Search Results (621)

The objective of this research is the development of a copper-based optical sensor for the detection of ovarian cancer marker CA 125 synthesized using low-temperature plasma. Optical materials produced with metals show unique advantages due to their ability to interact with light. There are different methods currently used for the synthesis of optical materials that can be associated with longer processing times and low material yield. The novelty of this study is the development of copper-based optical material (CuPy) using low-temperature plasma and subsequent modification for the detection of CA 125. Introduction: Plasma consists of a mixture of fully and partially ionized gas. It comprises diverse, highly energized species of atoms, ions, electrons, excited molecules, and charged species. These energized species are used to create new materials, for surface modifications, and in medical applications. Plasma can create a controlled environment for the creation of novel materials. Using low-temperature plasma, it will be possible to have precise control of the chemical composition and structure due to the creation of excited molecules, ions, and free radicals. Method: The CuPy material was synthesized using radio-frequency-assisted low-temperature plasma. Prior to synthesis, the plasma chamber was cleaned using radio frequency (RF) plasma without any reagents or gases. RF plasma was used for the synthesis of CuPy for 10 min and subsequent hydrogen plasma (50 sccm) for another 10 min. Two types of products were extracted from the chamber (one in water and another in methanol). These two products were analyzed using UV–visible absorbance spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). The methanol extracted samples were further modified with CA 125 antibody. Zeta potential measurements were performed to confirm the binding of the CA 125 antibody to the sensor. The sensing efficacy of the sensor towards CA 125 antigen was monitored using fluorescence spectroscopy. Results: The absorbance spectrum of methanol extracted CuPy shows absorbances around 251 nm, 282 nm, and 339 nm. The extracted product exhibited a red edge excitation emission in the visible region. The elemental composition and oxidation state of the sample were evaluated using XPS. CA 125 antibody conjugation with CuPy was confirmed using UV–visible absorbance spectroscopy, fluorescence spectroscopy, and FTIR spectroscopy. The antibody binding resulted in the fluorescence shifts towards higher wavelengths with an increase in the emission intensity compared with CuPy. Zeta potential measurements also confirmed the binding of the CA 125 antibody to the sensor. Different concentrations of CA 125 antigen resulted in the quenching of fluorescence. This change in the fluorescence intensity was used for the detection of CA 125. Conclusions: A copper-based optical material was developed using low-temperature plasma, and it was found to be effective for the detection of CA 125 ovarian cancer marker. Full article

Dengue fever, a rapidly spreading mosquito-borne viral disease, poses a major global health issue, particularly in tropical and subtropical regions. The absence of a universally effective vaccine against all four dengue virus serotypes necessitates continued exploration of novel vaccine strategies. This study evaluated a PrM-derived multiple antigenic peptide (MAP) vaccine candidate in BALB/c mice. Molecular docking analyses predicted favourable interactions of the selected PrM-derived B-cell epitope with both human (human BCR Fab region PDB ID: 5IFH) and mouse (mouse BCR PDB ID: 8EMA) B-cell receptors, supporting its antigenic potential. ChemSketch was used to develop the PrM–MAP vaccine construct, which was then commercially synthesized. Mice received single, double, and triple booster doses of the vaccine, while control groups received either PBS or adjuvant alone. Humoral immune responses were measured by ELISA, and safety was evaluated through in vitro cytotoxicity assays on HEK293 cells and the in vivo histopathological examination of major organs. Mice immunized with a single booster dose produced significant levels of PrM-specific antibodies compared to subsequent booster doses, suggesting variability in the humoral response elicited. Optimal coating concentrations in ELISA demonstrated the importance of antigen concentrations in the sensitivity of the assay. The safety evaluation indicated high cell viability, up to 150 µg/mL of PrM–MAP, in HEK293 cells, and an absence of significant histopathological damage in treated animals. The PrM–MAP vaccine construct elicited a detectable humoral immune response while demonstrating a high safety profile in vitro and in vivo. These findings indicate the potential of MAP-based vaccine platforms as promising candidates for further development against the dengue virus. Full article

Biolayer Interferometry (BLI) has traditionally been used for characterization of protein–protein interactions (PPI) with proteins, such as antibodies and their antigens, through kinetic and quantitation assays. Limitations, for example in sensitivity and the availability of established assay formats, have restricted its adoption across other analytical applications. This article highlights three case studies which demonstrate the expansion of BLI into novel applications, spanning the areas of protein detection and viral vector characterization. The first case study details the use of a multi-step signal amplification assay to enable the detection of low abundant molecules, such as cytokines, at lower concentrations than can be detected using the standard one-step binding approach. Cytokines are sandwiched between biotinylated and HRP-conjugated antibodies, then dipped into 3-amino-9-ethylcarbazole (AEC) reagent, resulting in an enhancement of the cytokine detection sensitivity. The second case study uses BLI for the quantitation of mixed populations of a bispecific antibody (bsAb). Bridging and dual binding assay formats are evaluated for their assessment of bsAb antigen binding kinetics and ability to determine the ratio of correctly assembled bsAb within a sample. In the third case study, BLI detection principles are used to estimate the percentage full capsids in a mixed population of AAV particles. Collectively, these case studies demonstrate the versatility of Octet^® BLI and highlight its potential to support an increasing range of analytical workflows beyond its traditional applications. Full article

Campylobacteriosis is one of the leading foodborne bacterial illnesses worldwide, with poultry meat representing its principal source of human infection. Broiler chickens are a key reservoir for Campylobacter jejuni, where the bacterium persists in the gastrointestinal tract without disease. Understanding this host–pathogen interaction is essential to clarify mechanisms of persistence and food safety implications. This study investigated persistent C. jejuni colonisation and systemic immune responses in experimentally infected broiler chickens. Following oral challenge with the virulent ST-403 strain, animals were monitored under controlled conditions. Caecal bacterial loads were determined at 13, 27, 35 and 41 days post-infection, alongside serum total and antigen-specific IgY levels and circulating cytokines and chemokines using ELISA and multiplex assays. C. jejuni rapidly colonised the caecum and persisted throughout the study, although bacterial loads gradually declined over time but remained high. Total IgY increased early after infection and then stabilized. Antigen-specific antibodies were higher in infected chickens than in controls. Cytokine profiling revealed distinct temporal patterns, with increased IL-6 and MIP-1β, decreased IL-2 and MIP-3α, and persistently low IFN-γ levels. C. jejuni established stable intestinal colonisation with a detectable but non-clearing systemic immune response. These findings further support the role of broiler chickens as a persistent reservoir for human exposure to C. jejuni. Full article

Objective: To determine the in vitro anti-Aspergillus activity of monoclonal antibodies (mAb) 1D2 and 4E4 alone and in combination with voriconazole, posaconazole, amphotericin B and caspofungin and to assess whether selected antimicrobial agents interfere with 1D2/4E4 binding to Aspergillus antigens. Methods: The in vitro antifungal activity of 1D2, 4E4 and all antifungal drugs was determined in a microplate-based assay. The anti-Aspergillus activity of mAb 1D2 and 4E4 in vitro alone and in combination with voriconazole, posaconazole, amphotericin B and caspofungin was determined using a checkerboard assay. Finally, a competitive ELISA was used to assess whether selected antifungal and antibacterial agents interfered with the binding of 1D2 and 4E4 to immobilised Aspergillus antigens. Results: The in vitro interactions of 1D2 with voriconazole, posaconazole, amphotericin B and caspofungin against four different Aspergillus species were all additive, with a fractional inhibitory concentration index (FICI) between 0.5 and 1. Similarly, the in vitro interactions of 4E4 with these same four antifungals against Aspergillus fumigatus (A. fumigatus) were all additive. An additive interaction was also detected for 4E4 and caspofungin against all four Aspergillus species tested. However, no interaction was found for 4E4 combined with voriconazole, posaconazole and amphotericin B against Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Aspergillus terreus (A. terreus), with an FICI between 1 and 4. Moreover, none of the tested antifungal or antibacterial agents significantly interfered with 1D2/4E4 binding to Aspergillus antigens in the competitive ELISA. Conclusions: In this study, 1D2 and 4E4 showed in vitro growth-inhibitory activity against the tested Aspergillus isolates. Specifically, 1D2 showed additive interactions with the tested antifungal agents across all tested species, whereas 4E4 showed a more species- and drug-dependent interaction profile. The tested antimicrobial agents did not measurably interfere with 1D2/4E4 binding under the competitive ELISA conditions examined. Overall, these findings provide preliminary in vitro evidence supporting the further evaluation of 1D2 and 4E4. Full article

Porcine diarrheagenic Escherichia coli strains exhibit heterogeneous virulence profiles involving multiple factors, complicating broad-spectrum serological diagnosis. Here, we designed a multiepitope fusion antigen, MEAET, containing predicted B-cell epitopes from six major virulence factors, K88/F4, F18, LT, Stx2e, HlyA, and Tir, and developed an indirect ELISA for broad antibody detection. Structural modeling predicted a well-folded conformation with multiple discontinuous B-cell epitopes, and molecular docking suggested favorable interactions between the C-terminal dendritic cell-targeting peptide and the porcine immune receptor SLA1. Immunization of piglets with purified recombinant MEAET generated hyperimmune serum with a titer exceeding 1:102,400, which specifically recognized all six individual antigens by Western blot. The optimized ELISA demonstrated satisfactory repeatability (intra-assay CV ≤ 4.26%) and reproducibility (inter-assay CV ≤ 5.57%), showed no detectable cross-reactivity with antibodies against other tested porcine pathogens, and exhibited good analytical sensitivity, with positive sera detectable at dilutions up to 1:3200. The optimized ELISA showed good repeatability and reproducibility, with intra-assay and inter-assay coefficients of variation of ≤4.26% and ≤5.57%, respectively, no detectable cross-reactivity with the tested heterologous controls, and good analytical sensitivity, with positive sera remaining detectable at dilutions up to 1:3200. The S/P ratio cut-off was 0.203. In field sera, positivity rates were 74.5% (35/47) in diarrheic pigs and 34.0% (36/106) in asymptomatic pigs. These primary results indicate that the MEAET-based indirect ELISA represents a promising tool for serological detection of antibodies against multiple virulence factors of diarrheagenic E. coli in pigs and for field surveillance in swine herds. Full article

Human anti-αGal antibodies (Abs), known for their marked polyreactivity, have been detected bound to the gray matter of the brains of Alzheimer’s disease (AD) patients, although their targets were unclear. Since αGal is a strictly xenogenic antigen absent in humans, this observation raised questions regarding the nature of the structures recognized by these antibodies. In this study, we investigated their potential interaction with gangliosides—glycan structures that are highly abundant in the central nervous system. Using a competitive inhibition ELISA, serum profiles of anti-αGal Abs isotypes and their indirect cross-reactivity with selected soluble gangliosides were analyzed in AD patients and healthy subjects (HSs). AD patients showed reduced levels of anti-αGal IgG and IgM, but increased IgA compared to HSs. Notably, pre-incubation with GM1, GM2, or GD1b did not reduce αGal–HSA binding in HS sera. In contrast, in AD sera, pre-incubation with GD1b reduced residual αGal–HSA binding for all antibody isotypes; additionally, GM1 inhibited IgM binding, and GM2 inhibited IgA binding. These results should therefore be interpreted as competitive inhibition patterns consistent with ganglioside-associated cross-reactivity rather than as direct evidence of antibody binding to immobilized gangliosides. Overall, the findings provide preliminary evidence that, in AD sera, a fraction of αGal–HSA-reactive antibodies can be competitively inhibited by selected gangliosides. This observation supports the presence of an altered humoral anti-carbohydrate signature in AD and identifies neuronal gangliosides as plausible candidate autologous targets that may help explain the previously reported binding of anti-αGal Abs to gray matter. However, given the indirect nature of the assay, these data should be considered hypothesis-generating and require confirmation by direct binding approaches. Full article

Rhodococcus equi (R. equi) primarily induces fatal pulmonary and extrapulmonary pyogenic granulomatous infections in foals, imposing substantial economic burdens on the equine industry. The emergence and spread of multidrug-resistant (MDR) R. equi have led to a therapeutic impasse in clinical settings. Although vaccination is a proven strategy against MDR pathogens, no commercial vaccine is currently available for R. equi. In this study, we employed a bioinformatics approach to systematically identify and prioritize antigenic epitopes derived from R. equi for multi-epitope vaccine design. Using ABCPred, NetMHCpan EL, and IEDB servers, 27 MHC-I and 9 MHC-II epitopes were selected from five previously validated R. equi vaccine candidates: ABC transporter, PBD2, NlpC/P60, Esterase, and M23. These epitopes were coupled with distinct peptide linkers to construct six multi-epitope vaccine constructs, designated V1–V6. The physicochemical properties, antigenicity, immunogenicity, and toxicity of the six vaccine constructs were analyzed, and the V3 and V4 constructs were ultimately selected. Using the HDOCK and Gromacs tools, the intermolecular interactions, binding affinity, and thermal stability of the V3 and V4 constructs with the equine MHC molecules EQCA-I and EQCA-II were evaluated. The results confirm that V3 and V4 exhibit strong binding affinity to EQCA-I and EQCA-II, with stable conformations following binding, indicating theoretical potential to induce humoral and cellular immunity in foals. Recombinant plasmids for V3 and V4 were constructed, and the V3 and V4 proteins were successfully prepared, confirming the feasibility of prokaryotic expression for these vaccine constructs. Immunization assays in SPF BALB/c mice showed that the multi-epitope vaccines elicited robust antigen-specific IgG antibody responses, reflecting preliminary humoral immunogenicity. However, these murine data have translational limitations, as they cannot fully represent equine immune responses. The findings establish a crucial theoretical foundation for the advancement of vaccines targeting R. equi while offering a reference for the design of vaccines against other drug-resistant microbial pathogens. Full article

Convergent evolution in protein antigens is common across pathogens, including SARS-CoV-2; the most likely reason is the need to evade the selective pressure exerted by previous infection- or vaccine-elicited immunity. There is a pressing need for automated analysis of convergent mutations. We developed ConvMut, a tool to identify patterns of recurrent mutations in SARS-CoV-2 evolution; we exploited the granular phylogeny-based lineage hierarchy developed by PANGO, allowing us to observe deltas, i.e., groups of mutations that are acquired with respect to the immediately upstream tree nodes. Deltas comprise amino acid substitutions, insertions, and deletions. ConvMut can perform individual protein analysis to identify the most common single mutations acquired independently in a given subtree. Lineages are then gathered into clusters according to user-selected sets of shared mutations. An interactive graph orders the evolutionary steps of clusters, details the acquired amino acid change for each sublineage, and allows us to trace the evolutionary path until a selected lineage. ConvMut also supports frequency analysis for a given nucleotide or amino acid changes at a given residue across a selected phylogenetic subtree. ConvMut facilitates the exploration of convergent evolutionary trends in SARS-CoV-2, providing insights that could support the development of broadly effective anti-Spike monoclonal antibodies and Spike-based vaccines. Full article

Background/Objectives: Antibody–drug conjugates (ADCs) have transformed the therapeutic landscape of breast cancer, expanding treatment opportunities across multiple disease settings. However, their increasing clinical use has revealed a heterogeneous spectrum of toxicities that extends beyond conventional chemotherapy-related adverse events. Emerging evidence suggests that ADC-associated toxicities are driven by a complex interplay between ADC structural characteristics, including target antigen expression, payload properties, linker stability, drug-to-antibody ratio, and patient-related susceptibility factors. This review aims to provide a comprehensive overview of ADC-related toxicities in breast cancer, integrating mechanistic insights with clinical management strategies and risk-adapted approaches. Methods: A narrative review of the literature was conducted focusing on clinical trials, real-world studies, translational investigations, and mechanistic evidence related to ADC-associated toxicities in breast cancer. Particular attention was given to the relationship between ADC design, toxicity mechanisms, patient-specific risk factors, and clinical management. Results: ADC-related toxicities encompass a broad range of adverse events, including hematologic toxicity, interstitial lung disease, gastrointestinal complications, hepatotoxicity, peripheral neuropathy, stomatitis, ocular toxicity, dermatologic adverse events, and cardiovascular manifestations. Current evidence indicates that toxicity profiles differ substantially across ADCs and are influenced by multiple factors, including payload class, linker chemistry, target biology, intracellular trafficking, bystander effects, systemic payload exposure, and host-related characteristics. While several toxicities can be anticipated through careful monitoring and early intervention, clinically significant variability remains, and validated predictive biomarkers are largely lacking. Emerging real-world evidence further highlights the importance of individualized toxicity assessment and multidisciplinary management. Conclusions: ADC-related toxicity should be viewed as a multifactorial biological process resulting from the interaction between ADC design and host susceptibility rather than as a uniform class effect. A mechanistic understanding of toxicity pathways may improve risk stratification, toxicity monitoring, and personalized management strategies. Future research should focus on the development of predictive biomarkers, pharmacologic risk models, and next-generation ADC platforms with improved therapeutic indices. This review proposes an integrated framework linking ADC structural determinants, toxicity mechanisms, and clinical management to support safer and more individualized use of ADCs in breast cancer. Full article

Background: While vaccines are designed to elicit targeted immune responses, in some cases, the immunogenic molecules employed can inherently interact with broader host cellular pathways as a secondary consequence. This phenomenon can be exemplified by COVID-19 vaccines. COVID-19 vaccines, including mRNA platforms, use the SARS-CoV-2 spike protein as an immunogen to induce the production of neutralizing antibodies. The spike protein binds the ACE2 (angiotensin-converting enzyme 2) receptor on human cells, mediating viral entry and infection. ACE2 is widely expressed across multiple tissues and is a key component of the renin–angiotensin–aldosterone system (RAAS) that acts as a homeostatic regulator of systemic and local blood flow, blood pressure, cardiac function, fluid balance and immunity. Some studies have proposed the interaction between the spike protein and ACE2 as a possible contributing factor to rare adverse effects observed following COVID-19 vaccination, including myocarditis, pericarditis, thrombosis, and reported alterations in blood pressure, though these mechanisms remain to be fully elucidated. Objectives: As a proof-of-concept approach in vaccine antigen development, we engineered SARS-CoV-2 spike mutants with impaired binding to the host receptor ACE2. Methods: By rational design, we produced and validated in vitro and in vivo spike point mutants that do not effectively bind ACE2. Results: The engineered spike mutants do not effectively bind the human entry receptor ACE2 while retaining the immunogenic properties equal to or better than the wild type spike and thus generate a protective response in animals when used as a vaccination agent. Conclusions: By establishing a straightforward molecular strategy for rational vaccine design, this work demonstrates the feasibility of limiting specific antigen–host receptor interactions while maintaining immunogenicity. This approach may be applicable to future vaccination strategies where antigen interaction with host cells could potentially interfere with physiological pathways. Full article

Central tolerance is provided by the autoimmune regulator (AIRE) expressing medullary thymic epithelial cells through high-avidity recognition of self-antigens. Peripheral mechanisms regulate adaptive immunity by deleting autoreactive T-cells that escape thymic selection, or by inducing their functional unresponsiveness through interaction with antigen-presenting cells, exposing cognate antigens. Multiple types of extrathymic AIRE-expressing cells residing in secondary lymphoid organs have been consistently described. We investigated whether AIRE binds to promoters of known autoantigens in human peripheral blood mononuclear cells by chromatin immunoprecipitation from four healthy donors using an anti-AIRE monoclonal antibody. Quantitative real-time PCR was used to detect AIRE occupancy at promoters of selected autoantigens. Accordingly, we revealed promoter amplicons of all tested autoantigen genes, and their corresponding transcripts. Expression of AIRE was further confirmed at both transcriptional and protein levels. Overall, although the role of AIRE in regulating autoantigen expression in thymic epithelial cells has been well reported, our study provides preliminary descriptive evidence of AIRE occupancy at promoters of known autoantigens in human bulk peripheral blood mononuclear cells, suggesting the presence of AIRE-mediated regulatory events in peripheral blood. Our data provide a rationale for future investigations aimed at elucidating the underlying molecular and immunological mechanisms. Full article

The continuous evolution of the SARS-CoV-2 virus, marked by the emergence of new variants, poses a significant threat to the efficacy of existing vaccines. However, a promising approach to addressing vaccine failure caused by viral mutations (particularly in the spike protein) is the development of a variant-proof (conserved), non-spike, multiepitope universal nanostructure vaccine with multifunctionality, biocompatibility, self-adjuvanticity, and structural similarity to pathogens in terms of size and shape. This study aimed to design a self-assembled nanostructure vaccine (SANV) featuring pentameric and trimeric coiled-coil peptide motifs, as well as other functional motifs, including epitopes, TAT, PADRE, and adjuvant. The cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B lymphocyte (BL) epitopes of SANV were screened from the IEDB with more than 50% individual predicted population coverage (PPC) and fused using linkers to enable self-assembly. The multimerization of the 24 SANV monomers was modeled using the GalaxyHomomer and AlphaFold web servers. Subsequently, the leading SANV constructs with (SANVa9) and without (SANVb6) adjuvant were analyzed for their physicochemical profiles and assessed for antigenicity, allergenicity, solubility, and antioxidant potential. Furthermore, the molecular interactions, specificity, and stability of SANVa9 and SANVb6 with the broadly neutralizing sarbecovirus antibody 5817 and toll-like receptors (TLR2, TLR3, and TLR7) were analyzed using molecular docking and simulation over a 100-nanosecond time scale. Finally, the comparative immune simulation profiles of SANVa9 and SANVb6 with controls indicated stronger, broad-spectrum immune responses that could be translated into in vitro and in vivo studies and warrant further evaluation before clinical use. Full article

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

Dengue Virus (DENV) circulates as four antigenically distinct serotypes whose dominance fluctuates over time in many endemic regions, a phenomenon known as serotype shift that is frequently associated with large outbreaks and increased disease severity. This review, through a synthesis of epidemiological, virological, immunological, entomological, and environmental evidence, observes that serotype shift likely arises from the interaction of multiple determinants rather than solely from viral evolution, with population immunity playing a central role. The accumulation of serotype-specific herd immunity, together with short-lived cross-protection and Antibody-Dependent Enhancement (ADE), reshapes population susceptibility and creates ecological space for heterologous serotypes with higher transmission potential. The synthesis of global dengue studies indicates that these immune dynamics interact with viral genetic diversity, vector competence, climate variability, and human factors such as demography, socioeconomic status, population density and mobility to drive cyclical and sometimes abrupt changes in serotype dominance. Notably, the review indicates that serotype changes often precede or coincide with more clinical severity and patterns of outbreaks, with direct implications for the process of forecasting outbreaks, vaccine performance, and preparedness to respond with appropriate health measures. On the whole, this review confirms the opinion that the change of dengue serotype occurrence becomes a consequence of interconnected biological and ecological processes involved in the transmission of dengue serotype shifts in hyperendemic areas. Full article