Search Results (185)

Monkeypox virus (MPXV) has caused 148,892 confirmed cases and 341 deaths from 137 countries worldwide, as reported by the World Health Organization (WHO), highlighting the urgent need for effective vaccines to prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time consuming, and susceptible to reversion to virulence. Alternatively, a reverse vaccinology approach offers a rapid, efficient, and safer alternative for MPXV vaccine design. Here, MPXV proteins associated with viral infection were analyzed for immunogenic epitopes to design multi-epitope vaccines based on B-cell, CD4+, and CD8+ epitopes. Epitopes were selected based on allergenicity, antigenicity, and toxicity parameters. The prioritized epitopes were then combined via peptide linkers and N-terminally fused to various protein adjuvants, including PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and the cholera toxin B subunit (CTB). All vaccine constructs were computationally validated for physicochemical properties, antigenicity, allergenicity, safety, solubility, and structural stability. The three-dimensional structure of the selected construct was also predicted. Moreover, molecular docking and molecular dynamics (MD) simulations between the vaccine and the TLR-4 immune receptor demonstrated a strong and stable interaction. The vaccine construct was codon-optimized for high expression in the E. coli and was finally cloned in silico into the pET21a (+) vector. Collectively, these results could represent innovative tools for vaccine formulation against MPXV and be transformative for other infectious diseases. Full article

Consumption of contaminated drinking water is known to cause waterborne diseases such as diarrhoea, dysentery, typhoid, and hepatitis. This study applied whole-genome sequencing (WGS) to detect, identify, compare, and profile diarrhoeagenic pathogens (Vibrio cholerae, Shiga toxin-producing Escherichia coli, and Escherichia coli O157:H7) from 3168 water samples and 135 faecal samples (human and animal). Culture-based methods, MALDI-TOF mass spectrometry, and PCR were employed prior to WGS for identification of pathogens. Culture-based results revealed high presumptive prevalence of STEC (40.2%), V. cholerae (37.1%), and E. coli O157:H7 (22.7%). The MALDI-TOF confirmed 555 isolates with V. cholerae identified as Vibrio albensis. Shiga toxin-producing Escherichia coli (STEC) was more prevalent in wastewater (60%), treated water (54.1%), and groundwater (36.8%). PCR detected 46.4% of virulence genes from the water isolates and 66% of virulence genes from the STEC stool isolates. WGS also revealed STEC (92.9%) as the most prevalent species and found common virulence (e.g., hcp1/tssD1 and hlyE) and resistance (e.g., acrA and baeR) genes in all three types of samples. Five resistance and thirteen virulence genes overlapped among treated water and stool isolates. These findings highlight the diarrhoeagenic pathogens’ public health risk in water sources and underscore the need for better water quality monitoring and treatment standards. Full article

(1) Background: The COVID-19 pandemic highlights the critical necessity for the development of mucosal vaccines. (2) Objective: In this study, we aimed to develop mucosal vaccines based on the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. (3) Methods: We engineered the RBD of the Spike protein by incorporating ten lysine residues (K10), thereby enhancing its positive charge under physiological conditions. (4) Results: Although this modification did not directly augment the immunogenicity of the antigen, its combination with the mucosal adjuvant cholera toxin B subunit (CTB) and administration via the pulmonary route in BALB/c mice resulted in the induction of robust neutralizing antibody titers. Antigen-specific antibody responses were observed in both serum and bronchoalveolar lavage fluid. Importantly, serum IgG antibody titers remained above 10⁴ six months following third immunization, suggesting the establishment of sustained long-term immunity. Additionally, the incorporation of five lysine residues (K5) into the RBD, in conjunction with CTB, significantly increased serum IgG and IgA antibody titers. (5) Conclusions: Adding poly-lysine to RBD and combining it with CTB can stimulate robust mucosal and humoral immune responses in mice. These findings offer valuable insights for the design of subunit mucosal vaccines. Full article

The performance of heterophase immunoassays is often limited by the kinetics of analyte binding. This problem is partially solved by bead-based assays, which are characterized by rapid diffusion in the particle suspension. However, at low analyte concentrations, the binding rate is still low. Here, we demonstrate a further improvement of analyte binding kinetics in bead-based immunoassays by simultaneously concentrating both an analyte and magnetic beads in a compact spatial region where binding occurs. The analyte is electrophoretically concentrated in a flow cell where beads are magnetically retained and dragged along the channel by viscous force. The flow cell is integrated with a microarray-based signal detection module, where beads with bound analyte scan the microarray surface and are retained on it by single specific interactions, assuring ultra-high sensitivity of the method. Thus, a continuous flow assay system is formed. Its performance is demonstrated by simultaneous detection of model pathogen biomarkers, cholera toxin (CT) and staphylococcal enterotoxin B (SEB), with a detection limit of 0.1 fM and response time of under 10 min. The assay is capable of real-time online sample monitoring, as shown by a 12 h long continuous flow analysis of tap water for SEB and CT. Full article

Non-O1, non-O139 Vibrio cholerae (NOVC) extraintestinal infections are rare, but recently, several clinical incidents have been reported worldwide. Toxigenic V. cholerae is a well-known etiological agent of cholera, responsible for acute dehydrating watery diarrhea. Outbreaks occur in an epidemic seasonal pattern, particularly in countries with poverty and poor sanitation. Strains of NOVC are usually not involved in causing the epidemic or pandemic outbreaks seen with potential strains of V. cholerae serogroup O1 and O139. However, they can still cause severe sporadic cases of intestinal as well as extraintestinal infections. In this study, we investigated a case of extraintestinal infections associated with the NOVC serogroup isolated from a deep closed wound abscess. The isolate was screened for the presence of three major virulence genes, toxR, ctxA, and tcpA. The strain tested positive for the toxR gene encoding the regulatory protein and cholera toxin (ctx) gene and tested negative for the toxin-coregulated pilus (TCP) gene, which is essential for the colonization of the human intestine, causing the severe diarrheal disease cholera. To the best of our knowledge, this is the first case of extraintestinal infection caused by toxigenic Vibrio cholerae non-O1/non-O139 in a hospitalized patient in Saudi Arabia. Full article

The first immune response controls many bacterial and viral inflammatory diseases. Oral immunization with cholera toxin (CT) elicits antibodies and can prevent cholerae in endemic environments. While the IgG immune response to the toxin is well-documented, the IgA and IgM epitopes responsible for the initial immune reaction to the toxin remained uncharted. In this study, our objective was to identify and characterize immunologically and structurally these IgA and IgM epitopes. We conducted SPOT synthesis to create two libraries, each containing one hundred twenty-two 15-mer peptides, encompassing the entire sequence of the three chains of the CT protein. We could map continuous IgA and IgM epitopes by testing these membrane-bound peptides with sera from mice immunized with an oral vaccine (Schankol™). Our approach involved topological studies, peptide synthesis, and the development of an ELISA. We successfully identified seven IgA epitopes, two in CTA, two in CTB, and three in protein P. Additionally, we discovered eleven IgM epitopes, all situated within CTA. Three IgA-specific and three IgM-specific epitopes were synthesized as MAP4 and validated using ELISA. We then used two chimeric 45-mer peptides, which included these six epitopes, to coat ELISA plates and screened them with sera from immunized mice. This yielded sensitivities and specificities of 100%. Our findings have unveiled a significant collection of IgA and IgM-specific peptide epitopes from cholera toxins A, B, and P. These epitopes, along with those IgG previously identified by our group, reflect the immunoreactivity associated with the dynamic of the immunoglobulins switching associated with the cholera toxin vaccination. Full article

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) represents a major global public health challenge due to its ability to cause diverse clinical infections, including urinary tract infections, bacteremia, neonatal meningitis, and sepsis. The growing prevalence of multidrug-resistant (MDR) ExPEC strains, which rapidly erode antibiotic efficacy, underscores vaccine development as a critical priority. Bioconjugate vaccines have emerged as a promising approach to mitigate ExPEC-associated infections. Methods and Results: In this study, we utilized protein glycan coupling technology (PGCT) based on oligosaccharyltransferase (OST) PglL to engineer a tetravalent bioconjugate vaccine targeting four predominant ExPEC serotypes (O1, O2, O6, and O25). We conducted a series of experiments to demonstrate the efficacy of the conjugate vaccine in eliciting humoral immune responses and inducing the production of specific antibodies against Escherichia coli O1, O2, O6, or O25 serotypes. Conclusions: This work establishes the first application of the O-linked PGCT system for engineering bioconjugate vaccines against ExPEC infections. Full article

Background/Objectives: EPICERTIN, a biotherapeutic candidate for mucosal healing in inflammatory bowel disease (IBD) and other mucosal disorders, was subjected to an extensive long-term stability program to evaluate its molecular stability and physicochemical properties. Additionally, a forced degradation assessment was conducted to identify EPICERTIN’s degradation products under various conditions, including thermal stress, pH variations, agitation, and oxidation. Methods: The stability of EPICERTIN drug substance (DS), formulated in phosphate-buffered saline (PBS) at 1 mg/mL and stored at 5 °C and 25 °C/60% relative humidity (RH), was monitored over a 2-year period, referencing relevant regulatory guidelines. Evaluations of EPICERTIN DS over the 24-month period included assessment of purity by SDS-PAGE and size exclusion high performance liquid chromatography (SEC-HPLC), identity by electrospray ionization mass spectrometry (ESI-MS) intact mass analysis and Western blotting, and potency by GM1-binding KDEL-detection ELISA (GM1/KDEL ELISA). The forced degradation patterns were analyzed by assessing purity (using SEC-HPLC and SDS-PAGE), potency (via GM1/KDEL ELISA), and intact mass (via ESI-MS). Results: The results overall support that EPICERTIN DS remains stable for 2 years under the tested conditions. The forced degradation assessment effectively identified degradation products, particularly under conditions of high temperatures (above 40 °C for 24 h), low pH values (pH 1 and 4), and oxidation upon exposure to 2% H₂O₂. Conclusions: These findings highlight EPICERTIN’s robust long-term stability in PBS formulation, reinforcing its potential as a viable drug candidate for the treatment of IBD. Full article

This study aimed to immobilise sucrase A (SacA) from Bacillus subtilis in E. coli using the AIDA-I system for the whole-cell biocatalysis to transform sucrose to lactate. The pAIDA-sacA plasmid, containing the sacA gene, was fused to the signal peptide of the toxin subunit B from Vibrio cholerae (ctxB) and the autotransporter of the aida gene, encoding a connector peptide and the β-barrel domain of the AIDA-I system. This plasmid was employed to transform E. coli strains W3110, WDHFAK, and WDHFAP, which are unable to naturally use sucrose. These strains were anaerobically cultured in batch fermentations using 10 g L⁻¹ sucrose as the sole carbon source. All strains successfully hydrolysed and fermented sucrose, exhibiting a homolactic profile. Among them, WDHFAP/pAIDA-sacA achieved the highest lactic acid titre of 9.84 ± 0.15 g L⁻¹ and a yield of 0.89 ± 0.02 g g⁻¹. Deletion of the mgsA gene in WDHFAP/pAIDA-sacA confirmed that lactic acid production occurred via the methylglyoxal bypass pathway, as lactic acid titres were reduced by over 80%, while ethanol production increased to 4.27 ± 0.26 g L⁻¹. Adaptive laboratory evolution of WDHFAK/pAIDA-sacA was conducted to improve its capacity and fermentation efficiency under elevated sucrose concentrations. The resultant strain, designated as WDHFAK^EV/pAIDA-sacA, consumed up to 65 g L⁻¹ sucrose, achieving 64.61 ± 1.65 g L⁻¹ lactic acid with a yield of 0.99 ± 0.03 g g⁻¹. These findings underscore AIDA-I-mediated SacA immobilisation as a robust strategy for whole-cell biocatalysis, enabling E. coli strains to efficiently ferment sucrose. Full article

Coastal lagoons have undergone changes due to anthropogenic activities, the presence of wastewater discharges, and unsustainable practices that alter water quality, favoring the presence of pathogenic microorganisms such as Vibrio. This study identified the presence of the genes for zinc metalloproteinase (HA) Vibrio sp. and choleric toxin (ctx) Vibrio cholerae, associated with the sources of contamination in the Mandinga Coastal Lagoon (MCL). During 2017, samplings were carried out in which sources of contamination associated with anthropogenic activities were identified. At the same time, water samples were collected from which DNA was extracted and the presence/absence of the HA and ctx genes was detected with a PCR analysis. The HA gene was identified in the three seasons of the year, while the ctx gene was only present in the dry and rainy seasons. The prevalence of both genes in the study area was independent of the presence of the pollution sources identified in the area. The absence of the ctx gene during the northern season is associated with the variability of the physicochemical parameters typical of the season. Full article

Background/Objectives: We generated a novel recombinant Vibrio cholerae ghost (rVCG)-based subunit vaccine incorporating the A1 subunit of cholera toxin (CTA1) and a multiepitope Chlamydia trachomatis (CT) antigen (MECA) derived from five chlamydial outer membrane proteins (rVCG-MECA). The ability of this vaccine to protect against a CT transcervical challenge was evaluated. Methods: Female C57BL/6J mice were immunized thrice at two-week intervals with rVCG-MECA or rVCG-gD2 (antigen control) via the intramuscular (IM) or intranasal (IN) route. PBS-immunized mice or mice immunized with live CT served as negative and positive controls, respectively. Results: Vaccine delivery stimulated robust humoral and cell-mediated immune effectors, characterized by local mucosal and systemic CT-specific IgG, IgG2c, and IgA antibody and IFN-γ (Th1 cytokine) responses. The elicited mucosal and systemic IgG2c and IgA antibody responses persisted for 16 weeks post-immunization. Immunization with rVCG-MECA afforded protection comparable to that provided by IN immunization with live CT EBs without any side effects, irrespective of route of vaccine delivery. Conclusions: The results underline the potential of a multiepitope vaccine as a promising resource for protecting against CT genital infection and the potential of CTA1 on the VCG platform as a mucosal and systemic adjuvant for developing CT vaccines. Full article

Biological hazards in products of animal origin pose a significant threat to human health. In Cambodia, there are few comprehensive data and information on the causes of foodborne diseases or risks. To date, there has been no known published study similar to this review. This systematic review is aimed to investigate the prevalence of biological hazards and their indicators in products of animal origin from 2000 to 2022. The main objective of this study was also to contribute to strengthening Cambodia’s food control system. This review followed the established “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) guidelines. In total, 46 studies were retained for complete review. Most studies (n = 40) had been conducted by or with external researchers, reflecting the under-resourcing of the National Food Control System in terms of surveillance; areas outside the capital were relatively understudied, reflecting evidence found in Ethiopia and Burkina Faso. Five categories of hazards were reported with the highest number of studies on fish parasites. Marketed fish, often originating from different countries, had a higher mean value of parasite prevalence (58.85%) than wild-caught fish (16.46%). Viral pathogens in bat meat presented a potential spillover risk. Many potentially important hazards had not yet been studied or reported (e.g., Norovirus, Shigella, toxin-producing Escherichia coli, and Vibrio cholerae). The findings of our review highlighted significant urgencies for national competent authorities to enhance food hygiene practices along the production chain, tackle import control, and enforce the implementation of a traceability system, alongside more research collaboration with neighboring countries and key trading partners. It is crucial to conduct more extensive research on food safety risk analysis, focusing on the identification and understanding of various biological hazards and their associated risk factors in food. Full article

The H9N2 subtype avian influenza viruses mainly cause respiratory symptoms, reduce the egg production and fertility of poultry, and result in secondary infections, posing a great threat to the poultry industry and human health. Currently, all H9N2 avian influenza commercial vaccines are inactivated vaccines, which provide protection for immunized animals but cannot inhibit the spread of the virus and make it difficult to distinguish between the infected animals and vaccinated animals. In this study, a trimeric consensus H9 hemagglutinin (HA) subunit vaccine for the H9N2 subtype avian influenza virus based on a baculovirus expression system was first generated, and then the effects of three molecular adjuvants on the H9 HA subunit vaccine, Cholera toxin subunit B (CTB), flagellin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) fused with H9 HA, and one synthetic compound, a polyinosinic–polycytidylic acid (PolyI:C) adjuvant, were evaluated in mice by intranasal administration. The results showed that these four adjuvants enhanced the immunogenicity of the H9 HA subunit vaccine for avian influenza viruses, and that GM-CSF and PolyI:C present better mucosal adjuvant activity for the H9 HA subunit vaccine. These results demonstrate that we have developed a potential universal H9 HA mucosal subunit vaccine with adjuvants in a baculovirus system that would be helpful for the prevention and control of H9N2 subtype avian influenza viruses. Full article

Competition between bacterial species is a major factor shaping microbial communities. It is possible but remains largely unexplored that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by actin-specific toxins, namely, Vibrio cholerae MARTX actin crosslinking (ACD) and Rho GTPase inactivation (RID) domains, Photorhabdus luminescens TccC3, and Salmonella’s own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin-binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays and by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. These results suggest that bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria. Full article

Numerous gene therapy drugs for cancer have received global approval, yet their efficacy against solid tumors remains inadequate. Our previous research indicated that the fiber protein, a component of the adenovirus capsid, can propagate from infected cells to neighboring cells that express the adenovirus receptor. We hypothesize that merging this fiber protein with an anti-cancer protein could enable the anti-cancer protein to disseminate around the transfected cells, presenting a novel approach to cancer gene therapy. In our study, we discovered that the knob region of the adenovirus type 5 fiber protein is the smallest unit capable of spreading to adjacent cells in a receptor-specific manner. We also showed that the recombinant knob protein infiltrates cells after dispersing to surrounding cells. To assess the potential of the knob protein to augment gene therapy for solid tumors in mice, we expressed a fusion gene of the A subunit of cytotoxic cholera toxin and the knob region in mouse tumors. We found that this fusion protein only inhibited tumor growth in receptor-expressing mouse melanomas, and this inhibitory effect persisted even in mice with anti-knob antibodies. Our study’s findings propose a novel cancer gene therapy strategy that enhances therapeutic effects by specifically delivering therapeutic proteins, expressed from in vivo administered genes, to target molecules. This outcome offers a fresh perspective on gene therapy for solid cancers, and we anticipate that knob proteins will serve as a platform for this method. Full article