and 95% similarity, with an almost identical structural overlap, RMSD 0.291 Å. Three epitopes, VPFPRLHFF, NDLVSEYQQYQDATI, and GQSGAGNNWAKGHYTEGAELIDS, exhibited high predicted antigenicity, with the 9-mer and 16-mer peptides displaying structurally compatible docking poses within the binding grooves of MHC class I and class II molecules, respectively, while B-cell epitope potential was inferred from sequence-based property predictions. Normal mode analysis, used as an exploratory approach, suggested comparable flexibility profiles for the parasitic- and human-derived peptide–MHC complexes. These findings provide an exploratory structural framework supporting a potential role of β-tubulin epitopes in molecular mimicry processes implicated in the development of chagasic megaviscera. Full article

Background: Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy where endogenous steroid excess may foster immune evasion. However, whether this hormonal axis directly modulates the antigen presentation machinery remains unclear. Methods: We applied an immunoinformatics approach to the TCGA-ACC cohort (n = 79) to investigate relationships among steroid phenotype, HLA expression, tumor microenvironment (TME), and patient outcome. Key findings were assessed in an independent validation cohort (ENSAT-ACC, n = 44) using C1A/C1B molecular subtypes corresponding to the steroid phenotypes. Results: Stratification by steroid phenotype revealed two distinct immunological profiles. The high steroid production (HSP) phenotype was associated with suppressed HLA expression and a lymphocyte-depleted “cold” TME. In contrast, the low steroid production (LSP) phenotype displayed elevated HLA expression, enriched T-cell infiltration, and upregulation of immune checkpoints (e.g., PDCD1, CTLA4), consistent with an inflamed but exhausted TME. The core signature of HLA downregulation in the HSP-like phenotype (C1A) and the significant survival advantage of the LSP-like phenotype (C1B) were confirmed in the validation cohort, demonstrating biological robustness despite platform and sample size differences. Conclusions: These findings identify the steroid phenotype as a critical regulator of immune escape in ACC. Our results support incorporating this stratification as a biomarker for patient selection, identifying LSP tumors as the subgroup most likely to benefit from immune checkpoint blockade due to their “hot” yet exhausted microenvironment. Full article

Background: mRNA LNP technology is now being widely applied as a highly effective vaccine platform. Antigen multimerization is a well-established approach to enhance the antibody titers and protective efficacy of several protein subunit vaccines. However, this approach has been less explored for mRNA LNP vaccines. Methods: Here, within the context of mRNA LNP vaccination, we used mStrawberry (mSb) as a model antigen to conduct a comprehensive, head-to-head comparison of the ability of the foldon (3-mer), IMX313 (7-mer), and ferritin (24-mer) multimerization domains to enhance immunogenicity in mice. Results: We compared multimerized antigen to monomeric secreted antigen and monomeric surface-displayed antigen and observed that the IMX313 domain efficiently multimerized mSb protein and significantly enhanced anti-mSb antibody titers, whereas the foldon and ferritin domains failed to multimerize or improve antibody levels. Conclusions: Our results extend the observation of improved immunogenicity from antigen multimerization to mRNA LNP vaccines and indicate that the 7-mer forming IMX313 multimerization domain may be an ideal candidate for multimer formation in the context of mRNA LNP vaccination. Future studies are needed to evaluate the multimerization of pathogen-derived antigens, in the mRNA LNP format, for the enhancement of neutralization and protective efficacy. Full article

Background/Objectives: Toxoplasma gondii (T. gondii) dense granule antigen 14 (GRA14) is a parasitophorous vacuole membrane protein that plays a critical role in the development of chronic-stage cysts. However, its potential as a vaccine antigen and long-term immunity have not been evaluated using a virus-like particle (VLP) platform. Methods: influenza matrix protein (M1)-based VLPs displaying GRA14 were generated. Female BALB/c mice were intranasally immunized with the VLP vaccine and orally challenged with lethal ME49 cysts either 10 weeks or 32 weeks after prime vaccination for short-term and long-term immunity evaluation, respectively. Results: GRA14 VLP vaccination elicited higher levels of T. gondii-specific IgG, IgG1, and IgG2a antibody responses in sera compared to non-immunized controls. Upon challenge infection, elevated IgG- and IgA-secreting plasma cells, germinal center B cells, and memory B cells were observed, and CD4⁺, CD8⁺ T-cells, as well as both Th1 (IFN-γ) and Th2 (IL-4, IL-5) cytokines, were also increased. For the short-term immunity study, vaccinated mice exhibited suppressed cerebral inflammation, significantly reduced brain cyst burdens, maintained stable body weight, and achieved 100% survival. For the long-term study, GRA14 VLPs sustained elevated IgG and IgG1 levels as well as conferred partial yet significant protection, with lower cyst loads and 83% survival. Conclusions: GRA14 VLPs induce durable, balanced humoral and cellular immunity and provide both short-term and long-term protection against lethal chronic toxoplasmosis, supporting their potential as promising vaccine candidates. Full article

The COVID-19 pandemic accelerated vaccine innovation but also exposed weaknesses in global access and manufacturing. Yeast-based platforms, particularly Saccharomyces cerevisiae and Pichia pastoris, also known as Komagataella phaffii, offer a practical complement to vector systems. These eukaryotic microorganisms combine safety, scalability, and cost-effectiveness with the ability to express complex antigens and assemble virus-like particles. Building on the success of the recombinant hepatitis B vaccine, recent advances in glycoengineering, CRISPR-based host optimization, and surface display technologies have expanded the utility of yeast-based platforms for the rapid development of vaccines. Yeast-derived SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines, such as Corbevax and Abdala (CIGB-66), demonstrate that affordable, immunogenic, and thermostable products are feasible at scale. Emerging innovations in glycan humanization, thermostable formulations, and oral or mucosal delivery highlight the potential of yeast-based vaccines for decentralized manufacturing and equitable pandemic preparedness. This review summarizes recent technical and clinical progress in yeast-based vaccine research, positioning these platforms as accessible and adaptable tools for future outbreak responses and global immunization strategies. Full article

The manufacturing process contributes significantly to the proliferation, metabolic state, and functional persistence of chimeric antigen receptor (CAR)-T cells. However, how different culture systems regulate CAR-T cell metabolism and thereby influence their long-term antitumor activity remains poorly understood. In this study, we compared dynamic cultivation using a wave bioreactor with static expansion systems (gas-permeable and conventional T-flasks) for the production of CD99-specific CAR-T cells. CAR-T cells expanded by the wave bioreactor exhibited faster proliferation and stronger cytotoxicity during culture. Upon repeated antigen stimulation, they retained these enhanced functional properties and showed the reduced expression of immune checkpoint molecules, preferentially preserved memory-like subsets, and displayed transcriptional features consistent with memory maintenance and exhaustion resistance. Targeted metabolomic profiling revealed enhanced Tricarboxylic Acid (TCA) cycle activity and features consistent with sustained oxidative phosphorylation, supporting mitochondrial-centered metabolic reprogramming. In a Ewing sarcoma xenograft model, wave bioreactor-cultured CAR-T cells showed a greater percentage of memory-like tumor-infiltrating lymphocytes. Collectively, these results indicate that wave bioreactor-based dynamic cultivation promotes mitochondrial metabolic reprogramming, which is characterized by an enhanced TCA cycle and sustained oxidative phosphorylation, thereby sustaining CAR-T cell functionality and providing a robust platform for the manufacturing of potent and durable cellular therapeutics. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) exhibits high immune infiltration yet remains clinically aggressive. Although immune checkpoint blockade benefits a subset of patients, the molecular programs enabling concurrent immune activation and immune evasion in TNBC are not fully defined. This study aimed to identify TNBC-specific tumor-intrinsic and tumor-extrinsic molecular features that may explain this paradox. Methods: Publicly available single-cell RNA-sequencing data from primary breast tumors were analyzed to characterize subtype-specific transcriptional programs across epithelial and stromal compartments. Tumor-intrinsic findings were independently validated using bulk transcriptomic and clinical data from the METABRIC cohort. Tumor microenvironment remodeling was evaluated using multiplexed tissue imaging of TNBC tumors. Functional analyses were done included Gene Ontology enrichment, Hallmark gene set enrichment analysis, and SERPINB3-centered protein–protein interaction network analysis using STRING. Results: Single-cell analysis identified SERPINB3 as a TNBC-enriched epithelial gene relative to ER+ and HER2+ tumors. This subtype-restricted pattern was validated in the METABRIC cohort and associated with pathways related to epithelial–mesenchymal transition, interferon signaling, and antigen presentation. TNBC tumors also displayed a humoral immune signature characterized by B-cell and plasmablast enrichment, as well as ectopic immunoglobulin gene expression in cancer-associated fibroblasts, endothelial cells, and myeloid populations. Multiplex imaging revealed coordinated associations between immune suppression, stromal activation, and tumor proliferation. Network analysis placed SERPINB3 within interconnected immune-regulatory and stromal signaling modules. Conclusions: Together, these data indicate that TNBC exhibits co-existing immune activation and immune-suppressive features. The identified epithelial and stromal signatures represent candidate biomarkers that may inform future studies of immune regulation and therapeutic stratification in TNBC. Full article

Background/Objectives: New vaccine platforms that rapidly yield low-cost, easily manufactured vaccines are highly desired, yet current approaches lack key features. We developed the Killed Whole-Cell/Genome-Reduced Bacteria (KWC/GRB) platform, which uses a genome-reduced Gram-negative chassis to enhance antigen exposure and modularity via an autotransporter (AT) system. Integrated within a Design–Build–Test–Learn (DBTL) framework, KWC/GRB enables rapid iteration of engineered antigens and immunomodulatory elements. Here, we applied this platform to the HIV-1 fusion peptide (FP) and tested multiple antigen engineering strategies to enhance its immunogenicity. Methods: For a new vaccine, we synthesized DNA encoding the antigen together with selected immunomodulators and cloned the constructs into a plasmid. The plasmids were transformed into genome-reduced bacteria (GRB), which were grown, induced for antigen expression, and then inactivated to produce the vaccines. We tested multiple strategies to enhance antigen immunogenicity, including multimeric HIV-1 fusion peptide (FP) designs separated by different linkers and constructs incorporating immunomodulators such as TLR agonists, mucosal-immunity-promoting peptides, and a non-cognate T-cell agonist. Vaccines were selected based on structure prediction and confirmed surface expression by flow cytometry. Mice were vaccinated, and anti-FP antibody responses were measured by ELISA. Results: ELISA responses increased nearly one order of magnitude across design rounds, with the top-performing construct showing an ~8-fold improvement over the initial 1mer vaccine. Multimeric antigens separated by an α-helical linker were the most immunogenic. The non-cognate T-cell agonist increased responses context-dependently. Flow cytometry showed that increased anti-FP-mAb binding to GRB was associated with greater induction of antibody responses. Although anti-FP immune responses were greatly increased, the sera did not neutralize HIV. Conclusions: Although none of the constructs elicited detectable neutralizing activity, the combination of uniformly low AlphaFold pLDDT scores and the functional data suggests that the FP region may not adopt a stable native-like structure in this display context. Importantly, the results demonstrate that the KWC/GRB platform can generate highly immunogenic vaccines, and when applied to antigens with well-defined native tertiary structures, the approach should enable rapidly produced, high-response, very low-cost vaccines. Full article

This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel^®, AH; Adju-phos^®, AP; and CpG-1018^®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing. Full article

Background/Objectives: African swine fever (ASF) causes massive global swine industry losses with no effective vaccine available. This study constructed T7 phages displaying key ASFV proteins to evaluate their potential as an ASF vaccine by assessing viral shedding and immune responses in pigs. Methods: Five ASFV proteins were displayed on T7 phages to form VLPs (ASFV-SC-T7 group), with soluble proteins (ASFV-SC group) and PBS as controls; 9 piglets were immunized, boosted at 28 days, challenged with virulent ASFV, and assessed via ELISA, flow cytometry, and real-time PCR. Results: ASFV-SC-T7 induced more high-titer antibodies and elevated monocytes/CD8⁺ T cells, but all groups developed ASF lesions, with ASFV-SC-T7 having higher lung/mesenteric lymph node viral loads and no survival improvement (only delayed fever). Conclusions: T7 phage-displayed ASFV proteins activate strong immunity, confirming T7 phages as a viable delivery platform, but failed to protect against virulent ASFV, requiring future optimization of antigens and regimens. Full article