Search Results (423)

Apolipoprotein E4 (apoE4) is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). Yet the molecular mechanisms underlying its contribution to AD remain to be fully elucidated. Here, we developed and characterized a set of apoE-specific single-domain antibodies (nanobodies) as a molecular toolbox to investigate intracellular apoE4. The nanobodies bind human apoE with nanomolar to sub-nanomolar affinity and recognize both apoE3 and apoE4. Domain-level epitope mapping revealed nanobodies that selectively bind either an N-terminal (residues 1–173) or C-terminal (residues 170–299) apoE4 fragment. Several nanobodies were validated as endoplasmic reticulum-targeted intrabodies that bind apoE4 intracellularly and promote its intracellular retention. These nanobodies constitute a versatile toolbox for probing and manipulating apoE4 in cellular models. As an exploratory application of this nanobody toolbox, we examined cytosolic apoE4, motivated by previous studies suggesting that cytosolic apoE4 fragments may influence AD-related cellular processes. Cytosolic expression of apoE4 resulted in perinuclear protein assemblies and the appearance of a ~25 kDa apoE4 fragment. Using a nanobody-based nuclear relocalization assay, we showed that cytosolic apoE4 remains accessible for nanobody binding but was not relocated to the nucleus by a nuclear localization signal-equipped nanobody. Altogether, this study introduces a nanobody-based toolbox to investigate apoE4 in distinct intracellular contexts, which can be relevant to AD. Full article

Background: Brucella outer membrane proteins (Omps) are an important part of its cell wall and major virulence-related factors. Omp16 and Omp19 proteins are the advantageous antigens of Brucella and have been widely used in research on vaccines against brucellosis. As an emerging vaccine, the mRNA vaccine has unique advantages in the fight against intracellular parasitic bacteria. Methods: In this study, mRNA encoding the omp16 and omp19 genes of Brucella. melitensis (B. melitensis) was synthesized using in vitro transcription. The target mRNA was transfected into HEK 293T cells to evaluate protein expression levels and assess its immunogenicity. Finally, bioinformatic approaches were employed to analyze potential antigenic epitopes. Results: In this study, the successfully constructed recombinant plasmids pIVTRup-omp16 and pIVTRup-omp19 were utilized to synthesize omp16-mRNA and omp19-mRNA, each approximately 600 nt in length. Western blot analysis detected the expression of proteins with molecular weights of 16 kDa and 19 kDa in HEK 293T cells at 24 h post-transfection with mRNA. Purified rOmp16 and rOmp19 had good immunogenicity, which could specifically bind to serum antibodies of brucellosis patients. rOmp16 had stronger immunogenicity than rOmp19. Epitope prediction showed that Omp16 contained seven epitopes and Omp19 contained six epitopes. In addition, Omp16 and Omp19 could form stable complexes with target receptors. Simulated immunization with Omp16 and Omp19 proteins significantly activated both CD4⁺ and CD8⁺ T cells. Conclusions: The immunogenic proteins were successfully expressed in cells based on the mRNA fragments synthesized from omp16 and omp19 genes of B. melitensis, which was a preliminary exploration for the preparation of B. melitensis mRNA vaccine. Full article

Mycoplasma pneumoniae (M. pneumoniae) is the crucial factor of global acquired respiratory infections. Currently, there are no specific disease modification treatments or vaccines available, and the vaccine development for this pathogen lags behind due to the complexity and variability of its antigens. A novel vaccine with broad-spectrum characteristics is essential to provide comprehensive protection against continuously evolving wild-type strains. Here, a broad-spectrum muti-epitope vaccine against M. pneumoniae had been designed through immunoinformatics methods. To ensure its broad-spectrum, we generated consistent sequences from all the antigen proteins of different strains, and then identified potential T cell epitopes. The multi-epitope vaccine (MEV) of M. pneumoniae incorporated 16 CTLs and 7 HTLs from the HMW1–3 and p1 adhesin proteins, which comprised 458 amino acids with adjuvant. The vaccine evaluation showed that the MEV had ideal physicochemical properties, high antigenicity, high immunogenicity, and was non-toxic. Furthermore, there was a strong and stable binding interaction between this vaccine and the toll-like receptors, which could be supported by the normal mode analysis. Finally, codon optimization resulted in the optimal GC content and higher CAI value. The vaccine candidate is expected to induce strong cellular immune responses and may provide protective immunity against the pathogen. We provided a novel in silico vaccine design strategy for vaccine design, which could provide a technical framework for the development of vaccines against other pathogens. Full article

Carbapenemase-producing bacteria, particularly those expressing the KPC-3 variant, pose a critical global health threat due to their resistance to nearly all β-lactam antibiotics, including carbapenems. Rapid and reliable detection tools are urgently needed to improve infection control and guide patient management. Nanobodies (VHHs) present a promising alternative to conventional antibodies thanks to their high stability, small size, and capacity to access cryptic epitopes. Here, we report the generation and characterization of a nanobody specifically targeting KPC-3. An immune VHH phage display library was constructed, with over 90% of clones containing correctly sized inserts. After three rounds of biopanning, high-specificity binders were identified by ELISA screening. Sequencing identified a nanobody with hallmark VHH features, which was expressed and validated by ELISA and Western blot. Although kinetic assays showed no inhibition of KPC-3 enzymatic activity, interestingly, the nanobody demonstrated high-binding recognition of both KPC-2 and KPC-3 in periplasmic extracts from clinical strains. Structural modeling further supported these results, highlighting favorable interaction surfaces. This study provides the first evidence of a nanobody raised against KPC-3 that recognizes a conserved epitope shared by KPC-3 and KPC-2, underscoring its promising use as a molecular tool for detecting KPC variants and establishing a basis for future affinity maturation toward therapeutic applications. Full article

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies. Full article

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing. Full article

Background/Objectives: Polysaccharide-protein conjugate vaccines have proven highly effective, yet they remain limited by manufacturing complexity, cost, and variable performance across serotypes, while carrier proteins can add unwanted immunological and production burdens. To address these constraints, we explored a carrier-protein-free conjugate vaccine concept in which a broadly MHC class II-binding helper epitope (PADRE) replaces the conventional protein carrier to provide T-cell help for a pneumococcal capsular polysaccharide antigen. Methods: Using serotype 15C CPS as a model, we generated CPS–PADRE conjugates and compared designs with or without a putative cleavable motif (RR) at the junction, alongside a conventional protein conjugate as a benchmark. Results: In mice, the CPS–protein conjugate induced the strongest CPS-specific IgG response, whereas CPS–PADRE conjugates elicited clear but overall lower antibody levels. Notably, incorporation of the cleavable motif did not improve immunogenicity and instead reduced humoral responses relative to the non-cleavable design. Conclusion: These findings support the feasibility of carrier-protein-free polysaccharide-peptide conjugate vaccines, while highlighting that cleavable junctions are not universally advantageous and must be empirically optimized for polysaccharide-helper epitope architectures. Full article

The continuous evolution of SARS-CoV-2 affects its infectivity and ability to evade the immune system. The XBB.1.5 subvariant carries numerous mutations compared to previous Omicron variants and exhibits significant evasion of polyclonal neutralizing antibodies. In this study, the mechanistic effects of mutations in the XBB.1.5 spike protein on structural stability, antigenic markers, and antibody epitopes were analyzed using homology modeling, epitope prediction, protein stability analysis, coarse-grained dynamic simulations, and chain-specific interface mapping. Thirty-eight amino acid substitutions were identified relative to Wuhan-Hu-1, including 22 in the receptor-binding region. The prefusion trimeric fold was conserved, with localized rearrangements in the N-terminal domain, receptor-binding domain, and S1/S2 region. Linear B-cell epitope prediction yielded similar epitope counts and length distributions in wild-type and XBB.1.5, but only moderate residue-level overlap (Jaccard ≈ 0.40–0.62), indicating epitope turnover and alteration of four conserved antigenic determinants. Functional screening suggested that ~45% of substitutions could affect protein function. Chain-specific interface analysis of the A–B protomer interface indicated preserved inter-protomer coupling with modest repacking of the polar/directional contacts. Overall, XBB.1.5 appears to maintain ACE2 engagement while redistributing antibody targets, underscoring the need for updated vaccine formulations and therapeutic antibodies. Full article

Canine distemper virus (CDV) remains a highly contagious and lethal pathogen, posing a severe global threat to domestic dogs and wild carnivores. To address the urgent need for effective interventions, we utilized a proprietary Vero-SLAM cell platform to isolate a wild-type CDV strain and generate neutralizing polyclonal antibodies. Subsequently, phage display technology was employed to screen for single-chain variable fragments (scFvs) targeting the CDV hemagglutinin protein (CDV-H). This approach led to the identification of a specific scFv with virus-binding affinity comparable to commercial antibodies, which effectively blocks CDV infection in Vero-SLAM cells. Molecular docking and molecular dynamics simulations were conducted to elucidate the interaction mechanism, suggesting that this scFv binds to a novel and unique epitope on the CDV-H. These findings not only expand our understanding of the antigenic properties of the CDV H protein but also provide a theoretical foundation and a promising candidate molecule for the development of future CDV diagnostics and antiviral strategies. Full article

Nanobodies (VHHs or single-domain antibodies) are powerful affinity reagents, but their routine use is often limited by production constraints and by the lack of a conserved Fc region for secondary detection. We describe a simplified strategy in which functional GST–nanobody fusion proteins are expressed directly in the cytoplasm of Escherichia coli Origami^TM 2 (DE3), a strain that supports disulfide bond formation through trxB/gor mutations. Using well-characterized nanobodies against GFP (Lag2) and mCherry (C11), we designed N-terminal GST fusions and confirmed by AlphaFold3-based modeling that both constructs preserve the GST fold and the VHH (Variable domain of the Heavy-chain antibody of Heavy-chain-only antibodies) β-sandwich with defined CDR loops and a predicted intradomain disulfide bond. Following IPTG induction and purification by glutathione affinity and size-exclusion chromatography, we obtained soluble GST-nb-GFP and GST-nb-mCherry at ~8–12 mg/L. Isothermal titration calorimetry showed nanomolar binding to their antigens (K_d ~123 nM for GFP and ~199 nM for mCherry). Consistent with conformational epitope recognition, GST-nanobodies were reactive in native-state dot blots but not in denaturing Western blots under the conditions tested. The GST moiety enabled indirect immunofluorescence via anti-GST antibodies, yielding specific labeling of GFP- or mCherry-tagged TGN38 in HeLa and H4 cells. Finally, we demonstrate “GST-nanobody pulldown” as a robust method for affinity capture from cell lysates. Together, this platform provides a low-cost, versatile route to functional nanobody reagents without requiring tag removal, and complements other nanobody designs (e.g., VHH-Fc fusions) in an application-dependent manner. Full article

The broadly neutralizing monoclonal antibody 3D1 potently neutralizes SADS-CoV by targeting a conserved epitope within the heptad repeat 1 (HR1) domain of the viral spike protein. Structural and biophysical analyses demonstrate that 3D1 binds with high affinity to a specific linear β-turn motif (residues A804–N809) in HR1. High-resolution crystallography reveals that this motif sits within a deep, electrostatically complementary paratope groove. Critically, 3D1 binding competitively inhibits the essential interaction between HR1 and HR2. Notably, its recognition is not dependent on HR1’s native helical conformation, as it maintains strong binding to conformationally constrained, stapled helical peptides. Collectively, the data indicate that 3D1 neutralizes by capturing a pre-hairpin intermediate state of HR1—a transition state between prefusion and postfusion forms—thereby sterically blocking the formation of the stable postfusion six-helix bundle that is essential for membrane fusion. This work defines a precise, structure-dependent neutralizing epitope and elucidates a mechanism of action that involves trapping a key fusion intermediate, offering a valuable template for the design of broad-spectrum coronavirus therapeutics. Full article

Background: Norovirus GII.4 is a major global health threat, yet no licensed vaccines exist due to the virus’s rapid evolution and high mutation rates. Objective: To rationally design and experimentally validate multi-epitope vaccine candidates against Norovirus GII.4 using computational immunoinformatics and in vivo evaluation. Methods: We employed reverse vaccinology to screen optimal norovirus GII.4 epitopes and systematically designed four construction strategies to evaluate different epitope topologies and adjuvants. Candidates underwent molecular dynamics simulations and were expressed in E. coli. Immunogenicity was assessed in BALB/c mice via ELISA and ELISPOT to evaluate humoral and cellular responses. Results: Three candidates (NV1, NV4, NV5) were successfully produced and induced cross-reactive antibodies against authentic GII.4 virus-like particles. Notably, the construction strategy influenced the immune response: NV5 (repetitive epitopes and HSP as adjuvant) elicited the highest antigen-specific antibody titers, NV1 (all types of epitopes and TLR as adjuvant) induced the strongest cellular response, and NV4 (repetitive epitopes and TLR as adjuvant) achieved the most rapid immune response. Consistently, in silico analysis showed that the NV1-TLR3 complex exhibits tighter interaction, higher binding energy, and greater structural stability, supporting its superior capacity to trigger cellular immunity. Conclusions: A rational multi-epitope vaccine design workflow successfully realized the translation from computational design to functional vaccines. Optimizing adjuvant selection and epitope construction is critical for eliciting immune responses in next-generation norovirus vaccines. Full article

This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel^®, AH; Adju-phos^®, AP; and CpG-1018^®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing. 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: Norovirus is a leading cause of epidemic acute gastroenteritis worldwide, associated with significant morbidity, mortality, and economic loss. Despite its global impact, no licensed vaccine is currently available, and vaccine development remains challenging. Methods: We explored avian immunoglobulin Y (IgY) antibodies as a low-cost countermeasure against norovirus infection. We generated recombinant protruding (P) domain proteins from the capsid protein (VP1) of noroviruses, representing two GII.4 variants and the GII.6 genotype. These were combined into a single immunogen to immunize laying hens to produce norovirus VP1-specific IgY antibodies. Results: Immunization of laying hens with the P domain proteins elicited high-titer (>1:450,000) P domain-specific IgY antibodies. The yolk-derived IgY effectively inhibited binding of various norovirus P particles to their histo-blood group antigen ligands, with 50% blocking titers (BT50) up to 1:8533 against homotypic GII.4 and 1:667 against heterotypic G1.1 Norwalk virus P particles. Importantly, the IgY neutralized replication of GII.4 norovirus in the human intestinal enteroid (HIE) system at a high titer of over 1:2500, equivalent to 0.70 µg/mL of total IgY. We also produced norovirus shell (S) domain proteins and corresponding IgY antibodies, which neutralized GII.4 norovirus replication in the HIE model at a titer of ~1:800, equivalent to 2.98 µg/mL of total IgY. This provides the first evidence that the S domain contains neutralizing epitopes. Conclusions: Our findings support the potential of IgY targeting norovirus P or S domains as a scalable, cost-effective strategy for preventing norovirus infection and disease. Full article