Cross-Reacting Material 197 (CRM197), a non-toxic mutant of diphtheria toxin, is structurally similar and immunologically cross-reactive with the native toxin. It is extensively utilized as a carrier protein in conjugate vaccines to enhance T-cell-dependent immunity and also displays antitumor properties via interaction with heparin-binding epidermal growth factor (HB-EGF). Nevertheless, the structural instability of CRM197 restricts its broader use, causing accelerated in vivo degradation, diminished immunogenicity, and challenges in recombinant expression and purification. Here, we engineered a CRM197 mutant with enhanced stability and superior immunogenicity. As a diphtheria immunogen or conjugate carrier, it elicits rapid, potent, and sustained immunity surpassing the wild-type, and maintains effective HB-EGF-binding activity for tumor growth inhibition. Structural insights reveal a rigidified activation site loop (residues 38-52), a stabilized receptor domain through additional hydrogen bonds at site 511, and strengthened adjuvant adsorption from an increased negative charge as the underlying mechanisms.

Reverse vaccinology has enabled sequence-based antigen discovery, but it overlooks the rich semantic knowledge embedded in the biomedical literature. Here we establish Semantic Vaccinology (SemVac), a paradigm that leverages large language models (LLMs) to predict protective antigens directly from scientific text. Benchmarking 14 state-of-the-art LLMs on a curated antigen dataset shows that text-reasoning-based approaches match or exceed specialized deep learning models in precision, while offering superior robustness on functionally ambiguous proteins. Intriguingly, explicit reasoning modes (e.g., chain-of-thought) increase recall but consistently reduce precision, revealing an over-reasoning pitfall in biological discovery tasks. Applied to the complete proteome of Mpox virus, SemVac recapitulates known protective antigens and identifies previously unrecognized candidates such as B20R, which our semantic analysis links to immune evasion and structural exposure. This work establishes literature-driven semantic reasoning as a powerful complement to conventional vaccinology, with broad implications for AI-aided scientific discovery.

Abstract Therapeutic antibodies are challenged by rapidly evolving pathogens that exploit glycosylation to shield epitopes. SARS-CoV-2 JN.1 exemplifies this, escaping antibodies through the N354-linked glycan. However, targeting glycosylated epitopes remains vacant, as scarce and heterogeneous glycan structures render existing approaches ineffective. Here, we introduce the Antibody Evolution Nexus with Causal-Driven Simulation (AENCS), integrating molecular simulation with causal inference. Applying AENCS to restore S309 efficacy against JN.1, we identified ACC01, exhibiting ∼24-fold improved neutralization. With limited prior knowledge of the N354 glycosylation site, ACC01 stabilized this glycan conformation, facilitating the determination of its cryo-EM structure. Causal dissection revealed how this glycan shield is functionally inverted into a binding anchor through multi-layered interactions. This mechanistic conversion, combined with the conservation of N354 glycosylation, enabled ACC01 to maintain potent activity against the latest variant NB.1.8.1. Collectively, AENCS demonstrates causal-driven antibody engineering can illuminate cryptic glycosylated epitopes, providing viable paradigms for exploring this vacant frontier.

Viral vectors and protein nanoparticles represent important platforms in vaccine delivery, yet their potential synergy to overcome fundamental biological barriers remains largely unexplored. To address the challenge of pre-existing immunity against viral vectors, we rationally engineered a cationic human ferritin nanoparticle, termed (+)hF, designed to electrostatically assembles with adenovirus serotype 5 (Ad5) into a supramolecular complex. This construct was generated via structure-guided point mutations that introduced positive surface charges while retaining the innate self-assembly capability of the ferritin nanocage. The resulting (+)hF formed nanocomplexes with Ad5, which enhanced transgene expressions both in vitro and in vivo and effectively reduced sensitively to anti-Ad5 neutralization. In murine models with pre-existing immunity, intranasal immunization with (+)hF-complexed Ad5-based vaccines (encoding antigens for SARS-CoV-2 or Hendra virus) elicited significantly higher antigen-specific IgG and neutralizing antibody titers. A key advantage of (+)hF is its endogenous origin, which ensures high biocompatibility by preventing the induction of anti-carrier antibodies and any associated immune burden. This study presents a new paradigm that engineers programmable biointerfaces to synergize viral vectors with protein nanocages, offering a general strategy to circumvent critical biological barriers for enhanced vaccine and gene delivery.

Brucellosis is a widespread zoonotic disease caused by Brucella, which is a facultative intracellular pathogen. Brucellosis poses a significant challenge to vaccine development due to the ability of Brucella to evade innate immunity, primarily through its atypical and low-toxicity lipopolysaccharide (LPS). To improve the suboptimal immunogenicity of subunit vaccines, a pathogen-mimicking nanovaccine was engineered to deliver the antigen and monophosphoryl lipid A (MPLA, a detoxified LPS analogue) in the present study. A fusion protein (SO) of the outer membrane protein 19 (Omp19) and Cu/Zn superoxide dismutase (SOD) acted as the antigen. SO was conjugated with an octaarginine peptide to absorb anionic MPLA (a TLR4 agonist), followed by self-assembly into uniform nanoparticles by electrostatic and hydrophobic interactions. The resultant vaccine (SOMs) enhanced the uptake by APCs, promoted the maturation of dendritic cells in vitro, and effectively activated the TLR4 signaling pathway. Immunization with SOMs induced a robust and balanced immune response in BALB/c mice, as reflected by substantially elevated antigen-specific IgG1 and IgG2a antibody titers and potent cellular immunity. The vaccine protected BALB/c mice against Brucella melitensis strain M5, along with no apparent systemic toxicity. This work validates a pathogen-mimicking vaccine strategy that counteracts the immune stealth of Brucella by replenishing critical TLR4 signaling. This strategy is promising for developing effective vaccines against intracellular pathogens.

BackgroundHendra virus (HeV) is a bat-adapted zoonotic henipavirus belonging to the Paramyxoviridae family. It is classified as a biosafety level 4 (BSL-4) pathogen owing to its broad host range and high fatality rate. Currently, no vaccines or therapeutics are approved for human use. Viral entry is mediated by the attachment (G) and fusion (F) glycoproteins; the heavily glycosylated G protein is responsible for receptor binding.MethodsThe extracellular domain of HeV-G was expressed in Expi293F cells and its glycosylation sites and glycan composition were identified by mass spectrometry. A series of functional assays-including viral entry, receptor binding, cell-cell membrane fusion, antibody neutralization and immunogenicity-were performed to delineate the role of each N-linked glycosylation site.ResultsGlycan profiling of HeV-G identified seven N-linked and multiple O-linked glycosylation sites, revealing that the stalk residues (N72, N159) predominantly carry high-mannose glycans, whereas the head-domain N-glycan sites (N306, N378, N417, N481, N529) are primarily modified with complex glycans. Notably, among the head-domain sites, N481 also harbors a substantial proportion of high-mannose glycans. Functional assays revealed that removal of N-glycans at N159, N306 and N417 markedly reduced membrane fusion. The N159 residue is a key site for fusion triggering, and its function is tolerant to specific amino acid substitutions, which may contribute to stabilizing or facilitating the conformational cascade required for F protein-mediated membrane fusion. The N529Q mutant specifically decreased EB3 binding by 2.6-fold, correlating with reduced infectivity. Binding assays with neutralizing antibodies showed that most N-glycan deletions had negligible effects, except that N159Q and N481Q reduced affinity to nAH1.3. Immunization studies in mice demonstrated that N-glycans had minimal impact on humoral immunity, with only minor site-specific differences.ConclusionThese findings provide a comprehensive characterization of HeV-G glycosylation, reveal site-specific roles of N-glycans in viral entry, receptor binding and membrane fusion, and offer new insights for vaccine and antibody development.

The invasion of host cells by the henipavirus is facilitated through the interaction between viral attachment (G) and fusion (F) glycoproteins with receptors on the cell surface. Langya henipavirus (LayV) was newly identified in China in 2022. The G proteins of LayV and Mojiang virus (MojV) exhibit high amino acid homology (86%), while they are located in a unique evolutionary clade within the Henipavirus genus. In this study, the crystal structure of the LayV G protein was resolved at a 3.37 Å resolution, revealing a head domain with six β-propeller-like domains distinct from other henipavirus G proteins, such as those of Nipah virus (NiV) and Hendra virus (HeV). Furthermore, the prominent loop in the center cavity of the LayV G protein showed unique structural features. In the ELISA and SPR assays, the LayV G protein was unable to bind to the existing henipavirus-neutralizing antibodies or the ephrin-B2 receptor. Immunogenicity studies in mice demonstrated robust antibody responses elicited by the LayV G protein. These antibodies exhibited strong reactivity against both LayV and MojV G proteins. However, only weak cross-reactivity was observed with other henipaviruses. Moreover, eight monoclonal antibodies targeting the LayV G protein were generated, two of which exhibited broad binding activity across different henipavirus G proteins. These findings underscore the need for tailored vaccines and therapeutics for LayV and related novel henipaviruses.

The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants highlights the need to update coronavirus 2019 disease (COVID-19) vaccine components. Epitope-based vaccine designs targeting conserved and immunorecessive regions of SARS-CoV-2 are critically needed. Here, we report an engineered epitope-focused immunogen design based on a novel horseshoe-shaped natural protein scaffold, named ribonuclease inhibitor 1 (RNH1), that can multiply display of conserved neutralizing epitopes from SARS-CoV-2 S2 stem helix. The designed immunogen RNH1-S1139 demonstrates high binding affinity to S2-specific neutralizing antibodies and elicits robust epitope-targeted antibody responses either through homologous or heterologous vaccination regimens. RNH1-S1139 immune serum has been proven to have similar binding ability against SARS-CoV, SARS-CoV-2 and its variants, providing broad-spectrum protection as a membrane fusion inhibitor. Further studies showed that RNH1 has the potential to serve as a versatile scaffold that displays other helical epitopes from various antigens, including respiratory syncytial virus (RSV) F glycoprotein. Our proposed immunogen engineering strategy via tailored horseshoe-shape nano-scaffold supports the continued development of epitope-focused vaccines as part of a next-generation vaccine design.

Lassa virus (LASV) belongs to the Arenaviridae family and causes severe hemorrhagic fever in humans. Although many vaccine candidates for Lassa fever exist, no vaccines have been approved for clinical use currently. The precursor glycoprotein complex (GPC), which is expressed as a trimer on the viral surface, is the main target for vaccine development. However, it has been a significant challenge to elicit effective neutralizing antibodies against LASV. In this study, we designed and produced a prefusion GPC trimer antigen of LASV, named GPCv2. Based on the structural information of GPC, we made modifications by replacing the amino acid at position 328 with proline and appending the trimerization domain. This resulted in a highly expressed prefusion trimeric form of GPCv2 that retained important conformational epitopes and stimulated higher levels of neutralizing antibodies. Moreover, vaccination with GPCv2 protected mice from LASV pseudovirus challenge. Additionally, immune repertoire sequencing showed that the induced immune clones in the trimeric group were more convergent and has its own unique V-J pairing bias compared with monomeric group. These findings demonstrate the potential of GPCv2 as a promising candidate antigen for an effective vaccine against LASV.

Antigen-encoding mRNA has emerged as a potent approach to prevent or treat various diseases, yet its clinical application necessitates consideration of reactogenicity and tolerability issues associated with existing delivery systems. The development of novel LNP-alternative delivery platforms represents a critical endeavor. Here, by screening and modifying functionalized motifs of an endogenous retrovirus-like protein, we developed a single-component engineered peptidyl-based vehicle, H3M1-5, which enables desirable biocompatibility, flexible modularity, and efficient mRNA delivery both in vitro and in vivo. H3M1-5 mediated potent delivery of linear mRNA, circular mRNA, and plasmid DNA, with an efficacy comparable to that of the leading commercial transfection reagent. The peptidyl-based mRNA vaccines efficiently facilitated germinal center responses and elicited robust immune responses. Notably, H3M1-5@M1R mRNA significantly protected mice in a lethal challenge model of orthopoxvirus, and H3M1-5@OVA mRNA delayed tumor growth in both prophylactic and therapeutic B16-OVA melanoma models. Importantly, in contrast to LNP formulations, the peptidyl-based mRNA vaccines didn’t trigger a detectable pro-inflammatory response and had a restricted transgene expression in the injection site. This work demonstrates a promising biomimetic strategy for designing simple and efficient peptidyl-based vehicles for mRNA vaccines with favorable safety profiles.

Although vaccines have been designed to be safe and effective, it still takes years to move vaccine development from benchside to bedside. Novel strategies are needed to accelerate the vaccine design process. Artificial intelligence (AI) has started to play an important role in the life science industry. In this review, we summarized progress of AI-assisted vaccine designs in three areas, including de novo protein subunit design that focuses more on the immunogenic part of the antigen, viral vectored-based vaccine innovation that combats pre-existing immunity, and mRNA vaccine platform optimization that improves stability and translational efficiency. Additionally, we also address the multifaceted challenges associated with integrating AI into vaccine development, encompassing technical, ethical, and regulatory considerations, and offer strategic recommendations to navigate these complexities. AI-driven methodologies hold the promise of revolutionizing next-generation vaccine design by improving safety profiles, stability, immunogenicity, and overall efficacy, thereby strengthening global preparedness against emerging infectious threats.

Vaccines are the most effective tool in preventing and managing infectious diseases. One of the critical challenges in vaccine development is the selection of suitable target antigens from the thousands of proteins produced by pathogens. Artificial intelligence is anticipated to play a significant role in addressing this challenge. In this study, we develop a framework termed PLGDL for protective antigen prediction that employs Protein Language and Geometric Deep Learning models. This framework leverages both primary sequence features and three-dimensional structural features of protein antigens, thereby reducing the biases associated with manually curated features. Our integrated model exhibits robustness across both constructed and public datasets and is applicable to viruses, bacteria, and eukaryotic pathogens. Notably, when applied to the ongoing Mpox outbreak, our model not only quickly identifies multiple known antigens but also discovers a protective antigen: G10R. Here, our study provides a high-performance screening tool for protective vaccine antigen prediction by synergistically utilizing the capabilities of protein language and geometric deep learning models, providing substantive insights and methodological advancements for rapid vaccine development.

Klebsiella pneumoniae (K. pneumoniae) has been detected on space stations. Microgravity is a key environmental factor in spaceflight, however, research on the phenotypic, genetic, and metabolic changes K. pneumoniae undergoes due to long-term exposure to microgravity is still limited. K. pneumoniae was cultured under normal gravity (NG) and simulated microgravity (SMG) for 56 days, showing phenotypic changes like slower growth, larger and rounder cell morphology, and increased biofilm formation in SMG. RNA sequencing analysis revealed that the DEGs were associated primarily with metabolic and growth pathways, including those involved in biofilm formation. Metabolomic analysis revealed changes in the activity of the phenylalanine metabolic pathway, which was the most significantly enriched pathway, due to the interaction between the mhp gene cluster and related DEMs. Co-expression network analysis revealed intricate relationships between DEGs and DEMs, notably in arginine and proline metabolism. This study provides insights into K. pneumoniae's response mechanisms to microgravity.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic henipaviruses within the Paramyxoviridae family, causing severe respiratory and neurological diseases in humans and animals with fatality rates up to 75%, and no licensed human vaccines or therapeutics. In this study, we identified a unique vulnerable epitope on the NiV attachment glycoprotein (G) recognized by the potent neutralizing antibody 14F8, which targets a receptor-binding site and neutralizes NiV effectively. Using the 2.8 Å crystal structure of the 14F8 Fab–NiV-G complex as a guide, we reconstructed this epitope on HeV-G via a single amino acid substitution (S586N), creating the HeV-GS586N mutant. Immunization with HeV-GS586N in BALB/c mice and cynomolgus monkeys elicited robust, broadly neutralizing antibody responses against both NiV and HeV, achieving higher NiV-neutralizing titers post-prime compared to wild-type HeV-G, as confirmed by pseudovirus and live-virus assays. Crystal structures of HeV-GS586N (3.3 Å) and its 14F8 complex (3.2 Å) showed the S586N substitution induced a 9 Å conformational rearrangement in β-propeller blade 6, reshaping the molecular skeleton and solvent-accessible surface without direct N586–14F8 interaction, thus mimicking the NiV epitope. These findings position HeV-GS586N as a promising broad-spectrum antigen for henipavirus prevention and demonstrate the value of structure-guided epitope reconstruction in universal vaccine design for emerging viral threats.

Trained immunity serves as a de facto memory for innate immune responses, resulting in long-term functional reprogramming of innate immune cells. It enhances resistance to pathogens and augments immunosurveillance under physiological conditions. Given that innate immune cells typically have a short lifespan and do not divide, persistent innate immune memory may be mediated by epigenetic and metabolic changes in long-lived hematopoietic stem cells (HSCs) in the bone marrow. HSCs fine-tune their state and fate in various training conditions, thereby generating functionally adapted progeny cells that orchestrate innate immune plasticity. Notably, both beneficial and maladaptive trained immunity processes can comprehensively influence HSC state and fate, leading to divergent hematopoiesis and immune outcomes. However, the underlying mechanisms are still not fully understood. In this review, we summarize recent advances regarding HSC state and fate in the context of trained immunity. By elucidating the stem cell-intrinsic and extrinsic regulatory network, we aim to refine current models of innate immune memory and provide actionable insights for developing targeted therapies against infectious diseases and chronic inflammation. Furthermore, we propose a conceptual framework for engineering precision-trained immunity through HSC-targeted interventions.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

>Dear Editor,In the era of COVID-19, the prevalence of viruses from other families poses new threats to humans. Despite the eradication of smallpox in 1980, the global outbreak of monkeypox in 2022(Lum et al., 2022; Nuzzo et al., 2022) alerted that the re-emerging orthopoxvirus(OPXV)remained a persistent threat to global health. OPXV includes a number of viruses with a broad host range, including variola virus(VARV), monkeypox virus(MPXV), vaccinia virus(VACV),

The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.

The Junín virus (JUNV) is one of the New World arenaviruses that cause severe hemorrhagic fever. Human transferrin receptor 1 (hTfR1) has been identified as the main receptor for JUNV for virus entry into host cells. To date, no treatment has been approved for JUNV. Herein, we investigated 12 anti-hTfR1 VHH (variable domain of the heavy chain of heavy-chain antibody) antibodies and confirmed their interaction with hTfR1. Most of them could bind to the hTfR1 apical domain, which is the glycoprotein 1 (GP1) binding domain of JUNV. Among them, 18N18 exhibited neutralizing activity against both the human immunodeficiency virus (HIV)-vectored lentiviral Junín pseudoviruses and the recombinant vesicular stomatitis virus (VSV)-vectored Junín pseudoviruses. We also verified that 18N18 blocked the interaction between hTfR1 and JUNV GP1. In addition, 18N18 could neutralize another New World arenavirus, the Machupo virus. Using AlphaFold 3-based simulation of 18N18–hTfR1 docking, we determined that 18N18’s binding epitope was located at the JUNV GP1 binding epitope. 18N18 represents a candidate for JUNV treatment and provides a potential approach that could be applied to New World arenaviruses.

Background Lassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited. Methods To enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences. Results The results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV. Conclusion This study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.