Search Results (54)

Intestinal infections caused by Salmonella enterica serovar Typhi (S. Typhi) remain a global health concern, making preventive strategies and diagnostic tools essential. This study aimed to identify mimotope peptides of the Vi antigen using phage display and assess their recognition by rabbit and 46 human sera, as well as their potential for diagnosis and immunogen design. Rabbits were immunized with the Vi antigen (AgVi) from S. Typhi ATCC 6539, and sera-derived IgG was used for phage biopanning. DNA sequences from selected phagotopes were synthesized as Salmonella mimotope peptides (SMPs), either linear or KLH-conjugated. Their reactivity was tested with ELISAs against AgVi and SMPs, using both rabbit sera and 46 human serum samples. Ten phagotopes were identified, with a consensus motif (D/G–A/V–x–P–x–x–G–x–x–x–x–x), suggesting α-helix structures. Immunization with KLH-conjugated peptides generated specific antibodies, particularly SMPVi/5 and SMPVi/10, which recognized AgVi and their respective peptides. Competitive inhibition assays confirmed that SMPVi/5 reduced the anti-AgVi binding in a dose-dependent manner. In human sera, AgVi recognition occurred in 52% of samples, while SMPVi/5 and SMPVi/10 were recognized in 45%. Overall, SMPVi/5 demonstrated immunogenicity and functional mimicry, supporting its use as a synthetic reagent for serological assays and as a candidate for immunogen design. Full article

Background/Objectives: Spotty liver disease (SLD), caused by Campylobacter hepaticus, is an emerging disease that leads to substantial production losses in the egg industry. The shift toward antibiotic-free and cage-free production systems has further intensified the impact of SLD. The current control measures largely rely on autogenous killed vaccines; however, their use is constrained by the slow and fastidious growth of C. hepaticus and inconsistent efficacy. To overcome these limitations, this study aimed to identify immunogenic mimotopes as vaccine candidates and express them on the surface of an avian pathogenic Escherichia coli (APEC) vector. Methods: To identify immunogenic mimotopes, Ph.D.-12 phage display peptide library was screened using the hyperimmune serum raised against killed whole-cell C. hepaticus in specific pathogen-free chickens. Subsequently, the outer membrane protein C (OmpC) of E. coli was used as a scaffold for constructing a surface display library. A single restriction site, PstI, located in the seventh external loop of OmpC, was strategically utilized to insert each 12-amino-acid mimotope with a six-histidine (6xHis) tag sequence at its N-terminus, generating ompC + mimotope fusion constructs. These constructs were cloned into the inducible expression vector pTrc and electroporated into an E. coli DH5α ∆ompC strain, which lacked ompC. The surface expression of the mimotopes was confirmed in vitro. The verified ompC + mimotope constructs were subsequently subcloned into the pYA3422 constitutive expression vector and electroporated into the APEC PSUO78 ∆aroA ∆asd vaccine vector strain. A chicken vaccination–challenge trial was conducted using nine groups of chickens, including an unvaccinated challenged control and an unvaccinated–unchallenged negative control. Each experimental group received a mixture of two recombinant E. coli strains carrying different mimotopes at a dose of 1 × 10⁹ CFU, which were administered orally twice at 16 and 18 weeks of age. Results: Fourteen immunogenic mimotopes corresponding to 13 different C. hepaticus proteins were identified as potential vaccine candidates. The expression of these mimotopes on the surface of the E. coli was successfully demonstrated using the OmpC-mediated surface display system. Of the 14 mimotopes tested, two flagellar-related peptides and one major outer membrane protein (MOMP)-derived peptide elicited significant immune responses and conferred protection against the C. hepaticus challenge. Conclusions: We successfully developed a functional E. coli surface display system that was capable of expressing 12-amino-acid mimotopes of C. hepaticus, providing a robust platform for evaluating vaccine candidates against SLD. Immunogenicity and efficacy studies in chickens demonstrated that three identified mimotopes conferred protection against C. hepaticus colonization of the bile and liver. Future in vivo investigations are necessary to develop and evaluate the immunogenicity and protective efficacy of a multivalent mimotope vaccine consisting of three identified mimotopes against both C. hepaticus and APEC, utilizing the ΔaroA Δasd APEC PSU078 strain as the vaccine vector. Full article

Background: Three-finger toxins (3FTxs) are a major axis of functional diversification in advanced snake venoms, with canonical paralytic activity mediated through muscle-type nicotinic acetylcholine receptors (nAChRs) and a broader set of non-nicotinic targets. This review integrates evidence bearing on coevolution between 3FTxs and target receptors, spanning toxin origin, diversification, receptor evolution, and ecological context. Methods: The synthesis draws on comparative genomic and transcriptomic studies of 3FTx gene-family evolution, codon-model analyses of selection, structural characterisation of toxin–receptor interfaces, and functional assays (including receptor-mimicking peptide binding) that link sequence variation to binding and toxicity. Results: Across lineages, 3FTx diversification is repeatedly structured by strong constraint on the disulphide-rich scaffold with accelerated change concentrated in solvent-exposed loops, alongside birth–death dynamics and exon/segment-level innovation that expand binding specificity. On the receptor side, resistance-associated variation is most intensively characterised for the nAChR α₁ orthosteric site and includes convergent, mechanistically distinct solutions such as electrostatic repulsion and glycosylation-mediated steric interference. Within the predominantly elapid systems currently examined, integrative datasets indicate that prey-selective binding and geographically variable susceptibility can arise from modest substitutions at toxin–receptor interfaces, but they also reveal substantial taxonomic and target-specific biases. Conclusions: Current evidence supports adaptive diversification in both toxins and receptors, while broader evolutionary interpretations are limited by uneven sampling and the frequent lack of matched toxin and receptor variants analysed within a common evolutionary framework. Development of predictive models will require joint pipelines linking genomics, structure-informed evolutionary inference, scalable functional assays, and explicit ecological network context. Full article

Mimotope-based immunoassays offer an eco-friendly alternative to chemically synthesized antigens for the quantitative analysis of small molecules, but their use for practical on-site and high-throughput residue monitoring remains limited. Herein, we report the selection, production, and application of a phage display–derived mimotope targeting an anti-uniconazole monoclonal antibody (UCZ-mAb), with the aim of developing two complementary immunoassays that enable sensitive, eco-friendly detection of UCZ residues in agricultural samples. A 12-mer phage-displayed peptide library was screened to identify UCZ-specific mimotopes, and a selected sequence was genetically fused to SpyTag and expressed in Escherichia coli to generate a SpyTagged mimotope. Leveraging the SpyCatcher/SpyTag self-assembly system, the SpyTagged mimotope was directionally conjugated onto SpyCatcher-functionalized time-resolved fluorescence beads (TRFBs) and subsequently used as a signal-labeled competitive antigen in a lateral flow immunoassay (LFIA) designed for rapid on-site screening. In parallel, a wash-free magnetic separation immunoassay (MSIA) suitable for green, high-throughput screening in routine laboratories was established using self-assembled mimotope-TRFB probes. The LFIA and MSIA exhibited half-maximal inhibitory concentrations (IC₅₀) of 3.70–6.72 μg/kg and 16.4–18.3 μg/kg, respectively, in real samples. Spiked-sample recoveries ranged from 91.1 to 107.8% for LFIA and 92.6–115.7% for MSIA, demonstrating acceptable accuracy and precision. These results indicate that the SpyTagged mimotope–based LFIA and MSIA provide complementary, reliable, and sensitive platforms for on-site screening and high-throughput monitoring of UCZ residues in agricultural samples, while avoiding the drawbacks associated with traditional chemical antigen synthesis. Full article

Mesenchymal stem cells have been widely investigated in the field of regenerative medicine and also used as a model to study the differentiation-induction properties of a variety of biomaterials. This study evaluates the osteoinductive potential of novel hydroxyapatite scaffolds functionalized with a phage-displayed peptide (SC1) selected via biopanning for its similarity to bone matrix proteins. The peptide, identified through sequence alignment as a mimotope of osteonectin (SPARC), was used to functionalize scaffolds. Results from SC1 were gathered at different time points (14, 28 and 46 days) and compared with those from nonfunctionalized hydroxyapatite (HA) scaffolds. In vitro experiments, by seeding human adipose-derived stem cells (hASCs), indicated satisfactory biocompatibility for both types of scaffolds. Histochemical observations showed that SC1, better than HA scaffolds, was able to improve hASC osteogenic differentiation, as evaluated through Alizarin Red staining (showing on average a darker staining of 100%). An increase was also observed, especially at early stages (14 days), for osterix (up to 60% increase) and osteonectin immunoexpression (up to 50% increase). In in vivo experiments, cell-free scaffolds of both types were subcutaneously implanted into the backs of mice and analyzed after 2, 4, 8 and 16 weeks. Also, in this case, SC1 more effectively promoted the osteogenic differentiation of infiltrated resident cells. In particular, increased immunoexpression of osterix and osteonectin (+30% and 35%, respectively) was found already at 2 weeks. It can be concluded that SC1 scaffolds may represent a valuable tool to address critical-sized bone defects. Full article

Immunotherapies, including checkpoint inhibitor antibodies, have precipitated significant improvements in clinical outcomes for melanoma. However, approximately half of patients do not benefit from approved treatments. Additionally, apart from Tebentafusp, which is approved for the treatment of uveal melanoma, there is a lack of immunotherapies directly focused on melanoma cells. This is partly due to few available targets, especially those expressed on the cancer cell surface. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface molecule overexpressed in human melanoma, with restricted distribution and low expression in non-malignant tissues and involved in several cancer-promoting and dissemination pathways. Here, we summarize the current understanding of the expression and functional significance of CSPG4 in health and melanoma, and we outline immunotherapeutic strategies. These include monoclonal antibodies, antibody–drug conjugates (ADCs), chimeric-antigen receptor (CAR) T cells, and other strategies such as anti-idiotypic and mimotope vaccines to raise immune responses against CSPG4-expressing melanomas. Several showed promising functions in preclinical models of melanoma, yet few have reached clinical testing, and none are approved for therapeutic use. Obstacles preventing that progress include limited knowledge of CSPG4 function in human cancer and a lack of in vivo models that adequately represent patient immune responses and human melanoma biology. Despite several challenges, immunotherapy directed to CSPG4-expressing melanoma harbors significant potential to transform the treatment landscape. Full article

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 10¹³ phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis’ gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output. Full article

Traditional vaccines use inactivated or weakened forms of pathogens which could have side effects and inadequate immune responses. To overcome these challenges, phage display has emerged as a valuable tool for identifying specific epitopes that could be used in vaccines. This review emphasizes the direct connection between epitope identification and vaccine development, filling a crucial gap in the field. This technique allows vaccines to be engineered to effectively stimulate the immune system by presenting carefully selected epitopes. Phage display involves screening libraries of random peptides or gene/genome fragments using serum samples from infected, convalescent, or vaccinated individuals. This method has been used to identify epitopes from various pathogens including SARS-CoV-2, Mycobacterium tuberculosis, hepatitis viruses, H5N1, HIV-1, Human T-lymphotropic virus 1, Plasmodium falciparum, Trypanosoma cruzi, and Dirofilaria repens. Bacteriophages offer advantages such as being immunogenic carriers, low production costs, and customization options, making them a promising alternative to traditional vaccines. The purpose of this study has been to highlight an approach that encompasses the entire process from epitope identification to vaccine production using a single technique, without requiring additional manipulation. Unlike conventional methods, phage display demonstrates exceptional efficiency and speed, which could provide significant advantages in critical scenarios such as pandemics. Full article

Leishmania amazonensis can cause cutaneous and visceral clinical manifestations of leishmaniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion’s average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis. Full article

Neuromyelitis optica spectrum disorder (NMOSD) is a central nervous system inflammatory demyelinating disease, the pathogenesis of which involves autoantibodies targeting the extracellular epitopes of aquaporin-4 on astrocytes. We neutralized the AQP4-IgG from NMOSD patient sera using synthesized AQP4 extracellular epitope peptides and found that the severe cytotoxicity produced by aquaporin-4 immunoglobin (AQP4-IgG) could be blocked by AQP4 extracellular mimotope peptides of Loop A and Loop C in astrocyte protection and animal models. ACT001, a natural compound derivative, has shown anti-tumor activity in various cancers. In our study, the central nervous system anti-inflammatory effect of ACT001 was investigated. The results demonstrated the superior astrocyte protection activity of ACT001 at 10 µM. Furthermore, ACT001 decreases the behavioral score in the mouse NMOSD model, which was not inferior to Methylprednisolone Sodium Succinate, the first-line therapy of NMOSD in clinical practice. In summary, our study showed that astrocytes are protected by specific peptides, or small molecular drugs, which is a new strategy for the treatment of NMOSD. It is possible for ACT001 to be a promising therapy for NMOSD. Full article

Combining adaptive and innate immunity induction modes, the repertoire of immunoglobulin M (IgM) can reflect changes in the internal environment including malignancies. Previously, it was shown that a mimotope library reflecting the public IgM repertoire of healthy donors (IgM IgOme) can be mined for efficient probes of tumor biomarker antibody reactivities. To better explore the interpretability of this approach for IgM, solid tumor-related profiles of IgM reactivities to linear epitopes of actual tumor antigens and viral epitopes were studied. The probes were designed as oriented planar microarrays of 4526 peptide sequences (as overlapping 15-mers) derived from 24 tumor-associated antigens and 209 cancer-related B cell epitopes from 30 viral antigens. The IgM reactivity in sera from 21 patients with glioblastoma multiforme, brain metastases of other tumors, and non-tumor-bearing neurosurgery patients was thus probed in a proof-of-principle study. A graph representation of the binding data was developed, which mapped the cross-reactivity of the mixture of IgM (poly)specificities, delineating different antibody footprints in the features of the graph—neighborhoods and cliques. The reactivity graph mapped the major features of the IgM repertoire such as the magnitude of the reactivity (titer) and major cross-reactivities, which correlated with blood group reactivity, non-self recognition, and even idiotypic specificities. A correlation between an aspect of this image of the IgM IgOme, namely, small cliques reflecting rare self-reactivities and the capacity of subsets of the epitopes to separate the diagnostic groups studied was found. In this way, the graph representation helped the feature selection in its filtering step and provided reduced feature sets, which, after recursive feature elimination, produced a classifier containing 51 peptide reactivities separating the three diagnostic groups with an unexpected efficiency. Thus, IgM IgOme approaches to repertoire studies is greatly augmented when self/viral antigens are used and the data are represented as a reactivity graph. This approach is most general, and if it is applicable to tumors in immunologically privileged sites, it can be applied to any solid tumors, for instance, breast or lung cancer. Full article

“Point of care” (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL⁻¹, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL⁻¹ according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence. Full article

The activation of the maternal immune system by a prenatal infection is considered a risk factor for developing psychiatric disorders in the offspring. Toxoplasma gondii is one of the pathogenic infections associated with schizophrenia. Recent studies have shown an association between high levels of IgG anti-T. gondii from mothers and their neonates, with a higher risk of developing schizophrenia. The absence of the parasite and the levels of IgGs found in the early stages of life suggest a transplacental transfer of the anti-T. gondii IgG antibodies, which could bind fetal brain structures by molecular mimicry and induce alterations in neurodevelopment. This study aimed to determine the maternal pathogenic antibodies formation that led to behavioral impairment on the progeny of rats immunized with T. gondii. Female rats were immunized prior to gestation with T. gondii lysate (3 times/once per week). The anti-T. gondii IgG levels were determined in the serum of pregestational exposed females’ previous mating. After this, locomotor activity, cognitive and social tests were performed. Cortical neurotransmitter levels for dopamine and glutamate were evaluated at 60 PND in the progeny of rats immunized before gestation (Pregestational group). The maternal pathogenic antibodies were evidenced by their binding to fetal brain mimotopes in the Pregestational group and the reactivity of the serum containing anti-T. gondii IgG was tested in control fetal brains (non-immunized). These results showed that the Pregestational group presented impairment in short and long-term memory, hypoactivity and alteration in social behavior, which was also associated with a decrease in cortical glutamate and dopamine levels. We also found the IgG antibodies bound to brain mimotopes in fetuses from females immunized with T. gondii, as well as observing a strong reactivity of the serum females immunized for fetal brain structures of fetuses from unimmunized mothers. Our results suggest that the exposure to T. gondii before gestation produced maternal pathogenic antibodies that can recognize fetal brain mimotopes and lead to neurochemical and behavioral alterations in the offspring. Full article

The application of monoclonal antibodies (mAbs), targeting tumor-associated (TAAs) or tumor-specific antigens or immune checkpoints (ICs), has shown tremendous success in cancer therapy. However, the application of mAbs suffers from a series of limitations, including the necessity of frequent administration, the limited duration of clinical response and the emergence of frequently pronounced immune-related adverse events. However, the introduction of mAbs has also resulted in a multitude of novel developments for the treatment of cancers, including vaccinations against various tumor cell-associated epitopes. Here, we reviewed recent clinical trials involving combination therapies with mAbs targeting the PD-1/PD-L1 axis and Her-2/neu, which was chosen as a paradigm for a clinically highly relevant TAA. Our recent findings from murine immunizations against the PD-1 pathway and Her-2/neu with peptides representing the mimotopes/B cell peptides of therapeutic antibodies targeting these molecules are an important focus of the present review. Moreover, concerns regarding the safety of vaccination approaches targeting PD-1, in the context of the continuing immune response, as a result of induced immunological memory, are also addressed. Hence, we describe a new frontier of cancer treatment by active immunization using combined mimotopes/B cell peptides aimed at various targets relevant to cancer biology. Full article

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell itself. Such an in situ process can be realized by exploiting target-specific molecules as templates to direct proximity-enhanced assembly. Modified nucleic acids carrying inert precursor fragments can be designed to co-hybridize on a target-specific template nucleic acid, such that the enforced proximity accelerates assembly of a functional molecule for antibody recognition. We demonstrate the in vitro feasibility of this adaptation of nucleic acid-templated synthesis (NATS) using oligonucleotides bearing modified peptides (“haplomers”), for templated assembly of a mimotope recognized by the therapeutic antibody trastuzumab. Enforced proximity promotes mimotope assembly via traceless native chemical ligation. Nevertheless, titration of participating haplomers through template excess is a potential limitation of trimolecular NATS. In order to overcome this problem, we devised a strategy where haplomer hybridization can only occur in the presence of target, without being subject to titration effects. This generalizable NATS modification may find future applications in enabling directed targeting of pathological cells. Full article