Search Results (662)

Background: Virus-like particles (VLPs) represent key tools for the development of vaccines due to their ability to induce a potent immune response to epitopes presented on their surface. However, the decoration of VLPs with a complete heterologous protein on the surface remains a bottleneck for clinical translation due to the complexity of manufacture. We present a novel platform, EPIclip™, for the decoration of VLPs mediated by high-affinity protein binding partners, colicin E7 (ColE7) and immunity protein 7 (Im7), within a single prokaryotic host. We evaluate this approach using a modified hepatitis B core capsid protein and IL-31 as a model epitope. IL-31 is a prominent therapeutic target for the development of pruritic diseases. Methods: We explore the design and development of the platform, including the use of T-cell-stimulating peptides. We demonstrate several small-scale purification methods for the candidate VLP, as well as morphological analysis by transmission electron microscopy (TEM). Further, we vaccinate mice with IL-31-displaying VLPs to evaluate immunogenicity and the ability to prevent IL-31-induced pruritus in vivo. Results: Our results demonstrate that decorated VLPs dosed in mice elicit an IgG response against IL-31 with at least six months of durability. In addition, IL-31-displaying VLPs suppress the development of IL-31-induced pruritus, confirming in vivo target neutralisation. Notably, IL-31-displaying VLPs induce a strong T-cell response against the VLP capsid but not against the cytokine, confirming a B-cell-biased immune response and the absence of detrimental autoreactive T cells. We further demonstrate the translation of this system with an additional virus capsid: tomato aspermy virus (TAV). Conclusions: Taken together, the novel EPIclip™ platform may represent a promising therapeutic approach for pruritic diseases. Additionally, this modular system could be adapted for a wide range of research as well as human and veterinary therapeutic applications. Full article

Background: Dengue virus (DENV) is becoming a global public health problem, but the immunogenicity of DENV structural proteins is not fully understood. Methods: We predicted the epitope-based immunogenicity of DENV proteins from four serotypes in silico and evaluated their efficacy in vitro (T-cell proliferation assays) and in vivo (ELISpot, qRT-PCR, and plaque reduction neutralization tests using murine splenocytes). We focused on the envelope protein, which contains envelope domain III. Immunogenic B-cell epitopes were predicted using BepiPred-2.0, and regions that induce T cell-mediated immune responses were analyzed using the immune epitope database (IEDB), which validates peptides presented on HLA class I. Results: Nine-amino-acid peptide candidates were selected based on a score of >0.1. The best peptide candidates were tested in T-cell proliferation assays to confirm the in silico data. Subsequently, BALB/c mice were vaccinated with candidate peptides showing immunity in the proliferation assay, and their splenocytes were analyzed. ELISpot and qRT-PCR data showed that some candidate peptides highly regulated cytokines, including interferon-γ, tumor necrosis factor-α, and interleukin-4. Murine sera were collected after peptide boosting 2 weeks apart. Stimulation of cellular immunity was confirmed for some candidates in plaque reduction neutralization tests. Full article

The development of cancer immunotherapies has transformed cancer treatment paradigms, yet durable and tumour-specific responses remain elusive for many patients. Neoantigens, immunogenic peptides arising from tumour-specific genomic alterations, have emerged as promising cancer vaccine targets. Early-phase clinical trials using different vaccine platforms, including mRNA, peptide, DNA, and viral vector-based personalised cancer vaccines, have demonstrated the feasibility of targeting neoantigens, with early signals of prolonged survival in some patients. Most current vaccine strategies focus on canonical neoantigens, typically derived from exonic single-nucleotide variants (SNVs) and small insertions/deletions (INDELs), yet this represents only a fraction of the potential neoantigen repertoire. Evidence now shows that non-canonical neoantigens, arising mostly from alternative splicing, intron retention, translation of non-coding RNAs, gene fusions, and retroelement activation, broaden the antigenic landscape, with the potential for increasing tumour specificity and immunogenicity. In this review, we explore the biology of non-canonical neoantigens, the technological advances that now enable their systematic detection, and their potential to inform next-generation personalised cancer vaccines. 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

Infectious diseases remain a persistent global health threat, intensified by the rapid emergence of antibiotic-resistant pathogens. Despite the transformative impact of antibiotics, the escalating resistance crisis underscores the urgent need for alternative therapeutic approaches. Antimicrobial peptides (AMPs) have emerged as promising candidates due to their broad-spectrum antimicrobial and immunomodulatory activities. The present study investigated 82 honey bee antimicrobial peptides (BAMPs) representing seven families: abaecin, apamin, apisimin, apidaecin, defensin, hymenoptaecin, and melittin among eight honey bee species. Immunoinformatics analyses identified five peptides (P15450, A0A2A3EK62, Q86BU7, C7AHW3, and I3RJI9A) with high antigenicity and non-allergenic profiles. Structural modeling, molecular docking with TLR3 and TLR4-MD2, and molecular dynamics simulations revealed stable receptor-peptide interactions and favorable binding energetics, further supported by silico immune simulations. Overall, these findings suggest that the selected BAMPs exhibit strong immunogenic potential and may serve as effective adjuvants or carrier molecules in subunit vaccine design against drug-resistant pathogens; however, further experimental validation is essential to confirm their safety and immunological efficacy. Full article

Background: Nipah virus (NiV), a zoonotic paramyxovirus with high case fatality and pandemic potential, remains without a licensed vaccine for humans to date. Although there has been progress in vaccine development, it remains limited, and peptide vaccines have rarely been validated in vivo. Methods: Here, we report the rational antigen selection, synthesis, and preliminary immunogenicity evaluation of NiV fusion glycoprotein (NiV-F)-derived linear peptides as vaccine candidates. Candidate epitopes were identified by in silico, and a total of 18 B- and T-cell epitope-derived peptides were shortlisted for synthesis and antigenicity validation by ELISA. Results: Antigenicity evaluation showed that 9 of the synthesized peptides have A_450nm of over 1 (8 from the F11 group, A_450nm: 1.13–3.6; 1 from the F18 group, A_450nm: 1.51), with the peptide constructs F11-3 (A_450nm: 3.5) and F11-4 (A_450nm: 3.6) showing the highest antigenicity. Interestingly, peptides from F11 with amidation increased antibody binding (F11-4-NH2, A_450nm: 3.05; F11-4-9mer-1-NH2, A_450nm: 0.87). The lead peptide candidates, F11-3 and F11-4, were subsequently used for the immunization experiment, and mouse sera were assessed against their homologous peptide antigens or recombinant NiV-F protein. ELISA result showed detectable antibody reactivity against their homologous antigen for the intramuscular (IM) F11-3 vaccinated group (A_450nm: 0.30 ± 0.35), whereas increased binding was observed for both IM-administered F11-3 (A_450nm: 1.62 ± 0.97) and F11-4 (A_450nm: 2.0 ± 0.77) against NiV-F protein, albeit without statistical significance compared to the negative control (NC, p > 0.05), and were markedly lower compared to mice immunized with NiV-F recombinant protein (PC, p < 0.01), underscoring the need for further optimization procedures. Conclusions: Collectively, these results support an exploratory antigen discovery and prioritization framework for NiV-F-derived peptide candidates and provide a foundation for future studies aimed at optimizing immunogenicity and evaluating protective relevance in appropriate preclinical models. Full article

Cancer immunotherapy increasingly relies on nucleic acid-based vaccines, yet achieving efficient and safe delivery remains a critical limitation. Polyplexes—electrostatic complexes of cationic polymers and nucleic acids—have emerged as versatile carriers offering greater chemical tunability and multivalent targeting capacity compared to lipid nanoparticles, with lower immunogenicity than viral vectors. This review summarizes key design principles governing polyplex performance, including polymer chemistry, architecture, and assembly route—emphasizing microfluidic fabrication for improved size control and reproducibility. Mechanistically, effective systems support stepwise delivery: tumor targeting, cellular uptake, endosomal escape (via proton-sponge, membrane fusion, or photochemical disruption), and compartment-specific cargo release. We discuss therapeutic applications spanning plasmid DNA, siRNA, miRNA, mRNA, and CRISPR-based editing, highlighting preclinical data across multiple tumor types and early clinical evidence of on-target knockdown in human cancers. Particular attention is given to physiological barriers and engineering strategies—including size-switching systems, charge-reversal polymers, and tumor-penetrating peptides—that improve intratumoral distribution. However, significant challenges persist, including cationic toxicity, protein corona formation, manufacturing variability, and limited clinical responses to date. Current evidence supports polyplexes as a modular platform complementary to lipid nanoparticles in selected oncology indications, though realizing this potential requires continued optimization alongside rigorous translational development. Full article

Background: Lactate Dehydrogenase C (LDHC) is a promising therapeutic target due to its highly tumor-specific expression, immunogenicity, and oncogenic functions. We previously showed that LDHC silencing in triple-negative breast cancer (TNBC) cells enhances treatment response to DNA-damage response-related drugs, supporting its therapeutic potential. However, no selective LDHC inhibitors exist, highlighting the need for innovative targeting strategies. Methods: We assessed the physicochemical properties and evaluated the delivery efficiency, anti-tumor activity, and safety of four cell-penetrating peptides (CPPs)—R10, 10R-RGD, cRGD-10R, and iRGD-10R—for siRNA-mediated LDHC silencing in TNBC. Clonogenic assays were used to evaluate effects on olaparib sensitivity, and TNBC zebrafish xenografts were utilized to study in vivo anti-tumor activity. Results: All CPP:siRNA complexes formed uniform nanocomplexes (129–168 nm) with low polydispersity indices (<0.25) and positive zeta potentials (+6.47 to +29.6 mV). Complexes remained stable in human serum for 24 h and showed no significant cytotoxicity in TNBC and non-cancerous cell lines. The 10R-RGD and cRGD-10R:siLDHC complexes achieved 40% LDHC protein knockdown, reduced TNBC clonogenicity by 30–36%, and enhanced olaparib sensitivity. Treatment of TNBC zebrafish xenografts with 10R-RGD or cRGD-10R:siLDHC complexes significantly reduced tumor growth by approximately 50% without major toxicity. Conclusions: These results demonstrate that CPP-mediated siRNA delivery enables selective LDHC silencing with tumor growth inhibition in triple-negative breast cancer models. This approach represents a novel, effective, and safe proof-of-concept therapeutic strategy to target LDHC, with potential translational relevance as a standalone therapy or in combination with common anti-cancer drugs. Full article

Transmissible gastroenteritis (TGE), caused by the TGE virus (TGEV), is a highly contagious enteric disease characterized by vomiting, dehydration, and watery diarrhea. It mainly endangers piglets within two weeks of age, with a 100% mortality rate, inflicting severe economic losses on the global swine industry. Since enteric tropism of the virus and mucosa serves as the first line of defense against viral invasion, an oral vaccine inducing sufficient secretory immunoglobulin A (SIgA) antibodies in animals should be developed. Being a generally recognized as safe (GRAS) microorganism, Bacillus subtilis can form endospores under extreme environmental conditions, which confer resistance to the hostile gastric environment and have been widely employed as delivery vehicles for oral vaccines owing to their immunoadjuvant activity and non-specific antidiarrheal effects. In this study, the AD antigenic epitope of the TGEV S protein was selected as the immunogen. The mature peptide of the B subunit of the heat-labile enterotoxin from enterotoxigenic Escherichia coli served as a mucosal adjuvant, and B. subtilis WB800N was used as the delivery host to construct the recombinant strain pHT43-LTB-AD/WB800N. After confirming the successful expression of the target protein, oral immunization was performed using mice as a model. The results demonstrated that this recombinant strain induced robust mucosal, humoral, and cellular immunity, along with considerable levels of neutralizing antibodies. These findings indicate that recombinant B. subtilis could serve as an oral vaccine candidate to combat TGEV infections. Full article

Neoantigen-based immunotherapies harness somatic mutations as tumor-specific targets and represent a major advance in personalized cancer treatment. Since neoantigens are presented exclusively on cancer cells, they enable highly selective T-cell recognition with minimal off-tumor toxicity. Neoantigen vaccines are rapidly emerging as a versatile class of personalized cancer immunotherapies designed to prime tumor-specific T cells by targeting somatic mutations unique to each patient’s tumor. Multiple types of neoantigen vaccines, using peptide, mRNA, and DNA, have shown feasibility, safety, and immunogenicity across diverse solid tumors. Emerging comparative data indicate that the vaccines using peptide-pulsed dendritic cells (DCs) elicit higher per-epitope CD8⁺ T cell responses than mRNA-based vaccines, likely due to more efficient class I presentation of synthetic peptides and ex vivo-loaded DCs. In contrast, mRNAs, despite their capacity of targeting multiple neoantigen peptides simultaneously, often induce CD4⁺-dominant responses due to immunodominance patterns during antigen processing. Recent clinical trials in melanoma, glioblastoma, pancreatic cancer, and other types of cancer have demonstrated not only robust immune activation but also encouraging relapse-free outcomes when administered in adjuvant settings. Treatment timing strongly influenced immune responsiveness; patients with early-stage disease or those vaccinated after surgical resection generally exhibit more preserved systemic immunity and greater vaccine-induced T cell expansion compared to those with advanced disease. Future progress will rely on improved neoantigen prediction, including incorporation of post-translationally modified antigenic targets and acceleration of manufacturing pipelines to ensure timely, personalized vaccine delivery. Collectively, neoantigen vaccines offer substantial promise for integration into next-generation cancer treatment strategies. Full article

Biomolecule–photosensitizer conjugates have rapidly evolved into one of the most powerful strategies for improving the selectivity, efficacy, and translational potential of photodynamic therapy (PDT). By integrating photosensitizers (PSs) with carbohydrates, amino acids, peptides, aptamers, proteins, cofactors, vitamins or antibodies, these constructs overcome long-standing limitations of classical PDT, including poor solubility, insufficient tumour accumulation, and strong dependence on oxygen availability. Beyond enhancing receptor-mediated uptake and enabling precise interactions with the tumour microenvironment (TME), bioconjugation also modulates aggregation, photochemical properties, intracellular accumulation, and immune system activation. A particularly transformative trend is the emergence of supramolecular architectures in which photosensitizers form defined nanostructured aggregates with peptides or proteins. Once considered an undesirable phenomenon, aggregation is now recognized as a tenable feature that governs photochemical behaviour. Engineered aggregates can undergo environment-triggered disassembly to monomeric, photoactive states, or operate as semiconductor-like nanodomains capable of Type I reaction through symmetry-breaking charge separation. This shift toward oxygen-independent radical pathways offers a promising solution to the challenge of hypoxia, a hallmark of the TME that severely compromises conventional Type II PDT. Parallel advances in 3D experimental platforms such as tumour organoids and organ-on-chip systems provide physiologically relevant validation of these conjugates, enabling the assessment of penetration, subcellular localization, immunogenic cell death, and therapeutic synergy within realistic TME conditions. Collectively, the integration of biomolecular targeting with controlled supramolecular design is redefining the landscape of PDT. Future progress will depend on designing conjugates that retain high activity under hypoxia, engineering dynamic aggregate states, and systematically validating these systems in advanced TME-mimetic models. Together, these developments position biomolecule–photosensitizer conjugates as a versatile and increasingly less oxygen-dependent class of next-generation phototherapeutic agents. Full article

Background/Objectives: The Zika virus (ZIKV) represents an ongoing threat to public health due to its neurological and congenital complications. Even after 10 years since the first major outbreak, correlated with an increase in congenital ZIKV syndrome, there is still no vaccine or treatment for this infection. Among the various existing platforms, DNA vaccines combined with the use of immunoinformatics tools allow for the efficient selection of immunogenic epitopes and immunostimulatory molecules with greater flexibility, in addition to being simple to manufacture and having a higher cost–benefit ratio in production. Methods: In this work, we conducted an integrated approach, combining in silico analyses and in vivo experimental validations, for the development of multi-epitope DNA vaccines against ZIKV. The computational analyses confirmed structural stability, adequate solubility, absence of toxicity, and immune induction potential for constructs based on epitopes from the Envelope (E) and NS1 proteins. Therefore, we evaluated DNA constructs containing the ENV + NS1 epitopes, both with and without fusion to the ssPGIP signal peptide, in BALB/c mice. Results: Both vaccines increased the population of CD4⁺ and CD8⁺ T lymphocytes, in addition to the production of IgG antibodies associated with the Th1 profile. The fusion with ssPGIP broadened the response, stimulating the release of Th1, Th2, and Th17 cytokines, as well as enhancing antibody formation. In contrast, its absence was associated with a slight increase in CD4⁺ and CD8⁺ T cells, accompanied by restricted cytokine production. Conclusions: These results indicate that epitope-targeted techniques offer a viable and safe method for inducing robust immune responses, demonstrating that combining immunoinformatics methods with early preclinical testing is an effective strategy for ZIKV vaccine development. Furthermore, although the present study focused on initial immunogenic characterization, future studies involving viral challenge in a suitable animal model will be essential to conclusively determine the protective efficacy of these vaccine candidates. 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

Background: Being central players in the adaptive immunity, the study of T-cell responses is crucial in both natural infections and vaccine-induced immunity. In this study, we assessed the antigen-specific T-cell responses to dengue virus (DENV) to identify the most immunogenic antigen for evaluating dengue-specific T-cell responses. Methods: Patients with dengue disease and subjects vaccinated with the QDENGA (TAK-003) vaccine (before and three months after vaccination) were enrolled. The T-cell-specific response was measured by ELISPOT and Activation Induced Markers (AIM) assay following PBMC stimulation either with DENV1-4 CD4 and CD8 MegaPools (MP) or serotype-specific DENV peptide pools at different concentrations. Results: We found that both DENV1-4 CD4 MP (at 1 µg/mL) and CD8 MP (at 5 µg/mL), which encompass all four DENV serotypes, elicited specific T-cell responses in patients with dengue infection independent of the infecting serotype. In contrast, selected serotype-specific DENV peptide pools have a lower ability to induce a measurable T-cell response. Moreover, DENV1-4 CD4 and CD8 MPs, at the highest concentrations, are suitable candidates to evaluate the dengue-specific T-cell response in vaccinated subjects. Conclusions: These findings support the use of the MP approach to investigate dengue-specific T-cell response to monitor the response during the infection and after vaccine administration. Full article

Background: Short peptide epitopes are valuable for mechanistic studies, yet their intrinsic low immunogenicity and lack of commercial antibodies hinder rapid antibody generation. Methods: We developed a reproducible, sequence-level workflow combining cross-species/structural triage, independent MHC-I/II prioritization, and conservative heteroclitic-style substitutions to enhance predicted MHC affinity while preserving native epitope features. Using visfatin as a model, two optimized fragments were conjugated to KLH and tested in mice for antibody titers, isotype profiles, and binding kinetics. Results: Mutant peptides improved MHC-binding prediction, elicited stronger antibody titers, and promoted isotype maturation (increased IgG1). Importantly, antibodies maintained measurable binding to wild-type sequences, indicating preserved cross-recognition. Similar effects were reproduced with additional antigens. Conclusions: This proof-of-concept study, based on small exploratory mouse cohorts (n = 3 per group), demonstrates that strategic, minimal sequence edits can significantly enhance peptide immunogenicity while preserving native epitope recognition. This streamlined workflow provides a low-barrier route to generate epitope-directed antibodies when commercial reagents are unavailable. Full article