Search Results (279)

Daratumumab, a human IgG1 monoclonal antibody targeting CD38, is widely used in multiple myeloma and AL amyloidosis. Despite its clinical success, many patients fail to achieve durable responses or relapse, underscoring the importance of understanding resistance mechanisms. Drawing on experience from other better-studied monoclonal antibodies, resistance to daratumumab can be categorized into four main mechanisms: (1) reduced CD38 expression on plasma cells; (2) increased expression of complement inhibitory proteins (CD55/CD59), impairing complement-mediated cytotoxicity; (3) reduced drug bioavailability due to urinary loss in non-selective nephrotic syndrome; and (4) the development of neutralizing anti-daratumumab antibodies. Anti-drug antibodies (ADAs) may represent a potential mechanism of treatment failure through effects on pharmacokinetics, efficacy, and safety, even in patients on daratumumab therapy. Seven different trials have tested anti-daratumumab antibodies. Among them, anti-daratumumab antibodies were identified in only 0–2.4% of patients, and only in a small portion of these has it been proven to be neutralizing. Overall, ADAs appear rare, but these findings are likely underestimated due to short follow-up and suboptimal timing of assessment. In conclusion, standardized ADA monitoring, particularly months after treatment interruption or in cases of inadequate response or infusion-related reactions, may improve patient management and therapeutic outcomes. Full article

Neutralizing antibodies (NABs) play a critical role in assessing the immune response elicited by vaccines, providing insight into their protective efficacy. Despite their importance, there is a notable gap in research directly comparing NAB levels among individuals vaccinated with different vaccines, across diverse ethnicities, and between genders. The study aimed to compare NAB levels across variables such as vaccination status, vaccine type, age, gender, and ethnicity. The NAB levels among different study groups were measured using the Finecare RBD Antibody Test and the cPass kit (ELISA). The data was analyzed statistically using SPSS version 27. A total of 172 study subjects were analyzed. The mean age of participants was 45.03 ± 16.72 years, with 50.9% male and 77.3% of Malay ethnicity. Median NAB levels, assessed by both assays, were highest in females, vaccinated healthy participants, and those who received the Pfizer vaccine. Age-group comparisons revealed variations in median NAB levels across studied groups. Participants aged 10–25 years in the vaccinated healthy (VH) group exhibited the highest median antibody levels; on the other hand, the 26–45 year age group showed the highest median levels in the breakthrough infection (BI) and certain non-vaccinated categories. Ethnic group comparisons highlighted that Malays consistently had the highest median NAB levels. Significant differences in antibody levels were found across vaccination status, ethnicity, and vaccine type (p < 0.001). This study underscores the influence of vaccination status, demographic factors, and vaccine type on NAB levels. The Finecare RBD Antibody Test and cPass kit demonstrated comparable trends, highlighting their utility in evaluating vaccine-induced immunity. The findings of this study highlight the need for tailored immunization strategies to optimize protective immunity. Full article

The continuous evolution of the SARS-CoV-2 virus, marked by the emergence of new variants, poses a significant threat to the efficacy of existing vaccines. However, a promising approach to addressing vaccine failure caused by viral mutations (particularly in the spike protein) is the development of a variant-proof (conserved), non-spike, multiepitope universal nanostructure vaccine with multifunctionality, biocompatibility, self-adjuvanticity, and structural similarity to pathogens in terms of size and shape. This study aimed to design a self-assembled nanostructure vaccine (SANV) featuring pentameric and trimeric coiled-coil peptide motifs, as well as other functional motifs, including epitopes, TAT, PADRE, and adjuvant. The cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B lymphocyte (BL) epitopes of SANV were screened from the IEDB with more than 50% individual predicted population coverage (PPC) and fused using linkers to enable self-assembly. The multimerization of the 24 SANV monomers was modeled using the GalaxyHomomer and AlphaFold web servers. Subsequently, the leading SANV constructs with (SANVa9) and without (SANVb6) adjuvant were analyzed for their physicochemical profiles and assessed for antigenicity, allergenicity, solubility, and antioxidant potential. Furthermore, the molecular interactions, specificity, and stability of SANVa9 and SANVb6 with the broadly neutralizing sarbecovirus antibody 5817 and toll-like receptors (TLR2, TLR3, and TLR7) were analyzed using molecular docking and simulation over a 100-nanosecond time scale. Finally, the comparative immune simulation profiles of SANVa9 and SANVb6 with controls indicated stronger, broad-spectrum immune responses that could be translated into in vitro and in vivo studies and warrant further evaluation before clinical use. Full article

Mechanisms of primary and acquired resistance are responsible for treatment failure with the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors (EGFR-TKIs) in the majority of patients with advanced Non-Small-Cell Lung Cancer (NSCLC) carrying EGFR-activating mutations. MicroRNAs (miRNAs) are important modulators of EGFR signaling in lung cancer. Recent studies suggested the role of miR-23c as a tumor suppressor or oncogenic miRNA in different tumor types. However, the role of miR-23c in NSCLC carrying EGFR mutations and its involvement in resistance to EGFR-TKIs has not been explored yet. We found that miR-23c was strongly downregulated in H1975 and HCC827-Gefitinib-Resistant (GR) NSCLC cell lines with intrinsic and acquired resistance to gefitinib, respectively, as compared to gefitinib-sensitive cell lines. Moreover, we demonstrated that miR-23c mimic inhibited proliferation, migration, invasion, and epithelial–mesenchymal transition of resistant cells and that Interleukin-6 Receptor (IL-6R) is a direct target of miR-23c in H1975 and HCC827-GR cell lines. Importantly, miR-23c mimic re-sensitized NSCLC-resistant cells to gefitinib, whereas the combination of miR-23c mimic with a neutralizing IL-6R antibody potentiated the sensitivity to the drug. Collectively, our data demonstrated that miR-23c acts as a tumor suppressor in NSCLC cell lines carrying EGFR mutations and that the axis miR-23c/IL-6R might represent a potential target for the development of therapeutic approaches to overcome resistance to gefitinib. Full article

Background/Objectives: HXP-GPOVac is a locally produced, inactivated Newcastle disease virus-based (NDV-HXP-S) COVID-19 vaccine manufactured in Thailand. This phase II trial compared its safety and immunogenicity with the mRNA vaccine BNT162b2 in adults aged 18–75 years. Methods: In this randomized, double-blind, active-controlled trial registered with the Thai Clinical Trials Registry (TCTR20220819003), 300 participants were assigned 3:1 to receive HXP-GPOVac or BNT162b2 on Days 1 and 29. Solicited adverse events (AEs) were recorded for 7 days after each dose, AEs were summarized through 28 days after each dose, and serious adverse events (SAEs), medically attended AEs (MAAEs), and adverse events of special interest (AESIs) were collected through Day 197. Humoral immunogenicity was assessed by pseudovirus 50% neutralization titers (NT₅₀) and anti-spike IgG concentrations at baseline, Day 29, Day 43, and Day 197. Seroconversion was defined as a ≥4-fold increase from baseline. A predefined subset underwent interferon-γ (IFN-γ) and interleukin-5 (IL-5) ELISpot assays to assess cell-mediated immune responses. The primary immunogenicity analysis assessed non-inferiority of HXP-GPOVac compared with BNT162b2 based on the NT₅₀ geometric mean titer ratio, with a prespecified non-inferiority margin of 0.5. Results: Solicited AEs were predominantly mild and occurred more frequently after the first dose in both groups; one or more solicited local or systemic AEs were reported by 23.7% (95% CI: 18.3–29.8) of HXP-GPOVac recipients and 44.7% (95% CI: 33.3–56.6) of BNT162b2 recipients after the first dose. AEs through 28 days after vaccination and SAEs were uncommon; MAAEs occurred in 17.0% of HXP-GPOVac recipients and 22.4% of BNT162b2 recipients, and none were considered related to vaccination. In the HXP-GPOVac group, NT₅₀ geometric mean titers increased from 5.6 at baseline to 65.5 at Day 29 and 505 at Day 43, declining to 63.6 at Day 197. Anti-spike IgG geometric mean concentrations rose from 7.5 BAU/mL at baseline to 102.7 BAU/mL at Day 29 and 514.6 BAU/mL at Day 43, decreasing to 61.0 BAU/mL at Day 197. BNT162b2 induced higher antibody levels at all time points. The NT₅₀ GMT ratio (HXP-GPOVac/BNT162b2) at Day 43 was 0.51 (95% CI: 0.39–0.67); the lower bound did not exceed the prespecified non-inferiority margin of 0.5, and non-inferiority was not established. Seroconversion rates at Day 43 were 97.6% for HXP-GPOVac and 97.1% for BNT162b2 (neutralizing antibody) and 98.6% and 97.1%, respectively (anti-spike IgG). ELISpot analyses demonstrated increased IFN-γ responses after the second dose without evidence of Th2-dominant skewing. Conclusions: HXP-GPOVac was well tolerated and induced substantial humoral and cellular immune responses, with high seroconversion rates and balanced T-cell polarization. Although absolute antibody levels were lower than those induced by BNT162b2 and the prespecified non-inferiority criterion was not met, these findings support continued evaluation of the inactivated NDV-HXP-S vaccine platform. Full article

Background: Antigenic drift limits the protective efficacy of influenza vaccine. Glycosylation of hemagglutinin (HA) represents a promising immunofocusing strategy that enhances neutralizing antibody responses by masking immunodominant non-neutralizing epitopes. Methods: B-cell epitopes of influenza viruses were retrieved from the Immune Epitope Database and were mapped onto the HA structure of A/Puerto Rico/8/1934 (H1N1). Structure-guided analysis identified residues 136 and 137 as candidate sites for N-linked glycosylation (NLG). Single-site mutants (136NLG and 137NLG) were generated using reverse genetics and evaluated for stability, receptor binding, viral replication, and immunogenicity in a murine model with inactivated whole-virus vaccines. Results: Both mutants exhibited increased thermostability at 42 °C. Glycosylation reduced the HA–sialic acid affinity, resulting in decreased viral adsorption and internalization efficiency in MDCK cells, and delayed viral replication at low multiplicity of infection (MOI). In vivo, all vaccine groups provided complete protection against lethal challenge; notably, the 136NLG group exhibited reduced weight loss, indicating improved protective efficacy compared with wild-type (WT). Conclusions: Targeted glycosylation at residue 136 in the HA head domain effectively enhances the viral stability and elicits a 1.78-fold increase in hemagglutination inhibition titer (GMT) relative to the WT, thereby improving vaccine performance. These findings establish a rational and structure-based design strategy for developing more stable and effective influenza vaccines. Full article

Background: The emergence of SARS-CoV-2 variants necessitates the development of effective adjuvants to enhance subunit vaccine immunogenicity. Safe adjuvants are essential to enhance the immunogenicity of SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines. Traditional Chinese medicine polysaccharides are attractive candidates due to their immunomodulatory properties. Methods: Female BALB/c mice (6–8 weeks) were immunized on days 0, 7, and 21 with an RBD protein (20 μg) alone or formulated with Poria cocos polysaccharide fraction PCP-I or PCP-II (200 μg), Isatis indigotica polysaccharide, or aluminum adjuvant; PBS served as a control. RBD-specific total IgG and subclasses were quantified by ELISA on day 7 after the third immunization. Neutralizing antibody titers were measured by a pseudovirus assay on days 14, 28, and 56 after the first immunization. Splenic CD19⁺ B cells were analyzed by flow cytometry, and antigen-stimulated IFN-γ and IL-4 spot-forming cells were quantified by ELISpot. Results: PCP-II significantly increased RBD-specific total IgG and IgG1 compared with RBD alone and other formulations, whereas IgG2a and IgG2b remained unchanged. Both PCP-I and PCP-II increased neutralizing titers versus RBD alone, and PCP-II showed an earlier and sustained increase in neutralizing responses through day 56. PCP-II showed a non-significant increase in splenic CD19⁺ B cell frequency. PCP-I and PCP-II markedly increased IFN-γ-secreting splenocytes without increasing IL-4, indicating enhanced antigen-specific cellular responses. Conclusion: In this comparative evaluation of traditional Chinese medicine polysaccharide candidates in a SARS-CoV-2 RBD subunit vaccine model, PCP-II showed the most prominent adjuvant activity. PCP-II enhanced antigen-specific humoral immunogenicity, improved neutralizing antibody responses, and was associated with increased IFN-γ-related cellular responses, supporting its potential as a candidate polysaccharide adjuvant for protein subunit vaccines. Full article

Background: People living with HIV (PLWH) constitute a vulnerable population during the COVID-19 pandemic; however, it remains uncertain whether long-term suppressive antiretroviral therapy (ART) restores sufficient immune competence to support robust hybrid immunity. While vaccination followed by breakthrough infection—termed hybrid immunity—typically elicits potent humoral responses in immunocompetent individuals, the functional quality and breadth of these responses against evolving Omicron subvariants remain poorly characterized in PLWH. This study aimed to assess functional antibody responses, including neutralizing activity and Fc effector functions, in vaccinated and unvaccinated PLWH who experienced breakthrough infection with Omicron subvariants BA.4/5 or BF.7. Methods: We enrolled three cohorts between December 5 and December 20, 2022: 25 HIV-negative individuals with breakthrough infection (BTI-HC), 20 ART-experienced PLWH with breakthrough infection following three-dose COVID-19 vaccination (BTI-HIV), and 10 ART-experienced PLWH with primary infection without prior vaccination (PI-HIV). All HIV-positive participants were receiving suppressive ART with regimens based on non-nucleoside reverse transcriptase inhibitors or integrase strand transfer inhibitors for a median of 3.4 years. We measured receptor-binding domain (RBD)-specific IgG, neutralizing antibody titers against ancestral D614G, Delta, BA.1, BA.4/5, BF.7, XDV, KP.2, and KP.3 variants, and antibody-dependent cellular cytotoxicity (ADCC) responses. Results: Despite lower absolute CD4⁺ T cell counts, BTI-HIV participants mounted RBD-binding IgG, neutralizing antibody, and ADCC responses that were comparable to BTI-HC and significantly exceeded PI-HIV across all tested variants. Both breakthrough infection cohorts exhibited immunological imprinting, with higher neutralizing titers against ancestral D614G than infecting BA.4/5 or BF.7 variants. Emerging variants XDV, KP.2, and KP.3 demonstrated substantial neutralization escape in all groups. PI-HIV showed markedly diminished neutralization breadth and failed to generate enough responses against all tested Omicron strains. Conclusions: Suppressive ART enables PLWH to mount hybrid immunity—conferred by vaccination followed by BF.7 or BA.4/5 breakthrough infection—with neutralizing and ADCC responses comparable to HIV-negative individuals, and significantly exceeding those of unvaccinated PLWH with primary infection. This underscores the critical role of vaccination in establishing effective hybrid immunity in this population. However, we observed immunological imprinting, with higher titers against ancestral strains than against infecting variants, and substantial escape by emerging sublineages XDV, KP.2, and KP.3 across all groups. These findings support prioritizing updated variant-containing vaccines for HIV-positive populations and reinforce the essential role of vaccination in this vulnerable group. Full article

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in global swine production. The causative agent, PRRS virus (PRRSV), comprises two genetically distinct species—PRRSV-1 and PRRSV-2—that differ substantially in antigenic composition and immune recognition. Despite widespread use of modified live vaccines (MLVs), protection against heterologous and cross-species strains remains inconsistent and difficult to predict. This review synthesizes current knowledge of homologous, heterologous, and cross-species protection, with emphasis on humoral and cellular immune responses and the viral determinants that constrain breadth of immunity. Neutralizing antibodies can confer near-sterilizing homologous protection under controlled conditions; however, their delayed induction and narrow specificity limit efficacy against heterologous strains. T-cell-mediated responses are generally broader but remain highly strain- and context-dependent. Structural features of PRRSV envelope glycoproteins, including glycan shielding and immunodominant decoy epitopes, further restrict antibody-mediated cross-protection while providing targets for rational vaccine design. We also examine potential drawbacks of preexisting immunity, including antigenic mismatch and non-neutralizing antibody-dominated responses that may contribute to suboptimal outcomes following heterologous exposure. Collectively, these findings highlight the multifactorial nature of PRRSV protection and the need for next-generation vaccines capable of inducing broader and more durable immunity. Full article

Severe fever with thrombocytopenia syndrome (SFTS) is a newly discovered tick-borne disease caused by SFTS virus (SFTSV) infection. Patients present with high fever, thrombocytopenia, and multiple organ dysfunction, with a high mortality rate and a lack of specific treatment, all of which indicate that research on the deterioration mechanism and treatment of this disease is urgent. Currently, multiple studies have indicated that cytokine storm is one of the core factors contributing to the deterioration of the disease. SFTSV inhibits the host’s type I interferon response through its non-structural protein NSs, thereby promoting immune evasion and viral replication. Extensive viral stimulation leads to dysfunction and abnormal polarization of immune cells (including monocytes, macrophages, dendritic cells, T cells, and B cells), triggering the massive release of pro-inflammatory factors(such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β)), anti-inflammatory factors (such as interleukin-10 (IL-10)), and chemokines(such as interferon-gamma inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), and interleukin-8 (IL-8)). This cytokine storm exacerbates the imbalance between pro-inflammatory and anti-inflammatory factors, as well as immune paralysis, leading to vascular endothelial damage, microthrombosis, and ultimately, multi-organ failure, which determines the clinical outcome. Simultaneously, specific cytokines and immune cell phenotypes can serve as biomarkers for disease severity and prognosis. In terms of treatment, this article further summarizes the intervention strategies targeting the aforementioned immune links, including intravenous immunoglobulin (IVIG), tocilizumab (targeting the IL-6 receptor), inhibitors of Janus kinase (JAK) and nuclear factor-kappa B (NF-κB) signaling pathways, interferon, neutralizing antibodies, and other immunotherapy methods. By analyzing the dynamic changes and mechanisms of cytokine storm in the course of SFTS, and summarizing current potential immunotherapy methods, this article aims to provide a theoretical framework for the future treatment of SFTS. Full article

Circular RNA (circRNA) has emerged as an alternative RNA modality for vaccine development due to its covalently closed structure and enhanced molecular stability compared with linear messenger RNA (mRNA). Following the clinical success of mRNA vaccines, circRNA-based platforms have gained attention in both prophylactic and cancer immunization. Unlike linear mRNA, circRNA relies on cap-independent translation and is commonly produced through in vitro transcription coupled with ribozyme-mediated self-circularization. In prophylactic vaccination, circRNA vaccines have demonstrated sustained antigen availability, robust humoral and cellular immune responses, and flexibility in multivalent designs and targeted delivery strategies that support germinal center reactions and neutralizing antibody generation. In cancer vaccines, circRNA has been applied to tumor-associated antigens, neoantigens, and non-canonical peptides, with a primary focus on inducing potent antigen-specific CD8⁺ T cell immunity and enabling combination immunotherapy approaches. This review summarizes recent applications of circRNA-based vaccines in prophylactic and cancer settings, emphasizing in vitro transcription-compatible self-circularization strategies and discussing how methodological choices in RNA design, translation control, purification, and delivery shape immunological outcomes. Full article

Cedar virus (CedV), related to the highly pathogenic bat-borne henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), is non-pathogenic in small animal models, likely due to the inability to produce interferon-antagonist proteins. We evaluated the pathogenesis of recombinant CedV (rCedV) in the African green monkey (AGM) model and determined if prior infection conferred cross-protective immunity against a lethal challenge with NiV Bangladesh (NiV-B) or HeV. AGMs infected with rCedV remained asymptomatic, with no clinical signs of disease or detectable viremia. The rCedV infected animals developed homologous neutralizing antibody responses that failed to cross-neutralize NiV-B or HeV. At 42 days post-rCedV infection, AGMs were challenged with a lethal dose of NiV-B or HeV, and prior infection with rCedV failed to protect against NiV-B challenge, with all animals succumbing to NiV-B. Similarly, rCedV infection did not confer consistent protection against HeV, with 2/4 animals succumbing to lethal HeV. These findings confirm that CedV is non-pathogenic in the AGM model of NiV and HeV infection, justifying its classification as a BSL-2 agent. The findings also demonstrate that rCedV does not elicit a cross-protective immune response to prevent lethal disease from either NiV-B or HeV highlighting significant immunological differences between CedV and the pathogenic henipaviruses. Full article

Background: Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli (EHEC), is a highly potent exotoxin responsible for severe complications such as hemolytic uremic syndrome (HUS). Among its isoforms, Stx2 exhibits stronger cytotoxicity and poses greater clinical risk, yet no effective therapy currently exists. Methods: In this study, two human monoclonal antibodies, YG12-1 and YG12-2, were identified from a phage display library and systematically characterized using an integrated modeling–validation workflow. Results: Structural modeling with ImmuneBuilder and Rosetta revealed that YG12-2 possessed a longer CDRH3 topology, more short-range hydrogen bonds, and stronger electrostatic complementarity, corresponding to lower binding energy and higher apparent affinity in ELISA and SPR. Although YG12-2 had a better affinity, YG12-1 shows better protective activity in a murine model of acute peritoneal infection. This paradox highlights a non-linear relationship between structural affinity and biological efficacy, emphasizing the importance of functional epitope accessibility and pharmacokinetic behavior in determining neutralization outcomes. Conclusions: Overall, these results indicated that targeting Stx2 with YG12-1 and YG12-2 could serve as a promising protective strategy against E. coli O157:H7 infection. Full article

Background/Objectives: SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette–Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis, other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB (Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods: We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime–boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results: Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime–boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions: Our study highlights the potential of the prime–boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates. Full article

Background/Objectives: The emergence of immune-evasive SARS-CoV-2 variants highlights the need for adaptable vaccine strategies. Trimeric receptor-binding domain (tRBD) antigens offer structural and immunological advantages over monomeric RBDs, but DNA vaccine efficacy has been limited by inefficient antigen expression, particularly in non-dividing antigen-presenting cells. Although cytoplasmic transcription–based DNA platforms have been developed to overcome nuclear entry barriers, their utility for antigen structure–function optimization remains underexplored. This study evaluated whether integrating a rationally designed trimeric RBD with a T7-driven cytoplasmic transcription system could enhance immunogenic performance. Methods: A DNA vaccine encoding a tandem trimeric SARS-CoV-2 RBD was delivered using a T7 RNA polymerase-driven cytoplasmic transcription system. In vitro antigen expression was assessed following Lipofectamine 3000-mediated transfection. In vivo, mice were immunized with the SM-102-based Rpol/tRBD/LNP formulation, and immunogenicity was assessed by antigen-specific antibody titers, serum neutralizing activity, and T-cell response profiling, together with basic safety/tolerability evaluations. Results: The T7-driven cytoplasmic transcription system markedly increased antigen mRNA and protein expression compared with conventional plasmid delivery. Rpol/tRBD vaccination induced higher anti-RBD IgG titers, enhanced neutralizing antibody activity, and robust CD8⁺ T cell responses relative to monomeric RBD and plasmid-based trimeric RBD vaccines. Immune responses were Th1-skewed and accompanied by germinal center activation without excessive inflammatory cytokine induction, body-weight loss, or hepatic and renal toxicity. Conclusions: This study demonstrates that integrating rational trimeric antigen engineering with direct cytoplasmic transcription enables balanced and well-tolerated immune activation in a DNA vaccine context. The T7 autogene-based platform provides a flexible framework for antigen structure–function optimization and supports the development of next-generation DNA vaccines targeting rapidly evolving viral pathogens. Full article