Lassa virus (LASV), the causative agent of Lassa fever, must be handled under biosafety level 4 (BSL-4) conditions, requiring validated inactivation protocols to ensure laboratory and public safety. Although LASV is an enveloped virus theoretically susceptible to physical and chemical inactivation methods, quantitative data on its inactivation kinetics remain limited. This study systematically evaluated the efficacy of thermal treatment (56 °C, 70 °C, 95 °C), laboratory chemical inactivants (beta-propiolactone, formaldehyde, methanol, TRIzol), and five commercial disinfectants against infectious LASV. Viral infectivity was determined by titrating residual virus in Vero E6 cells, and complete inactivation was verified by three consecutive blind passages. Thermal inactivation was achieved at 56 °C for 40 min, 70 °C for 5 min, and 95 °C for 2 min. Both 0.1% and 0.05% beta-propiolactone completely inactivated LASV after 24 h at 4 °C, while 4% formaldehyde, 50% methanol, and 25% TRIzol achieved complete inactivation within 15 min, 10 min, and 2 min, respectively. For surface disinfection, 2% and 5% Micro-Chem Plus™ and 75% ethanol reduced viral titers by ≥4 log10 TCID50/mL within 30 s; 1% sodium hypochlorite and 0.25% Virkon required 1 min, whereas 3% hydrogen peroxide required 3 min to achieve the same reduction. These results provide quantitative, evidence-based parameters that can serve as a valuable reference for the safe handling of LASV under controlled BSL-4 laboratory conditions.

Zika virus (ZIKV) infection of the developing brain induces neuroinflammatory responses that can restrict viral replication but may also contribute to neurological injury when dysregulated. Although neuronal phospholipid homeostasis is critical for membrane integrity and synaptic function, how ZIKV infection reprograms lipid metabolism to drive neuropathogenesis remains unclear. Here, we identify calcium-independent phospholipase A2β (iPLA2β) as a key mediator of ZIKV-induced neuronal lipid remodeling. iPLA2β is rapidly upregulated in neurons and in the neonatal brain following infection, and its expression is promoted by the viral envelope protein, which interacts with iPLA2β in neurons. Genetic ablation of iPLA2β reduces viral burden in the developing brain and in primary neurons and disrupts ZIKV-induced phospholipid remodeling, characterized by altered arachidonic acid–containing phospholipids and depletion of docosahexaenoic acid-enriched species. Importantly, sustained iPLA2β activity promotes phospholipid remodeling that supports efficient ZIKV replication, thereby increasing viral burden and contributing to a pro-inflammatory lipid environment. This is associated with increased prostaglandin E₂ production, glial activation, and neuronal loss. Both genetic and pharmacological inhibition of iPLA2β partially restores phospholipid homeostasis and reduces viral burden, accompanied by attenuation of neuroinflammatory responses. Functionally, targeting iPLA2β improves survival and neurobehavioral outcomes and mitigates long-term cognitive deficits following neonatal ZIKV infection. Collectively, these findings support iPLA2β-dependent phospholipid remodeling as a host process that facilitates ZIKV replication and links infection to neuroinflammation and neurological injury, highlighting iPLA2β as a potential therapeutic target in ZIKV-associated neuropathogenesis.

The continued antigenic evolution of SARS-CoV-2 Omicron subvariants has progressively eroded vaccine-elicited protective immunity, driving demand for next-generation candidates that confer broad-spectrum protection against phylogenetically divergent strains. Here we report the design and preclinical evaluation of SV, an mRNA vaccine encoding a heterodimeric receptor-binding domain (RBD) antigen. In this construct, a previously optimized monomeric RBD (BSCOV06) is tandemly linked to the KP.3 RBD, presenting two antigenically distinct RBDs within a single immunogen. A two-dose SV regimen in BALB/c mice elicited high-titer neutralizing antibodies with potent cross-reactivity against BA.1, XBB.1.5, JN.1, KP.3, and the phylogenetically distant XDV variant. Integrated B cell receptor (BCR) and T cell receptor (TCR) repertoire profiling revealed that SV drives qualitatively distinct adaptive immune remodeling relative to BSCOV06. Key features included elevated class-switched somatic hypermutation, sustained naive B cell engagement, broad polyclonal T cell expansion, and extensive VJ gene-usage reprogramming across both lymphocyte compartments. In BALB/c and K18-hACE2 transgenic mice, SV conferred robust protection against JN.1 and XDV challenge, substantially reducing pulmonary viral loads and attenuating histopathological injury. Notably, SV achieved immunogenicity and cross-protective efficacy comparable to or exceeding those of the three-dose BSCOV06 schedule, supporting the potential of heterodimeric antigen design. These findings support SV as a promising broad-spectrum COVID-19 vaccine candidate. More broadly, they suggest that heterodimeric RBD architectures incorporating antigenically divergent variants may represent a generalizable platform for countering viral immune evasion, with implications for future SARS-CoV-2 variants and other rapidly evolving viral pathogens.

Chikungunya virus (CHIKV) is a re-emerging arbovirus causing acute febrile illness and chronic debilitating arthritis, thereby imposing significant global public health and economic burdens. While robust immune responses involving inflammatory cytokines and immune cell infiltration characterize acute infection, the cellular mechanisms underlying pathogenesis and chronicity remain incompletely defined. In this study, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively profile splenic and peripheral blood mononuclear cells (PBMCs) immune responses in rhesus macaques at day 7 post CHIKV infection, an acute phase. Splenic neutrophils marked recruitment, upregulation of S100A8/S100A9, downregulation of interferon-stimulated genes (ISGs), and functional activation marked by degranulation, enhanced anti-apoptotic pathways, and neutrophil extracellular traps (NETs) formation, as visualized by multiplex immunofluorescence. Pseudotime trajectories delineated progressive states transitioning from proliferation to effector functions. Splenic T and B cells showed increased abundance with innate-to-adaptive transition signature. Splenic CD4+ and CD8+ T cell subsets and B cell subsets exhibited enrichment for innate immune pathways and translational machinery. In PBMCs, CD8+ T cell subsets and B cell subsets exhibited activation of adhesion, cytokine signaling, and protein homeostasis pathways in infection group. Notably, CHIKV infection induced skewing of T cells toward states enriched for Th1 and Th17 differentiation (marked by NXPE3, LEF1, NFKBIZ expression) in PBMCs. These results delineate the spatiotemporal immune landscape during CHIKV acute phase, in this non-human primate model, identifying neutrophil-mediated inflammation, lymphocyte transcriptional adaptation, and Th1/Th17 polarization as hallmarks of acute phase, offering a detailed cellular map that may inform future investigations into CHIKV pathogenesis and therapeutic strategies.

The global emergence of monkeypox virus (MPXV) highlights the urgent need for a deeper understanding of host–pathogen interactions. Although transcriptional responses to MPXV infection have been characterized, the role of epitranscriptomic regulation particularly N6‑methyladenosine (m6A) modification remains largely unexplored. We performed an integrated analysis of time‑series transcriptomic and m6A methylome profiles using whole blood samples collected from MPXV‑infected rhesus macaques at 7, 14, and 21 days post‑infection, with distinct animals used at each time point. Host gene expression and m6A modification dynamics were examined over the course of infection. Differential expression and differential m6A modification analyses were conducted, followed by integrative pathway and immune cell signature profiling. MPXV infection induced sustained host reprogramming, characterized by suppression of immune pathways and activation of metabolic processes. A global increase in m6A modifications was observed, accompanied by upregulation of the methyltransferase METTL3 and downregulation of demethylases (FTO, ALKBH5) and readers (YTHDF1-3). Knockdown of METTL3 or YTHDF2 reduced viral replication, suggesting a proviral role for this regulatory circuit. Integrative analysis identified 38 genes with coordinated changes in both transcription and m6A modification across all three time points. Focusing on literature-curated pathogenic pathways, we further identified 11 dual-regulated host factors. Notably, DNAJB1 was the only gene shared between these two independent selection strategies. m6A peaks near transcription start sites and within 5′UTR positively correlated with gene expression, whereas coding region modifications showed weak negative correlations. Immune lineage signatures showed gradual declines in T cell, NK, and monocyte/macrophage signatures with a progressive increase in B cell signatures. Cross‑dataset comparison confirmed core m6A regulatory trends despite heterogeneity across tissues and viral strains. This study reveals m6A epitranscriptomic remodeling as a key correlate of the host response to MPXV infection and nominates DNAJB1 alongside the other 10 dual‑regulated genes as candidate host factors for further mechanistic investigation.

Purpose:Although studies have described the clinical characteristics of norovirus enteritis (NoVE) associated with convulsions, research on its molecular epidemiology remains limited. Therefore, this study aimed to investigate both the clinical manifestations and genotypic features of NoVE complicated by convulsions. Methods:NoVE children admitted to our hospital between November 2021 and May 2022 were divided into NoVE complicated with convulsion group and NoVE group. Then, we screened the risk factors of NoVE complicated with convulsion based on multivariate regression analysis. The stool samples were collected, followed by amplification of VP1 sequences of GI and GII and phylogenetic analysis of VP1 in both groups. Results:Logistic regression analysis showed that elevated serum CK-MB levels and fever were independent risk factors for convulsions in NoVE patients, while a lower frequency of diarrhea was an independent protective factor. GII.3 genotype was merely detected in 12 NoVE samples (14.81%) and was detected in 5 (15.19%) NoVE with convulsion samples. The GII.4 genotype was detected in 28 (84.85%) samples of the NoVE with convulsion group and 69 (85.19%) samples from the NoVE group. Conclusions:Elevated serum CK-MB concentration, fever, and frequency of diarrhea were independent influencing factors for NoVE complicated by convulsions.

This study systematically evaluated the humoral immune responses and cytokine profiles of 259 pediatric participants, comprising both healthy individuals and patients with Acute Lymphoblastic Leukemia (ALL), following either administration of an inactivated SARS-CoV-2 vaccine or natural infection. The data indicate that vaccination elicits measurable immune responses in both groups, supporting its relevance in immunocompromised pediatric populations. Although antigen-specific responses to the spike (S) protein and receptor-binding domain (RBD) were attenuated in the ALL cohort, levels of neutralizing antibodies and antibodies capable of inhibiting RBD-ACE2 binding were comparable to those in healthy controls, suggesting that functional antibody-mediated immunity can still be achieved in ALL patients. Notably, vaccinated ALL patients exhibited higher neutralizing antibody titers against the SARS-CoV-2 prototype strain and major variants of concern (Alpha, Beta, Delta) than their healthy counterparts. However, both groups demonstrated markedly reduced responses to the Omicron variant, underscoring its substantial immune escape potential. Cytokine analysis revealed dysregulated expression patterns in the ALL group, with minimal modulation following vaccination, in contrast to the immunosuppressive cytokine regulation observed in healthy individuals. Importantly, the JAK/STAT signaling pathway emerged as a key component in the immune response to SARS-CoV-2 among healthy subjects. These insights provide a scientific basis for the optimization of COVID-19 immunization strategies tailored to vulnerable pediatric groups.

Background: Rotavirus (RV) A is one of the major reasons which causes acute dehydration and diarrhea. It is also one of the highest morbid diseases in children. There are only a few reports about the changes in prevalence and VP4/VP7 genotype of RVs in southwest China. Here is the report about the prevalence of RVs from 2015 to 2020 in Yunnan, southwest China. Methods: The virus genes were extracted from RV positive samples, then VP4/VP7 genes were amplified, followed by sequencing and gene typing, phylogenetic analysis, antigenic epitope variation analysis, and selective pressure analysis were also performed. Results: A total of 135 VP4 gene sequences and 143 VP7 gene sequences were obtained from stool samples during 2015–2020. Of them, P[8] genotype accounted for 97.0% of the total, while the P[4] genotype accounted for 3.0%. As for the VP7 genotype, G9 genotype accounted for 86.0% of the total, the G3 genotype accounted for 9.1%, and the G2 genotype accounted for 4.9%. G9P[8] was identified as the predominant RV strain during the epidemic season in Yunnan during 2015–2020. Phylogenetic analysis showed that G9 genotype sequences were primarily similar to African strains (KJ753473, KY661937), while P[8] genotype sequences were close to Southeast Asian strains (JQ837878, KX362594). In antigenic epitope variation analysis, among 37 epitopes of P[8] genotype, the RotaTeq™ vaccine strain covers 31 amino acid positions, Rotarix™ covers 28 amino acid positions, while LLR covers only 9. In the representative sequence of the G9 genotype, RotaTeq™ vaccine strains cover 27 out of 29 amino acid positions, Rotarix™ covers 16 positions, and LLR covers 16 positions. The results of the selective pressure analysis indicated potential positive sites for the G9P[8] genotype located at vp7-44, vp7-100, vp7-221, vp7-278, vp4-3, and vp4-4. Conclusions: Our study shows that G9P[8] is the most dominant RV genotype in Yunnan, China. Consistent with the recent epidemic trend of RV strains in China, this study could provide new perspectives on vaccine research.

The emergence of XBB- and JN.1-lineages with remarkable immune evasion characteristics have led to rises in breakthrough infections within populations. In addition, the unfavorable impacts of immune imprinting, stemming from continuous exposure to antigens from circulated viruses, have been observed to incline immune response against earlier lineages, thereby declining the neutralization to newly emerged Omicron subvariants. In response to this, the advancement of next-generation vaccines against COVID-19 targeting components from new subvariants such as XBB-lineage is imperative. In the current study, a self-assembled trimeric recombinant protein (RBDXBB.1.5-HR) was generated by concatenating the sequences of the receptor binding domain (RBD) derived from XBB.1.5 with heptad-repeat 1 (HR1) and HR2 sequences from the spike S2 subunit. Adjuvanted-RBDXBB.1.5-HR induced robust humoral and cellular immune responses, characterized by elevated neutralization against JN.1-inculuded subvariants and a substantial population of antigen-specific T memory cells. Protective immunity conferred by RBDXBB.1.5-HR vaccine was preserved post-immunization, as evidenced by germinal center B (GC B) and T follicular helper (Tfh) responses, sustained neutralization potency, and an increase in memory B cells (MBCs) and long-lived plasma cells (LLPCs). The RBDXBB.1.5-HR vaccine showed a favorable boosting effect when administered heterologously after three doses of inactivated virus (IV) and mRNA vaccines. Significantly, it provided protection against live Omicron EG.5.1 viruses in vivo. The monovalent RBDXBB.1.5-HR vaccine showed favorable safety and immunogenicity, boosting neutralizing antibodies against JN.1- and XBB-lineage subvariants in individuals with prior COVID-19 vaccinations. These findings highlight its clinical potential in safeguarding against circulating Omicron subvariants.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is tick-borne bunyavirus with a fatality rate up to 30%, posing a severe public health threat. There is currently no therapeutics for SFTSV infection. Here, from SFTSV convalescent donors, we discovered an ultrapotent NAb ZS004-1C5 with subnanomolar neutralization titers (IC50=0.3 ng/mL). Cyro-EM structure revealed that ZS004-1C5 has a super long HCDR3 (23aa) to reach a unique “groove epitope” on SFTSV Gn. Next, mouse protection studies demonstrated that delayed (up to 48 hrs) therapeutic treatment of ZS004-1C5 100% protected STAT1-/- mice from lethal SFTSV challenge. In a non-human primate model, therapeutic administration of ZS004-1C5 significantly protected rhesus macaques from SFTSV-induced synptoms, viremia and inflammation. Lastly, using AI-guided algorithms, we identified ZS004-1C5-like NAbs from human BCR repertoire and discovered a germline IgM that can neutralize SFTSV. Our study is the first antibody protection study against SFTSV in monkeys. Our findings provided proof-of-concept data to support clinical development of this antibody for SFTSV therapy, and discovered a novel neutralizing epitope that could benefit future vaccine design works. This work was supported by the Education Foundation of Westlake University and the HRHI program of Westlake Laboratory of Life Sciences and Biomedicine 1010060220B1. Vaccines and Immunotherapy (VAC)

Mucosal immunity provides efficient protection against upper-airway infections, limiting viral shedding and transmission. However, currently, no nasal spray COVID-19 vaccines are approved by WHO for global use. Here we develop a two-component intranasal vaccine that combines an adenovirus vector expressing the spike protein of the XBB.1.5 variant (Ad5XBB.1.5) with a self-assembled trimeric recombinant protein derived from the receptor binding domain (RBDXBB.1.5-HR). This two-component vaccine elicits superior humoral and cellular immunity against XBB.1.5 variants compared with the individual components. It also provides protective immunity against live XBB.1.16 virus challenges in mice, and prevents XBB.1.5 virus transmission in a hamster model. Notably, the activation of the STING signalling pathway in mucosal dendritic cells is essential for the adjuvant effect of the adenovirus vector. We also incorporate another trimeric protein from the BA.5 variant (RBDBA.5-HR), creating a three-component vaccine (Ad5XBB.1.5 + RBDXBB.1.5-HR + RBDBA.5-HR) that shows enhanced broad-spectrum neutralization. The two-component vaccine demonstrates high tolerability and safety in humans, inducing enhanced mucosal immunity and high levels of neutralizing antibodies in all participants. Our findings underscore this strategy for clinical COVID-19 intranasal vaccine development. A combination of adenovirus-vectored and subunit protein intranasal vaccine enhances immune response against SARS-CoV-2 variants in animal models and humans.

Objectives:From June to November 2023, a severe dengue fever outbreak occurred in Xishuangbanna, China, a border area with Myanmar and Laos. This study aimed to identify the pathogen responsible for this outbreak and analyze the genomic characteristics of epidemic strains, providing reference data for dengue prevention and control. Methods:Serum samples from dengue virus (DENV) NS1-positive patients (July-October 2023) were collected. RNA extraction, serotyping via probe quantitative polymerase chain reaction, and whole-genome amplification using 18 primer pairs followed by sequencing were performed. Base and amino acid mutations were analyzed using DNAMAN. Phylogenetic trees (maximum likelihood for the whole genome and neighbor-joining for the E protein) were constructed using MEGA11. Protein secondary structures were compared via online tools provided by PRABI Lyon-Gerland. Results:Among 1465 samples, 833 were DENV-1 positive, with no other dengue serotype or flavivirus co-infection. Genomic analysis of 10 isolates showed high similarity to the 2023 Guangdong strain (PP540291). Non-structural proteins had higher base mutation rates than structural ones, with NS2 showing the highest (10.92%). Many unique 2013 Yunnan strain mutations were preserved. Phylogenetic trees clustered the epidemic strains with Guangdong and Southeast Asian isolates. NS1 RNA-binding sites remained stable. Conclusions:This study provides valuable insights for dengue control in the China-Myanmar-Laos border areas, as well as for viral pathogenesis research and vaccine development.

With the broad spread of the chikungunya virus (CHIKV), there is an increasing demand for more effective and broadly protective vaccines. Here, we designed CHIKV mRNA vaccines containing full-length structural proteins or part of structural proteins (envelope proteins) based on conserved sequences from 769 viral strains encompassing four lineages. The vaccine induced strong cellular and humoral immune responses in BALB/c mice and provided robust protection. Immunization of BALB/c mice with either of the two vaccines induced high levels of neutralizing antibodies against pseudoviruses from four distinct lineages, highlighting their potential for broad cross-lineage protective efficacy. Immunoglobulin repertoire analysis revealed two important BCR V-J gene combinations, IgHV1-4-IgHJ3 and IgHV1-4-IgHJ2, and lineage-specific immunity analysis revealed significant upregulation of TCRs containing V19 and V20. BCR and TCR immunodiversity may be a potential reason for the broad-spectrum protection against CHIKV afforded by the vaccine. In A129 mice, it elicited lower levels of neutralizing antibodies but prevented mouse mortality and cleared chronic infection. In the rhesus macaque model, both vaccines elicited a certain level of humoral and cellular immune responses and protected the rhesus macaques from the CHIKV challenge. In conclusion, the results from both mouse and rhesus macaque models indicate that the vaccine could be a candidate for clinical use against CHIKV.

Lassa fever (LF) is a fatal hemorrhagic disease caused by the Lassa virus (LASV), which mainly spreads in Africa. As China's interactions with Africa become more frequent, the risk of LF being imported into China also rises, making the study of LASV increasingly urgent. In this study, the Lineage IV LASV strain was successfully isolated from the first imported case in China. Compared with the LASV genome, the isolated strain may exhibit greater infectivity and interspecies transmission capabilities. We successfully established BALB/c, C57BL/6, and AG129 mouse infection models and found that intranasal inoculation was the most stable infection method. Select the anti-LASV drug LHF-535 for preliminary evaluation, further confirming the stability of the model. In summary, the isolated strain exhibits enhanced transmission capabilities and may spread between mice via the respiratory tract, meriting greater attention and emphasis. This study will bridge the gap in China's independent P4-level pathogen isolation, meet national biosafety and strategic needs, and provide certain support for LASV research.

In recent years, Dengue virus (DENV) has continued to pose significant health risks in tropical and subtropical areas worldwide, raising health alerts worldwide. It can cause hyperviremia in humans and can even lead to fatal clinical diseases. The life cycle of DENV is intricately linked to cellular lipids, and the virus selectively utilizes relevant enzymes involved in lipid metabolism to modulate the existing metabolic system in host cells during entry, replication, assembly, and other stages, thereby creating an environment conducive to its complete replication cycle. At present, there is a lack of effective and specific anti-DENV treatment measures. This review summarizes the recently identified lipid metabolism molecules and metabolic related diseases that affect DENV infection, explores the dependence of DENV on lipid metabolism and provides potential targets for the treatment of dengue fever (DF).

BACKGROUND:Our previous findings indicate Semaphorin 4D (Sema4D) as a potential pediatric leukemia biomarker, promoting leukemogenesis via PI3K/AKT and ERK pathways, but its upstream regulatory mechanisms remain unclear. This study was conducted to explore the potential regulatory relationship between Insulin growth factor 2 mRNA binding protein 3 (IGF2BP3) and Sema4D in acute myeloid leukemia (AML). METHODS:The expression levels of Sema4D and IGF2BP3 were analyzed in PBMCs from 41 newly diagnosed patients with pediatric acute myeloid leukemia (AML) and 35 healthy pediatric donors who presented no history of leukemia using western blotting and qRT-PCR. IGF2BP3 overexpression and knockdown models were established in Kasumi-1 and HL-60 cells via lentiviral infection. Cell proliferation, apoptosis, and cell cycle distribution were assessed using CCK-8 and flow cytometry. The stability of Sema4D mRNA and m6A methylation levels were evaluated via mRNA stability assay and qPCR. RESULTS:This study discovered that Sema4D and IGF2BP3 were overexpressed in the peripheral blood mononuclear cells (PBMCs) of AML patients, and their expression levels were positively correlated. IGF2BP3 overexpression enhanced cell proliferation and cell cycle progression, and inhibited apoptosis in AML cells, while knockdown had the opposite effect. Mechanistic exploration revealed that IGF2BP3 enhances the stability of Sema4D mRNA through m6A-dependent mechanisms. CONCLUSION:In this study, we demonstrate that the IGF2BP3/Sema4D axis is a crucial regulator in AML development, driving cell proliferation and survival through post-transcriptional regulation of Sema4D by IGF2BP3 in an m6A-dependent manner. Our findings highlight the potential of targeting this axis as a therapeutic strategy for AML treatment.

Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a growing global health threat with substantial mortality and no effective treatments. We report the discovery of ZS1C5, a human antibody that neutralizes SFTSV with subnanomolar potency by targeting a previously unknown interdomain epitope on glycoprotein Gn, mediated by an elongated CDRH3. A single dose of ZS1C5 conferred robust protection in both murine and, for the first time, non-human primate SFTSV infection models, demonstrating rapid viral control and immune restoration. Importantly, we applied a structure-guided mining approach to human B cell repertoires and identified germline-encoded ZS1C5-like antibodies, underscoring the potential for rapid recall of protective immunity. Together, these findings establish ZS1C5 as a promising clinical candidate and provide a blueprint for developing therapeutics and vaccines targeting interdomain epitopes in emerging bunyaviruses.

Urgent research into innovative severe acute respiratory coronavirus-2 (SARS-CoV-2) vaccines that may successfully prevent various emerging emerged variants, particularly the Omicron variant and its subvariants, is necessary. Here, we designed a chimeric adenovirus-vectored vaccine named Ad5-Beta/Delta. This vaccine was created by incorporating the receptor-binding domain from the Delta variant, which has the L452R and T478K mutations, into the complete spike protein of the Beta variant. Both intramuscular (IM) and intranasal (IN) vaccination with Ad5-Beta/Deta vaccine induced robust broad-spectrum neutralization against Omicron BA.5-included variants. IN immunization with Ad5-Beta/Delta vaccine exhibited superior mucosal immunity, manifested by higher secretory IgA antibodies and more tissue-resident memory T cells (TRM) in respiratory tract. The combination of IM and IN delivery of the Ad5-Beta/Delta vaccine was capable of synergically eliciting stronger systemic and mucosal immune responses. Furthermore, the Ad5-Beta/Delta vaccination demonstrated more effective boosting implications after two dosages of mRNA or subunit recombinant protein vaccine, indicating its capacity for utilization as a booster shot in the heterologous vaccination. These outcomes quantified Ad5-Beta/Delta vaccine as a favorable vaccine can provide protective immunity versus SARS-CoV-2 pre-Omicron variants of concern and BA.5-included Omicron subvariants.

The immune escape capacities of XBB variants necessitate the authorization of vaccines with these antigens. In this study, we produce three recombinant trimeric proteins from the RBD sequences of Delta, BA.5, and XBB.1.5, formulating a trivalent vaccine (Tri-Vac) with an MF59-like adjuvant at a 1:1:4 ratio. Tri-Vac demonstrates immunogenicity in female NIH mice, inducing cross-neutralization against various SARS-CoV-2 variants, including pre-Omicron and Omicron BA.2.75, BA.5, and XBB lineages. It elicits measurable antigen-specific T cell responses, germinal center B cell responses, and T follicular helper responses, effectively protecting against live Omicron XBB.1.16 challenges. Protective immunity is maintained long-term, with sustained neutralizing antibodies and T cell responses, as well as memory B cells and long-lived plasma cells observed by day 210 post-immunization. Tri-Vac also serves as a candidate booster for enhancing immunity after three doses of inactivated virus or mRNA vaccines. A phase 1 investigator-initiated trial was initiated to assess safety and immunogenicity in humans, focusing on the primary endpoint of adverse reactions within 7 days and key secondary endpoints including the geometric mean titers (GMTs) of serum neutralizing antibodies within 30 days and 6 months post-vaccination, as well as adverse events within 30 days and serious adverse events within 6 months post-vaccination. Preliminary data indicate Tri-Vac has good safety and immunogenicity, improving neutralization against multiple variants, including JN.1, in previously vaccinated individuals, highlighting its clinical potential for protecting against SARS-CoV-2 variants. The registration number of this clinical trial is ChiCTR2200067245.