Search Results (1,066)

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: Pathogenic flaviviruses pose a serious threat to human health, and vaccines are an effective means of prevention and control. Although related vaccines have achieved significant progress, safety and efficacy limitations still exist, urgently requiring the development of novel vaccine platforms. The insect-specific flavivirus Chaoyang virus (CYV), with a structure similar to pathogenic flaviviruses and limited to insect cell replication, has potential as a safe vaccine vector. Methods: To systematically evaluate CYV’s potential as a universal flavivirus vaccine backbone and provide a vaccine candidate for type I Japanese encephalitis virus (JEV) prevention, this study constructed a chimeric JEV genotype I (GI) prME protein vaccine candidate CYV-JEV using CPER technology, systematically assessing its safety and immunoprotective effects. Results: Using the CPER method, CYV-JEV was successfully rescued, showing efficient replication in mosquito cells but defective replication in mammalian cells. As a vaccine backbone, CYV did not induce inflammatory responses or immune cell subset imbalances in IFNAR^−/− mice. CYV-JEV exhibited no pathogenicity in adult and suckling IFNAR^−/− mice. Immunisation of IFNAR^−/− mice with 10⁶ FFU twice provided complete protection against lethal challenge (100%) and effectively reduced paralysis rates (62.5%). Single-cell sequencing further revealed extensive T- and B-cell activation in the immune spleen. Conclusions: The results demonstrate that the CYV-based CYV-JEV candidate vaccine demonstrates both safety and efficacy, representing a promising alternative to attenuated JEV vaccines, with CYV showing potential as a safe and effective universal flavivirus vaccine backbone. Full article

Background/Objectives: The enhancement of antitumor immune responses by radiotherapy (RT) is partially attributed to the activation of the IFN-type-I pathway. However, the loss of HLA-class-I molecules, which occurs in a large percentage of non-small-cell lung cancers (NSCLCs), may block the cytotoxic effect of T-cells and immunotherapy (IO). Moreover, autophagy is also involved in HLA downregulation. We investigated the complex interactions between RT, HLA molecules, autophagy, and IFN-type-I responses. Methods: The A549, H1299, and ATG7-deficient NSCLC cell lines, along with the modified shLC3A H1299 cell line, were used for in vitro experiments. The effect of RT (8 and 3 × 8 Gy) on Interferon beta (IFNβ), IFN-stimulated genes (ISGs), and HLA-class-I expression in combination with IFN-type-I-response inhibitors (Ruxolitinib, Tofacitinib, Amlexanox) targeting the JAK and TBK1 was studied with Flow cytometry and RT-PCR. Results: RT significantly induced HLA-class-I expression. A parallel upregulation of IFNβ and ISGs mRNA levels was also documented. Although the IFN-type-I-response inhibitors suppressed the RT-induced IFNβ and ISGs expression, their effect on HLA-class-I expression was minimal. Blockage of LC3A autophagy (shLC3A cell line) significantly upregulated HLA-class-I basal levels, and RT further enhanced HLA expression. IFN-type-I-response inhibitors blocked the RT-inductive effect in the shLC3A H1299, but had no effect in the ATG7-deficient H1650 cell line. Conclusions: The current study supports the theory that baseline autophagy, RT-induced autophagy blockage, and IFN-type-I response enhancement define the HLA-class-I levels in NSCLC cells. This complex interplay emerges as a promising target for the development of radio-vaccination strategies to enhance the efficacy of radio-immunotherapy. Full article

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections. 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/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

Conventional cancer treatments often fail due to the immunosuppressive tumor microenvironment, immune tolerance, and chronic inflammation. Therefore, new therapeutic approaches are urgently needed. Cancer vaccines can stimulate natural killer cells and cytotoxic T-lymphocytes, and induce long-lasting memory responses that help overcome the immunosuppressive tumor microenvironment. Recent advances in nucleic acid, peptide, and dendritic cell-based vaccines have improved antigen delivery and immune activation, while combinations with immune checkpoint inhibitors and ablative therapies enhance therapeutic efficacy and durability. Preclinical and clinical studies targeting tumor-associated antigens have shown promising outcomes. With poor survival rates and limited treatment options, hepatocellular carcinoma (HCC) appears to be the most prevalent cause of cancer-related deaths worldwide. Advances in antigen discovery, vaccine delivery systems, and synergistic combination strategies are paving the way for more effective and durable immune responses. By integrating molecular insights with clinical innovation, cancer vaccines hold the potential not only to improve treatment outcomes but also to redefine long-term disease management and survival in HCC. Full article

Background/Objectives: SARS-CoV-2 continues to generate antigenically divergent variants that reduce the breadth of existing vaccine-induced antibody responses. Fc-fusion subunit vaccines offer advantages in stability, antigen display, and Fc-mediated immune engagement. This study aimed to design and evaluate a tetravalent RBD–Fc fusion construct incorporating RBDs from Wuhan-Hu-1 and Omicron BA.4/BA.5 and to determine whether this configuration can induce broad antibody recognition across SARS-CoV-2 variants. The objective was to assess its feasibility, biochemical properties, and initial immunogenicity. Methods: Immune responses to the construct were first assessed using the C-ImmSim simulation platform. The full-length fusion was synthesized, subcloned into pcDNA3.1(+), expressed in HEK293 cells, and purified by Protein G affinity chromatography. Protein integrity was evaluated by reducing SDS–PAGE. BALB/c mice (female, 8 weeks) were immunized with a prime–boost–boost schedule, and sera were analyzed by ELISA, considering binding to Wuhan-Hu-1, Omicron BA.4/BA.5, and a panel of RBD variants. Results: In silico analysis predicted coordinated antigen clearance, class switching, memory B- and CD4⁺ T-cell formation, and transient cytokine induction. The recombinant protein was expressed efficiently, yielding a major ~56 kDa band and a ~23 kDa RBD fragment. Vaccinated mice generated strong IgG responses to Wuhan-Hu-1 and BA.4/BA.5 RBDs and showed broad binding to major variant RBDs. Conclusions: The tetravalent RBD–Fc fusion vaccine was successfully produced and elicited broad antibody binding across SARS-CoV-2 variants, supporting its potential as a versatile protein-based vaccine platform. Full article

Background: Individual differences in immune responses to African swine fever virus (ASFV), whether induced by vaccination or natural infection, may be linked to genetic variation in the genes involved in antigen presentation. Methods: A total of nine pigs from the 112-population were selected for RNA-seq analysis. To pinpoint key transcription factors (TFs) regulating gene expression in the lymph nodes, weighted Kendall’s Tau rank correlation analysis was performed to link the TF binding potential with the extent of differential expression of target genes. Results: CD8⁺ T cells expressing a specific epitope of the ASFV p72 protein (ACD8⁺) accounted for 41% of the total CD8⁺ T cells in peripheral blood. A total of 2062 transcripts were identified as differentially expressed across the nine pigs (q-value < 1 × 10⁻⁸). Differential expression levels of the target genes for MECP2, ETS1, ZBTB33, ELK4, and E2F4 were significantly correlated with their TF binding potential (p < 0.05). Six SNPs were identified in the promoter region of ELK4. Analysis of the 112-pig population revealed that SNPs at S.-404A>G and S.-668C>T loci were significantly associated with ACD8⁺ levels (q-value < 0.01). Individuals with the AA genotype at S.-404A>G had significantly higher ACD8⁺ counts compared to those with AG and GG genotypes (q-value < 0.05). At the S.-668C>T locus, ACD8⁺ levels were highest in the CC genotype, followed by CT and TT genotypes, with CC showing notably higher ACD8⁺ counts (q-value < 0.05). Notably, the S.-404A>G site overlaps with potential binding sites for TFs FOXA2, GATAs, and TRPS1, while the S.-668C>T site lies within the binding regions for NR1H3, RARA, VDR, and NR1I3. Conclusion: These mutations may disrupt TFs binding to the ELK4 promoter, potentially reducing ELK4 expression and impairing antigen processing and presentation. Full article

Background/Objectives: Varicella-zoster virus (VZV) causes herpes zoster (HZ/shingles), particularly in older adults with weakened cell-mediated immunity (CMI), which is essential for controlling VZV reactivation and reducing HZ severity. Currently vaccines, like recombinant subunit or live-attenuated vaccine, showed shortcomings in eliciting CD8⁺ T-cell responses. Addressing this, we utilized the novel replication-defective chimpanzee adenovirus vector ChAdOx1 to construct the ChAdOx1-VZV (CVE) vaccine, using full-length glycoprotein E (gE) as antigen. This study evaluated the immunogenicity of a heterologous intramuscular (IM) prime/intranasal (IN) boost regimen with the aim of developing a novel VZV vaccine candidate. Methods: BALB/c mice were immunized with CVE using homologous or heterologous prime-boost regimens via IM or IN. And cynomolgus macaques were immunized intramuscularly with three doses of CVE. Cellular responses were assessed by intracellular cytokine staining (ICS) and IFN-γ ELISpot using splenocytes and PBMCs. Humoral responses were evaluated by serum gE-IgG ELISA and bone-marrow LLPC ELISpot. Memory subsets and tissue-resident T cells were analyzed by flow cytometry. Results: Heterologous IM prime/IN boost CVE regimen markedly enhanced both cellular and humoral responses, especially CD8⁺ T-cell responses. The induced LLPC and memory T cell responses indicate the potential for long-term protection against herpes zoster. In cynomolgus macaques, CVE induced robust serum gE-specific IgG responses and strong IFN-γ secreting T-cell activity, supporting the immunogenicity of CVE in a genetically distinct primate model and enhancing its clinical translational potential. Conclusions: CVE induces potent cellular and humoral immunogenicity, with IM prime/IN boost vaccination. Cross species immunogenicity observed in nonhuman primates further strengthens the translational relevance of this platform. These findings support CVE as a promising herpes zoster vaccine candidate and provide a rationale for continued evaluation in human-relevant systems. Full article

High-grade gliomas (HGGs) and diffuse midline gliomas (DMGs) in pediatric patients carry a poor prognosis, necessitating the rapid development of novel therapies. Peptide vaccines represent a safe, repeatable, and rational immunotherapeutic modality aimed at inducing potent, tumor-specific T-cell responses. In this review, we define the scope of current progress by arguing that immunogenicity in children with HGG/DMG hinges on three factors: appropriate antigen class (neoantigen vs. TAA), the use of potent immunoadjuvants, and successful navigation of immune suppression. To address the gap between biological promise and clinical reality, we analyze clinical trials targeting shared tumor-associated antigens (e.g., CMV pp65, Survivin) and specific shared neoantigens (H3.3K27M). Crucially, we highlight pivotal data from the PNOC007 trial, where the magnitude of H3.3K27M-specific T-cell expansion correlated directly with significantly longer overall survival (OS), establishing a causal link between pharmacodynamics and clinical benefit. However, the unique challenges of the immunosuppressive tumor microenvironment and the detrimental effect of necessary corticosteroids remain paramount barriers. Future success relies on multi-modal combination strategies, the development of next-generation personalized neoantigen vaccines, and the application of advanced neuroimaging to accurately assess treatment response. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

Background: Antigen format strongly influences the immunogenicity of gene-based vaccines. Full-length Spike is widely used in licensed COVID-19 vaccines, while truncated subunits such as S1 or the receptor-binding domain (RBD) may simplify vector design but risk reduced potency. We aimed to compare these antigen formats in an AAV9 delivery platform. Methods: BALB/c mice were immunized intramuscularly with recombinant AAV9 encoding full-length Spike, S1, or RBD at doses of 1 × 10¹⁰ or 1 × 10¹¹ viral genomes. Immune responses were assessed by serology, virus neutralization, T-cell profiling, and histopathology. Results: All constructs expressed antigen in vitro and in vivo. Only full-length Spike elicited robust neutralizing antibodies at both doses, with titers rising significantly by week 12. High-dose RBD induced neutralization in a minority of animals, whereas S1 failed to do so. Antigen-specific IgG responses scaled with insert length (Spike > S1 > RBD). Cellular immunity was dominated by CD8⁺ effector memory T cells, strongest in the Spike group, which also induced measurable CD4⁺ responses. Local transient myositis was observed at the injection site but resolved by week 24, with no systemic pathology. Conclusions: Full-length Spike outperforms truncated subunits in the AAV context, highlighting antigen structure as a critical factor for next-generation coronavirus vaccine design. Full article

Introduction/Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a threat to global public health with a mortality rate of up to 30%. However, there is currently no commercialized SFTSV vaccine. This study focused on the construction of DNA vaccines with different structures based on the surface glycoproteins Gn and Gc to identify the immunodominant conformations. Methods: The DNA vaccines encoding secretory proteins including Gn or Gc monomer, heterodimer of Gn and Gc (dimer), two forms of hexamer composed of the Gn and Gc heterodimer (hexamer-1 and hexamer-2) or ferritin nanoparticles of Gn, and non-secretory proteins including Gn (Gn-TM) and Gc (Gc-TM) were constructed. Western blot confirmed the expression level and the specificity of those DNA vaccines. After vaccinating mice with those DNA vaccines, its induced humoral and cellular immunity were comprehensively evaluated. Results: The DNA vaccines were constructed successfully. The DNA vaccines of Gn and polymers including dimer, hexamer-2, and ferritin nanoparticles inducing stronger binding antibody, neutralizing antibody, and antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing antibody induced by these constructs was also cross-recognized by other five SFTSV pseudovirus strains. However, the T cell response induced by Gc, dimer or hexamer-2 DNA vaccines were significantly higher than those in most other groups, including Gn. Conclusion: The DNA vaccines encoding dimer or hexamer-2 demonstrated superior immunogenicity over other conformations, after taking the results of humoral and cellular responses into account. This study revealed the advantages of using polymer conformations in SFTSV vaccine design and provided new targets in SFTSV vaccine development. Full article