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Keywords = Cholera Toxin B

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41 pages, 7499 KiB  
Article
Development of a Broad-Spectrum Pan-Mpox Vaccine via Immunoinformatic Approaches
by Japigorn Puagsopa, Panuwid Jumpalee, Sittichoke Dechanun, Sukanya Choengchalad, Pana Lohasupthawee, Thanawat Sutjaritvorakul and Bunyarit Meksiriporn
Int. J. Mol. Sci. 2025, 26(15), 7210; https://doi.org/10.3390/ijms26157210 - 25 Jul 2025
Viewed by 890
Abstract
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 [...] Read more.
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
(This article belongs to the Section Molecular Informatics)
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14 pages, 1366 KiB  
Article
A Poly-Lysine-Based RBD Mucosal Vaccine Induces Potent Antibody Responses in Mice
by Huifang Xu, Han Wang, Peng Sun, Tiantian Wang, Bin Zhang, Xuchen Hou, Jun Wu and Bo Liu
Vaccines 2025, 13(6), 582; https://doi.org/10.3390/vaccines13060582 - 29 May 2025
Viewed by 464
Abstract
(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 [...] Read more.
(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
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13 pages, 3541 KiB  
Article
Ultrasensitive Bead-Based Immunoassay for Real-Time Continuous Sample Flow Analysis
by Yuri M. Shlyapnikov and Elena A. Shlyapnikova
Biosensors 2025, 15(5), 316; https://doi.org/10.3390/bios15050316 - 15 May 2025
Viewed by 642
Abstract
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. [...] Read more.
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
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17 pages, 1223 KiB  
Article
Dynamics of IgM and IgA Antibody Response Profile Against Vibrio cholerae Toxins A, B, and P
by Salvatore Giovanni De-Simone, Paloma Napoleão-Pêgo, Guilherme Curty Lechuga, Joao Pedro Rangel Silva Carvalho, Sergian Vianna Cardozo, Alexandre Oliveira Saisse, Carlos Medicis Morel, David William Provance and Flavio Rocha da Silva
Int. J. Mol. Sci. 2025, 26(8), 3507; https://doi.org/10.3390/ijms26083507 - 9 Apr 2025
Cited by 1 | Viewed by 577
Abstract
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 [...] Read more.
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
(This article belongs to the Section Molecular Biophysics)
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17 pages, 11480 KiB  
Article
A Bioconjugate Vaccine Against Extra-Intestinal Pathogenic Escherichia coli (ExPEC)
by Linhui Hao, Wenhua Huang, Yan Guo, Xiankai Liu, Jun Wu, Li Zhu, Chao Pan and Hengliang Wang
Vaccines 2025, 13(4), 362; https://doi.org/10.3390/vaccines13040362 - 28 Mar 2025
Viewed by 788
Abstract
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 [...] Read more.
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
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16 pages, 3894 KiB  
Article
Preclinical Long-Term Stability and Forced Degradation Assessment of EPICERTIN, a Mucosal Healing Biotherapeutic for Inflammatory Bowel Disease
by Wendy M. Kittle, Micaela A. Reeves, Ashley E. Fulkerson, Krystal T. Hamorsky, David A. Morris, Kathleen T. Kitterman, Michael L. Merchant and Nobuyuki Matoba
Pharmaceutics 2025, 17(2), 259; https://doi.org/10.3390/pharmaceutics17020259 - 15 Feb 2025
Cited by 1 | Viewed by 809
Abstract
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 [...] Read more.
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 article belongs to the Section Biopharmaceutics)
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16 pages, 1662 KiB  
Article
Immobilisation of Sucrase A from Bacillus subtilis on the Surface of Escherichia coli Mediated by the AIDA-I Autotransporter: Application on the Homolactic Fermentation
by Jorge Sánchez-Andrade, Victor E. Balderas-Hernández, Ana P. Barba de la Rosa and Antonio De Leon-Rodriguez
Processes 2025, 13(2), 470; https://doi.org/10.3390/pr13020470 - 8 Feb 2025
Viewed by 935
Abstract
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 [...] Read more.
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 WDHFAKEV/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
(This article belongs to the Special Issue Application of Enzymes in Sustainable Biocatalysis)
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14 pages, 6300 KiB  
Article
H9 Consensus Hemagglutinin Subunit Vaccine with Adjuvants Induces Robust Mucosal and Systemic Immune Responses in Mice by Intranasal Administration
by Liming Lin, Shunfan Zhu, Beibei Yang, Xin Zhang, Huimin Wu, Shixiang Wu, Li Wu, Jianhong Shu, Yulong He and Huapeng Feng
Microorganisms 2024, 12(11), 2294; https://doi.org/10.3390/microorganisms12112294 - 12 Nov 2024
Viewed by 1435
Abstract
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 [...] Read more.
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
(This article belongs to the Topic Advances in Vaccines and Antimicrobial Therapy)
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17 pages, 3110 KiB  
Article
Antagonistic Effects of Actin-Specific Toxins on Salmonella Typhimurium Invasion into Mammalian Cells
by David B. Heisler, Elena Kudryashova, Regan Hitt, Blake Williams, Michelle Dziejman, John Gunn and Dmitri S. Kudryashov
Biomolecules 2024, 14(11), 1428; https://doi.org/10.3390/biom14111428 - 9 Nov 2024
Viewed by 1453
Abstract
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 [...] Read more.
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
(This article belongs to the Section Cellular Biochemistry)
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12 pages, 3824 KiB  
Article
Surface Display of Cholera Toxin B Subunit Recombinant Escherichia coli Ghosts Further Enhances Resistance to Chlamydia abortus Infection in Mice
by Huaiyu Zhang, Yunhui Li, Wei Li, Zhaocai Li, Jizhang Zhou and Dewen Tong
Microorganisms 2024, 12(8), 1656; https://doi.org/10.3390/microorganisms12081656 - 13 Aug 2024
Cited by 2 | Viewed by 1618
Abstract
Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that [...] Read more.
Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge. Full article
(This article belongs to the Section Medical Microbiology)
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17 pages, 5095 KiB  
Article
Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission
by Maria Ciuro, Maria Sangiorgio, Valeria Cacciato, Giuliano Cantone, Carlo Fichera, Lucia Salvatorelli, Gaetano Magro, Giampiero Leanza, Michele Vecchio, Maria Stella Valle and Rosario Gulino
Int. J. Mol. Sci. 2024, 25(13), 7059; https://doi.org/10.3390/ijms25137059 - 27 Jun 2024
Cited by 2 | Viewed by 1967
Abstract
Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression [...] Read more.
Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders. Full article
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21 pages, 3027 KiB  
Article
The JMU-SalVac-System: A Novel, Versatile Approach to Oral Live Vaccine Development
by Andreas Iwanowitsch, Joachim Diessner, Birgit Bergmann and Thomas Rudel
Vaccines 2024, 12(6), 687; https://doi.org/10.3390/vaccines12060687 - 20 Jun 2024
Viewed by 1994
Abstract
Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of [...] Read more.
Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a ΔtyrS/tyrS+-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene (tyrS) and the in trans complementation of tyrS on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans. Full article
(This article belongs to the Special Issue Advances in Oral Vaccine Development)
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10 pages, 4613 KiB  
Article
Borrelia burgdorferi 0755, a Novel Cytotoxin with Unknown Function in Lyme Disease
by Sam T. Donta
Toxins 2024, 16(6), 233; https://doi.org/10.3390/toxins16060233 - 21 May 2024
Viewed by 3681
Abstract
The pathophysiology of Lyme disease, especially in its persistent form, remains to be determined. As many of the neurologic symptoms are similar to those seen in other toxin-associated disorders, a hypothesis was generated that B. burgdorferi, the causative agent of Lyme disease, [...] Read more.
The pathophysiology of Lyme disease, especially in its persistent form, remains to be determined. As many of the neurologic symptoms are similar to those seen in other toxin-associated disorders, a hypothesis was generated that B. burgdorferi, the causative agent of Lyme disease, may produce a neurotoxin to account for some of the symptoms. Using primers against known conserved bacterial toxin groups, and PCR technology, a candidate neurotoxin was discovered. The purified protein was temporarily named BbTox, and was subsequently found to be identical to BB0755, a protein deduced from the genome sequence of B. burgdorferi that has been annotated as a Z ribonuclease. BbTox has cytotoxic activity against cells of neural origin in tissue culture. Its toxic activity appears to be directed against cytoskeletal elements, similar to that seen with toxins of Clostridioides difficile and Clostridioides botulinum, but differing from that of cholera and E. coli toxins, and other toxins. It remains to be determined whether BbTox has direct cytotoxic effects on neural or glial cells in vivo, or its activity is primarily that of a ribonuclease analogous to other bacterial ribonucleases that are involved in antibiotic tolerance remains to be determined. Full article
(This article belongs to the Section Bacterial Toxins)
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16 pages, 4368 KiB  
Article
Efficient Production of Self-Assembled Bioconjugate Nanovaccines against Klebsiella pneumoniae O2 Serotype in Engineered Escherichia coli
by Yan Zhang, Peng Sun, Ting Li, Juntao Li, Jingqin Ye, Xiang Li, Jun Wu, Ying Lu, Li Zhu, Hengliang Wang and Chao Pan
Nanomaterials 2024, 14(8), 728; https://doi.org/10.3390/nano14080728 - 21 Apr 2024
Cited by 4 | Viewed by 2492
Abstract
Nanoparticles (NPs) have been surfacing as a pivotal platform for vaccine development. In our previous work, we developed a cholera toxin B subunit (CTB)-based self-assembled nanoparticle (CNP) and produced highly promising bioconjugate nanovaccines by loading bacterial polysaccharide (OPS) in vivo. In particular, the [...] Read more.
Nanoparticles (NPs) have been surfacing as a pivotal platform for vaccine development. In our previous work, we developed a cholera toxin B subunit (CTB)-based self-assembled nanoparticle (CNP) and produced highly promising bioconjugate nanovaccines by loading bacterial polysaccharide (OPS) in vivo. In particular, the Klebsiella pneumoniae O2 serotype vaccine showcased a potent immune response and protection against infection. However, extremely low yields limited its further application. In this study, we prepared an efficient Klebsiella pneumoniae bioconjugate nanovaccine in Escherichia coli with a very high yield. By modifying the 33rd glycine (G) in the CNP to aspartate (D), we were able to observe a dramatically increased expression of glycoprotein. Subsequently, through a series of mutations, we determined that G33D was essential to increasing production. In addition, this increase only occurred in engineered E. coli but not in the natural host K. pneumoniae strain 355 (Kp355) expressing OPSKpO2. Next, T-cell epitopes were fused at the end of the CNP(G33D), and animal experiments showed that fusion of the M51 peptide induced high antibody titers, consistent with the levels of the original nanovaccine, CNP-OPSKpO2. Hence, we provide an effective approach for the high-yield production of K. pneumoniae bioconjugate nanovaccines and guidance for uncovering glycosylation mechanisms and refining glycosylation systems. Full article
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19 pages, 2897 KiB  
Article
Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity
by Muhammed Salah Uddin, Angelo Kaldis, Rima Menassa, José Ortiz Guluarte, Daniel R. Barreda, Le Luo Guan and Trevor W. Alexander
Vaccines 2024, 12(4), 375; https://doi.org/10.3390/vaccines12040375 - 1 Apr 2024
Cited by 2 | Viewed by 3056
Abstract
Background: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their [...] Read more.
Background: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. Methods: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. Results: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). Conclusions: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle. Full article
(This article belongs to the Special Issue Advances in Oral Vaccine Development)
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