Search Results (516)

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity. Full article

This study characterizes AP-20-A, a lytic podovirus infecting Sinorhizobium meliloti, isolated from agricultural chernozem. Its 49.4 kbp genome shows negligible intergenomic similarity with known rhizobiophages (<2%). Core structural proteins—the major capsid protein (MCP) and terminase large subunit (TerL)—show closest homology to podoviruses infecting Paenibacillus, rather than to alphaproteobacterial viruses, suggesting cross-phylum horizontal gene transfer. This exchange is ecologically plausible, as Paenibacillus and Sinorhizobium co-exist in the rhizosphere. Over 63% of predicted proteins are functionally uncharacterized, with structural homologs detected in bacteria, archaea, and eukaryotes. We report the first identification in a rhizobiophage of a Tad2-like domain, predicted to block the bacterial Thoeris type II anti-phage defense. AP-20-A infected 56% of native S. meliloti strains; agrocenose isolates showed higher resistance than phytocenose isolates, evidence of local co-evolution. Among susceptible strains, 60% entered putative pseudolysogeny (with one strain exhibiting growth stimulation), whereas a symbiotically elite inoculant strain was completely lysed within hours. Some host strains carry additional AbiE systems; whether these independent defense–counterdefense layers interact during infection remains unknown. We conclude that resident phages represent a selective force that can disrupt inoculant establishment, underscoring the need to integrate soil virome assessment into agricultural microbiome management. Full article

Biofilms are structured communities of microorganisms embedded in a self-produced extracellular polymeric substance (EPS) matrix, whose development significantly enhances microbial resistance to antibiotics, disinfectants, and host immune defenses, posing major challenges in clinical, industrial, and environmental settings. Compared with planktonic cells, biofilm-associated microorganisms can exhibit up to 10- to 1000-fold increased tolerance to antimicrobial agents, contributing to the persistence of biofilm-associated infections (BAIs). These infections remain difficult to eradicate due to reduced penetration, altered metabolic states, and the presence of dormant or persister cells. Anti-biofilm strategies can be broadly classified into physical approaches (e.g., ultrasound, mechanical stress, and light-based approaches) that target biofilm structure; chemical and enzymatic methods (e.g., EPS-degrading enzymes) that destabilize the matrix; and biological and molecular strategies (e.g., quorum-sensing (QS) inhibitors, anti-virulence agents, bacteriophages, phage-derived antimicrobial molecules, antimicrobial peptides, and natural bioactive compounds) that modulate biofilm development and integrity by targeting regulatory pathways and matrix stability through distinct mechanisms of action. Natural compounds, including lactoferrin, lactoferrin-derived peptides, and probiotic and postbiotic fractions of lactic acid bacteria (LAB), as well as plant-derived metabolites, have shown promising anti-biofilm effects, with efficacy often enhanced through complementary or potentially synergistic interactions. However, despite these advancements, clinical translation remains limited. For example, BAIs account for approximately 80% of chronic infections, with high recurrence rates and therapeutic failure reported in device-associated infections and chronic wounds. These limitations highlight the need for clinically translatable, multimodal approaches that integrate structural biofilm disruption, antimicrobial targeting, and host response modulation to design more effective and sustainable anti-biofilm strategies. Full article

Considering the therapeutic potential of the Proteus mirabilis PM16 podophage, the interaction between PM16, its host strain, and the mouse immune system was investigated. We evaluated how pre-existing humoral immunity to PM16 influences the immune response against P. mirabilis and the neutralization of the phage itself. Balb/c mice were divided into three groups and immunized two times with (1) 0.9% NaCl, (2) adjuvants, or (3) a mixture of PM16 and an adjuvant. Then, each group was subdivided into three subgroups: mock infection, infection with P. mirabilis, and infection with P. mirabilis followed by model phage therapy with PM16. The obtained results demonstrated that pre-immunization with PM16 enhanced the anti-P. mirabilis IgG antibody response upon bacterial challenge, indicating that the phage potentiates antibacterial immunity. In addition, pre-immunization elicited a significant anti-PM16 antibody response that resulted in in vitro neutralization of phage lytic activity. However, phage-neutralizing antibodies neither decreased the efficacy of phage therapy nor influenced bacteria-specific immune response. Thus, while PM16 can boost the host’s immune response against its bacterial host, the resulting humoral immunity also drives phage clearance through both direct and bacteria-mediated neutralization pathways, revealing a complex immunopharmacological relationship central to phage therapy. Full article

Background/Objectives: Antimicrobial resistance (AMR) in Escherichia coli is a growing public health concern, particularly in regions affected by environmental contamination and poor wastewater management. Data on locally isolated E. coli-targeting phages in Lebanon remain limited. This study aimed to isolate, characterize, and evaluate two lytic bacteriophages against AMR E. coli. Methods: Two phages, EPIMAM01 (gb:PQ493298) and EPIMRB01 (gb:PQ657784), were isolated from untreated sewage in Beirut using E. coli ATCC 25922. Characterization included double-layer agar assays, one-step growth analysis, and stability testing across temperature and pH ranges. Bacteriolytic activity was assessed in planktonic cultures and preformed biofilms. Host range and efficiency of plating (EOP) were evaluated using clinical isolates. Whole-genome sequencing and comparative analyses were performed. Results: Both phages produced clear plaques and showed a latent period of ~40 min. EPIMAM01 had a higher estimated burst size (140 PFU/infected cell) than EPIMRB01 (75 PFU/infected cell). Both phages remained stable between 4–50 °C and within a pH range of 5–10 but showed marked loss of activity at temperatures ≥60 °C and pH ≤3 or ≥12. EPIMAM01 effectively inhibited planktonic growth of E. coli ATCC 25922, whereas EPIMRB01 showed stronger biofilm-disrupting activity against preformed E. coli biofilms. Both phages lysed several of the 17 tested clinical E. coli isolates. Comparative analyses of gene presence/absence patterns, bacterial defense systems, and adsorption phenotypes among the tested E. coli strains identified mlaA, ydcQ, and ompD-2 as candidate adsorption-associated genes and suggested CRISPR systems may reduce susceptibility. Genomic analysis classified both phages as T4-like phages lacking lysogeny, virulence, or AMR genes. Conclusions: EPIMAM01 and EPIMRB01 are lytic phages with complementary antimicrobial properties, supporting their potential for further development as AMR control agents. Full article

The antimicrobial resistance crisis has led to the use of metals and bacteriophages as possible alternatives to antibiotics. Experimental studies have examined interactions between ionic/nano-silver and bacteriophages against multidrug-resistant bacteria. However, these approaches have often failed to examine whether silver affects the stability and infectivity of bacteriophages. Here, we utilized experimental evolution to evolve resistance to ionic silver in bacteriophage T7. High ionic silver concentrations that do not represent physiological exposure conditions were used to impose strong selective pressure. Evolution of ionic silver resistance in phage T7 was rapid, as evidenced by recovery of bacteriophage growth in E. coli following repeated exposures to ionic silver, enhanced infectivity of silver-selected populations relative to parallel control and ancestral populations under increasing ionic silver concentrations, and greater suppression of E. coli growth in standard medium. Furthermore, silver resistance evolved without loss of thermal or pH stability under the conditions tested. The genomic foundation of silver resistance was relatively simple, with positive and negative natural selection differentiating the silver-selected populations from the controls and ancestral populations across serial passages in silver. Support for replication-associated adaptation under ionic silver selection may be reflected in recurrent mutations identified in genes involved in transcription, DNA replication, and genome maintenance, including T7p07 (RNA polymerase), T7p10 (DNA ligase), and T7p29 (DNA polymerase I). These findings highlight the importance of evaluating phage –silver combination strategies within an evolutionary framework that accounts for the adaptive capacity of bacteriophages under silver selection. Full article

Bacteriophages (phages) represent promising therapeutic agents. Their clinical use is challenged by the rapid rise of resistant bacterial clones. To overcome this problem, phages can be trained in vitro to improve their ability to cope with the possible resistance that may arise. Here, we co-evolved phages with their hosts under different conditions and assessed their ability to infect an adapted bacterial panel using qPCR. The co-evolution experiment yielded a panel of bacterial clones adapted either to a phage, a competing phage, or a cocktail of both. Phages were adapted either in the continuous presence of an evolutionarily naïve host, in a cocktail with a competing phage, under both conditions, or under neither condition. We assessed each resulting phage for its ability to infect evolved bacterial clones in the panel we created, using qPCR for preliminary high-throughput assessment. This allowed us to evaluate 500 phages–bacteria interactions. Overall, qPCR-detected phage production following infection of different bacterial clones improved to varying degrees when evolutionary naïve hosts were added during the training. However, the screening suggests that optimal training conditions are phage-specific. For Enterobacter cloacae phages EC151 and EC152, the broadest increase in qPCR-estimated phage production in our experiments was observed when a competing phage and/or an evolutionarily naïve host was included during adaptation. For Stenotrophomonas maltophilia phages StM171 and StenM174, the presence of an evolutionarily naïve hosts appeared beneficial in both replicates; co-adaptation with a competing phage led to a complete loss of StM171 infectivity in both experiments but benefited StenM174. Phages passaged for ten passages consistently infected a broader range of bacterial clones than those sampled after five passages. Sequencing of eight EC152-derived phages identified recurring mutations in a transcriptional regulator, and in some cases, in baseplate and tail fiber genes. Full article

Background: The monkeypox virus (MPXV) has attracted considerable global attention due to its potential to cause widespread outbreaks, necessitating the development of rapid and accurate diagnostic methods of significant clinical importance. A29, a key envelope protein of MPXV, represents a promising diagnostic target. Methods: A novel monoclonal antibody, D10, was isolated from the human Tomlinson I+J phage display library by biopanning against the recombinant A29 protein. The D10 Fab fragment was expressed and purified, and its binding affinity was characterized by biolayer interferometry. Molecular docking was performed to predict potential interacting residues. Specificity and detection performance were evaluated by direct and competitive enzyme-linked immunosorbent assay (ELISA). Results: D10 possesses a unique complementarity-determining region sequence and exhibits strong binding affinity toward the A29 protein. Structural modeling analysis suggested potential interacting residues of A29, including Gln67, Arg74, Asn75, Arg81, and Asn84, which may primarily interact with Ser10, Thr5, Gly49, Gly47, and Glu97 in the heavy chain of D10. The binding affinity, determined by biolayer interferometry, showed a dissociation equilibrium constant of 6.44 nM, indicating strong binding capability. Furthermore, competitive ELISA demonstrated that D10 binds selectively to the A29 protein, with a half-maximal inhibitory concentration of 1.88 μg/mL and a limit of detection of 0.12 μg/mL. Conclusions: Overall, this monoclonal antibody provides a valuable tool for the immunological detection of MPXV and holds potential for future clinical diagnostic applications. Full article

The double-crested cormorant (Nannopterum auritum), a piscivorous bird endemic to North America, frequently forages in aquaculture ponds during migration and wintering, contributing to economic losses in catfish-producing regions of the southern United States. While interactions between cormorants and aquaculture systems are well documented, their associated microbial communities and genetic elements remain less characterized. In this exploratory study, Hi-C-enabled metagenomics was applied to fecal samples from two cormorants to generate a genome-resolved, descriptive analysis of gut microbial composition and to associate bacterial genomes with mobile genetic elements (MGEs), antimicrobial resistance genes (ARGs), and putative virulence-associated genes. Metagenome-assembled genomes (MAGs) included taxa reported in aquatic or animal-associated environments, including Edwardsiella tarda, Plesiomonas shigelloides, Clostridium perfringens, and Campylobacter volucris. ARGs were detected across multiple MAGs, with E. tarda harboring the greatest diversity. Hi-C-enabled linkage of plasmids and phages to putative hosts, providing structural insight into microbial organization. Analyses are descriptive (n = 2) and do not include statistical comparisons or diversity metrics. These findings demonstrate the utility of Hi-C for resolving gene–host associations and provide a framework for future studies of microbial connectivity in One Health contexts. Full article

Nanobodies have emerged as highly valuable biotherapeutic and diagnostic reagents due to their high specificity, low immunogenicity, and superior tissue penetration. However, traditional nanobody discovery methods rely on camelid immunization and phage display techniques, which are time-consuming and labor-intensive. Meanwhile, existing computational prediction methods for Nanobody–Antigen Interaction (NAI) suffer from several limitations: first, general Protein–Protein Interaction (PPI) models cannot adapt to the specific binding patterns of NAI; second, sequence-based models struggle to capture critical binding features, resulting in unsatisfactory prediction accuracy. To address these challenges, we propose an NAI prediction method based on Protein Language Models (PLMs). Specifically, a structure-aware PLM is first fine-tuned on PPI datasets to learn universal protein binding patterns. This model performs joint encoding of amino acid sequences and protein local structure-related sequences. It can implicitly learn spatial structural priors solely from sequence inputs, which alleviates the limitation of conventional sequence-based models in capturing structural binding characteristics. Subsequently, we use mean, max and min pooling to extract complementary global sequence features that a single pooling method cannot fully capture. We then apply voting fusion to reduce prediction bias and improve model robustness under class imbalance and small-sample scenarios. Evaluated on the NAI benchmark dataset constructed from SAbDab-nano, the proposed model outperforms the best baseline methods in key metrics including Accuracy, Recall, F1-score, AUC-ROC, and AUPR. It exhibits robust performance under class imbalance and small-sample scenarios, validating the effectiveness of the framework. Full article

Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here, we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed using X-ray crystallography. Functional effects on chromatin engagement were evaluated using an AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. The crystal structures of two Nbs (NbC03 and NbH10) confirmed there were canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and identifies one Nb capable of functionally disrupting the LEDGF–chromatin interaction. These Nbs serve as valuable tools for functional studies and structure-based drug design. Full article

Phage therapy has enormous potential in combating bacterial resistance in food animals. However, its application via the oral route remains limited due to challenges associated with the gastrointestinal tract (GIT) environment and a lack of rigorous clinical trial evidence. Therefore, we systematically searched in Google Scholar, PubMed, Scopus, and Web of Science databases following PRISMA guidelines and finally identified 111 articles on oral phage therapy in food animals from where we summarized the key physiological and chemical factors of the gut environment hindering the effectiveness of oral phage therapy (OPT), examined the methods used to evaluate phage stability in the GI environment, and highlighted potential strategies to mitigate these challenges. In addition, we performed quantitative analysis to visualize in vitro pH and thermal stability patterns of phages targeting bacteria isolated from food animals and variability in buffer and incubation period across stability studies. The GIT consists of several anatomically and functionally distinct segments, where complex interactions occur among digestive enzymes, gastric acids, electrolytes, commensal microbiota, and mucosal immune components. The acidic pH of the stomach is a major barrier to successful oral phage delivery. According to our analysis of pH stability testing data from the reviewed studies, most phages targeting antimicrobial-resistant bacteria in food animals remained stable at pH 5–9 and inactivated under highly acidic (pH ≤ 2) or highly alkaline (pH ≥ 11) conditions. In addition, phages are susceptible to high temperatures (above 60 °C), digestive enzymes (e.g., pepsin, trypsin, lipases), bile salts, and host immune responses. Several in vitro laboratory techniques are available to assess phage stability under simulated GI conditions, but variations occur in the assessment protocols. Microencapsulation using alginate and chitosan has been used to protect phages from the adverse GI environment. Additionally, enteric-coated capsules, antacids, co-encapsulation with acid-neutralizing agents, consumption of alkaline water, and daily phage administration are suggested to improve phage survival and efficacy. For the successful clinical implementation of OPT in food animals, future research should focus on elucidating the molecular and physicochemical determinants of phage stability, understanding the humoral immune response to OPT, standardizing laboratory protocol for assessing phage viability, improving the scalability of encapsulation methods, and exploring other potential delivery techniques. Full article

Background/Objectives: Malaria affects almost half of the world’s population and causes more than 600,000 deaths annually. Young children in malaria-endemic areas have the highest mortality rate because of their immature immune systems. Global efforts to control the disease have had limited success, with two WHO-recommended pre-erythrocytic malaria vaccines showing suboptimal efficacy; no vaccine has yet been approved against the blood stages of the parasite that causes the clinical symptoms of malaria. Therefore, there is an urgent need to identify new vaccine candidates against the parasite’s blood stages to achieve protection against the disease. Methods: Previous studies in our lab identified a few potential vaccine candidates expressed on the surface of malaria-parasite-infected RBCs using sera from disease-resistant children from malaria-endemic regions and a phage-displayed cDNA library generated from P. falciparum. In an innovative approach, we successfully immunized mice using live Plasmodium falciparum-infected red blood cells {Pf-iRBCs (L)}, the membrane fraction of P. falciparum-infected RBCs {Pf-iRBCs (M)}, and live uninfected human red blood cells (hRBCs) in suitable adjuvants. The polyclonal sera produced against Pf-iRBC immunizations were evaluated for specificity and parasite inhibition in vitro and used in a phage display biopanning assay to identify novel antigens on the surface of Pf-iRBCs. Results: Our data indicate that the polyclonal serum produced in BALB/cJ mice, against live Pf-iRBC (L) and their membrane fraction, specifically interacts with surface antigens of parasitic origin on Pf-iRBCs. Additionally, the anti-Pf-iRBC polyclonal serum exhibits significant parasite-killing activity in the in vitro growth inhibition assay (GIA). We have identified both known and novel antigens associated with the Pf-iRBC membrane using phage display cDNA library screening assays. Conclusions: As a proof of concept, our phage display screening identified antigens known to be associated with the Pf-iRBC membrane. Additionally, we identified several unknown Pf-iRBC antigens predicted to be associated with Pf-iRBC membrane (PlasmoDB), suggesting that our approach has the potential to identify novel antigens yet to be evaluated as vaccine candidates against falciparum malaria. In our follow-on studies, we will evaluate the newly identified antigen using an integrated in vitro and in vivo challenge experiment. These studies form the core supporting data for further evaluation of the vaccine potential of novel Pf-iRBC antigens and for follow-on vaccine trials in non-human primates, with an ultimate goal of a malaria vaccine for humans. Full article

Lymphocyte activation gene-3 (LAG-3) is a pivotal immune checkpoint receptor that exerts a negative regulatory effect on T-cell function. Although LAG-3-blocking antibodies have shown promising clinical potential, the inherent limitations of conventional monoclonal antibodies necessitate the development of novel antibody formats with enhanced biological and pharmacological properties. In this study, a panel of single-domain antibodies (sdAbs) targeting human LAG-3 was generated via phage display technology. Among these candidates, 2H-G7 was identified as a high-affinity sdAb that binds to LAG-3 with an equilibrium dissociation constant (K_D) in the nanomolar range. Notably, 2H-G7 potently blocks the interactions of LAG-3 with both of its key ligands, fibrinogen-like protein 1 (FGL1) and major histocompatibility complex class II (MHC-II). Its capacity to restore impaired T-cell function was validated by quantifying interleukin-2 (IL-2) secretion and CD69 expression in stimulated primary human peripheral blood mononuclear cells (PBMCs). Epitope mapping studies localized the binding site of 2H-G7 to the D1D2 extracellular domains of LAG-3, distinct from relatlimab, a clinically approved LAG-3-blocking antibody serving as the benchmark. In a xenogeneic mouse model of non-small-cell lung cancer (NSCLC), 2H-G7-Fc exhibited superior tumor growth inhibition efficacy compared with relatlimab. These findings demonstrate that 2H-G7 is a promising lead candidate for the development of next-generation LAG-3-targeted tumor immunotherapies. Full article

To address the emerging multidrug-resistance crisis caused by Klebsiella pneumoniae, we expressed the endolysin Lys59 derived from phage VB_KpP_HS106 and performed a comprehensive analysis of its antibacterial activity and structural features. Molecular modeling revealed that Lys59 carries a highly positively charged N-terminus and an amphipathic helix at the C-terminus. In vitro antibacterial assays showed that Lys59 exhibited significant bactericidal activity against K. pneumoniae with an approximately 4 log reduction at 50 µg/mL in 2 h. Meanwhile, Lys59 exhibited potent, broad-spectrum activity against both Gram-negative and Gram-positive bacteria. Stability analysis indicated that Lys59 retained high activity over a pH range of 3–9 and a temperature range of 4–55 °C. Notably, the antibacterial activity of Lys59 was found to be regulated by metal ions. Molecular docking indicated that K⁺ can enhance binding stability by interacting with ASN35 and VAL57. In contrast, Mg²⁺ and Ca²⁺ suppressed catalytic function by binding to the essential GLU17 residue. Furthermore, treatment with 200 µg/mL of Lys59 resulted in a 44.6% reduction in K. pneumoniae biofilm biomass. Overall, this study identified a phage-derived endolysin with broad-spectrum antimicrobial activity and demonstrated its potential as an antibacterial agent against multidrug-resistant K. pneumoniae. Full article