Search Results (47)

Coronaviruses continue to disrupt health and economic productivity worldwide. Porcine epidemic diarrhea virus (PEDV) is a devastating swine disease and SARS-CoV-2 is the latest coronavirus to infect the human population. Both viruses display a similar spike protein on the surface that is a target of vaccine development. Despite the availability of commercial vaccines for both viruses, there is still a high occurrence of infections and a great need for enhanced efficacy and lower costs. We previously produced the PEDV spike protein (S) using transgenic maize, enabling a low-cost supply of the vaccine candidate. In this study, we (1) test orally delivered PEDV vaccine candidates in pigs to optimize the mucosal immune response; (2) generate the SARS-CoV-2 S1 protein in maize; and (3) perform structural characterization of the S1 protein for PEDV and SARS-CoV-2. We demonstrated high expression levels in maize of the S1 subunit of the SARS-CoV-2 spike protein, both with and without fusion to the heat-labile enterotoxin B (LTB) subunit. We found that the LTB fusion protein from both coronaviruses preferentially assembles into higher molecular weight multimers, consistent with the formation of trimers. For PEDV, administering the spike protein fused to LTB to young pigs elicited a higher level of mucosal IgAs compared to maize grain containing the S1 protein alone or controls. This suggests that fusing the coronavirus spike protein with LTB may provide better protection. Full article

Diarrheal diseases caused by Shigella and enterotoxigenic Escherichia coli (ETEC) are significant health burdens, especially in resource-limited regions with high child mortality. In response to the lack of licensed vaccines and rising antibiotic resistance for these pathogens, this study developed a recombinant Shigella flexneri strain with the novel incorporation of the eltb gene for the heat-labile enterotoxin B (LTB) subunit of ETEC directly into Shigella’s genome, enhancing stability and consistent production. This approach combines the immunogenic potential of LTB with the antigen delivery properties of S. flexneri outer membrane vesicles (OMVs), aiming to provide cross-protection against both bacterial pathogens in a stable, non-replicating vaccine platform. We confirmed successful expression through GM1-capture ELISA, achieving levels comparable to ETEC. Additionally, proteomic analysis verified that the isolated vesicles from the recombinant strains contain the LTB protein and the main outer membrane proteins and virulence factors from Shigella, including OmpA, OmpC, IcsA, SepA, and Ipa proteins, and increased expression of Slp and OmpX. Thus, our newly designed S. flexneri OMVs, engineered to carry ETEC’s LTB toxin, represent a promising strategy to be considered as a subunit vaccine candidate against S. flexneri and ETEC. Full article

Triple-negative breast cancer (TNBC), which constitutes 10–20 percent of all breast cancers, is aggressive, has high metastatic potential, and carries a poor prognosis due to limited treatment options. LT-IIc, a member of the type II subfamily of ADP-ribosylating—heat-labile enterotoxins that bind to a distinctive set of cell-surface ganglioside receptors—is cytotoxic toward TNBC cell lines, but has no cytotoxic activity for non-transformed breast epithelial cells. Here, primary TNBC cells, isolated from resected human tumors, showed an enhanced cytotoxic response specifically toward LT-IIc, in contrast to other enterotoxins that were tested. MDA-MB-231 cells, a model for TNBC, were used to evaluate potential mechanisms of cytotoxicity by LT-IIc, which induced elevated intracellular cAMP and stimulated the cAMP response element-binding protein (CREB) signaling pathway. To dissect the role of ADP-ribosylation, cAMP induction, and ganglioside ligation in the cytotoxic response, MDA-MB-231 cells were exposed to wild-type LT-IIc, the recombinant B-pentamer of LT-IIc that lacks the ADP-ribosylating A polypeptide, or mutants of LT-IIc with an enzymatically inactivated A1-domain. These experiments revealed that the ADP-ribosyltransferase activity of LT-IIc was nonessential for inducing the lethality of MDA-MB-231 cells. In contrast, a mutant LT-IIc with an altered ganglioside binding activity failed to trigger a cytotoxic response in MDA-MB-231 cells. Furthermore, the pharmacological inhibition of ganglioside expression protected MDA-MB-231 cells from the cytotoxic effects of LT-IIc. These data establish that ganglioside ligation, but not the induction of cAMP production nor ADP-ribosyltransferase activity, is essential to initiating the LT-IIc-dependent cell death of MDA-MB-231 cells. These experiments unveiled previously unknown properties of LT-IIc and gangliosides in signal transduction, offering the potential for the targeted treatment of TNBC, an option that is desperately needed. Full article

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes reproductive failure and respiratory symptoms, leading to huge economic losses for the pig farming industry. Although several vaccines against PRRSV are available in the market; they show an overall low efficacy, and several countries have the need for vaccines covering the local, circulating variants. This project aims at developing a new chimeric antigen targeting specific epitopes from PRRSV and evaluating two test adjuvants to formulate a vaccine candidate. The test antigen was called LTB–PRRSV, which was produced recombinantly in Escherichia coli and consisted of the heat labile enterotoxin B subunit from E. coli (LTB) and four epitopes from PRRSV. LTB–PRRSV was rescued as inclusion bodies and methods for its solubilization, IMAC-based purification, and refolding were standardized, leading to mean yields of 18 mg of pure protein per liter culture. Layered double hydroxides (LDH) have been used as vaccine adjuvants given their biocompatibility, low cost, and positive surface charge that allows an efficient adsorption of negatively charged biomolecules. Therefore, LDH were selected as delivery vehicles of LTB–PRRSV. Pure LTB–PRRSV was adsorbed onto LDH by incubation at different LDH:LTB–PRRSV mass ratios (1:0.25, 1:0.5, 1:1, and 1:2) and at pH 9.5. The best adsorption occurred with a 1:2 mass ratio, and in a sucrose-tween solution. The conjugates obtained had a polydispersity index of 0.26, a hydrodynamic diameter of 192 nm, and a final antigen concentration of 64.2 μg/mL. An immunogenicity assessment was performed by injecting mice with LDH:LTB–PRRSV, Alum/LTB–PRRSV, or LTB–PRRSV in a scheme comprising three immunizations at two-week intervals and two dose levels (1 and 5 μg). LTB–PRRSV was capable of inducing strong humoral responses, which lasted for a longer period when LDH was used as the delivery vehicle/adjuvant. The potential of LDH to serve as an attractive carrier for veterinary vaccines is discussed. Full article

Nanoparticles (NPs) have been widely utilized in vaccine design. Although numerous NPs have been explored, NPs with adjuvant effects on their own have rarely been reported. We produce a promising self-assembled NP by integrating the pentameric Escherichia coli heat-labile enterotoxin B subunit (LTB) (studied as a vaccine adjuvant) with a trimer-forming peptide. This fusion protein can self-assemble into the NP during expression, and polysaccharide antigens (OPS) are then loaded in vivo using glycosylation. We initially produced two Salmonella paratyphi A conjugate nanovaccines using two LTB subfamilies (LTIB and LTIIbB). After confirming their biosafety in mice, the data showed that both nanovaccines (NP(LTIB)-OPS_SPA and NP(LTIIbB)-OPS_SPA) elicited strong polysaccharide-specific antibody responses, and NP(LTIB)-OPS resulted in better protection. Furthermore, polysaccharides derived from Shigella or Klebsiella pneumoniae were loaded onto NP(LTIB) and NP(LTIIbB). The animal experimental results indicated that LTIB, as a pentamer module, exhibited excellent protection against lethal infections. This effect was also consistent with that of the reported cholera toxin B subunit (CTB) modular NP in all three models. For the first time, we prepared a novel promising self-assembled NP based on LTIB. In summary, these results indicated that the LTB-based nanocarriers have the potential for broad applications, further expanding the library of self-assembled nanocarriers. Full article

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets. Full article

The antimicrobial application of carbon nanomaterials, such as carbon nanotubes (CNTs), capped CNTs, CNT_2–5, C₆₀, C₇₀, HO-C₆₀, [C₆₀]₂, and [C₆₀]₃ fullerenes, is increasing, owing to their low cytotoxicity properties compared to other nanomaterials such as metallic nanoparticles. Enhanced mechanical properties and antibacterial activity can be caused by the incorporation of CNTs in 3-dimensional (3D) printed nanocomposites (NCs). The interruption of the bacterial membrane resulting from the cylindrical shape and high aspect ratio properties has been found to be the most prominent antibacterial mechanism of CNTs. However, the unraveling interaction of CNTs, capped CNTs, CNT_2–5, C₆₀, C₇₀, HO-C₆₀, [C₆₀]₂, and [C₆₀]₃ fullerenes with virulence factors of the main bacterial pathogenesis has not yet been understood. Therefore, in the present study, interactions of these carbon-based nanomaterials with the eight virulence factors, including protein kinase A and (ESX)-secreted protein B of Mycobacterium tuberculosis, pseudomonas elastase and exotoxin A of Pseudomonas aeruginosa, alpha-hemolysin and penicillin-binding protein 2a of Staphylococcus aureus, and shiga toxin 2a and heat-labile enterotoxin of Escherichia coli, were evaluated with the molecular docking method of AutoDock Vina. This study disclosed that the binding affinity was highest for CNT_2–5 and [C₆₀]₃ toward alpha-hemolysin, with binding energies of −32.7 and −26.6 kcal/mol, respectively. The stability of the CNT_2–5–alpha-hemolysin complex at different times was obtained according to the normal mode analysis of ElNémo and iMOD servers. Full article

Background. Enterotoxigenic E. coli (ETEC) is a principal cause of diarrhea in travelers, deployed military personnel, and children living in low to middle-income countries. ETEC expresses a variety of virulence factors including colonization factors (CF) that facilitate adherence to the intestinal mucosa. We assessed the protective efficacy of a tip-localized subunit of CF antigen I (CFA/I), CfaE, delivered intradermally with the mutant E. coli heat-labile enterotoxin, LTR192G, in a controlled human infection model (CHIM). Methods. Three cohorts of healthy adult subjects were enrolled and given three doses of 25 μg CfaE + 100 ng LTR192G vaccine intradermally at 3-week intervals. Approximately 28 days after the last vaccination, vaccinated and unvaccinated subjects were admitted as inpatients and challenged with approximately 2 × 10⁷ cfu of CFA/I+ ETEC strain H10407 following an overnight fast. Subjects were assessed for moderate-to-severe diarrhea for 5 days post-challenge. Results. A total of 52 volunteers received all three vaccinations; 41 vaccinated and 43 unvaccinated subjects were challenged and assessed for moderate-to-severe diarrhea. Naïve attack rates varied from 45.5% to 64.7% across the cohorts yielding an overall efficacy estimate of 27.8% (95% confidence intervals: −7.5–51.6%). In addition to reducing moderate–severe diarrhea rates, the vaccine significantly reduced loose stool output and overall ETEC disease severity. Conclusions. This is the first study to demonstrate protection against ETEC challenge after intradermal vaccination with an ETEC adhesin. Further examination of the challenge methodology is necessary to address the variability in naïve attack rate observed among the three cohorts in the present study. Full article

Introduction: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrhea in travelers as well as for children living in low- to middle-income countries. ETEC adhere to intestinal epithelium via colonization factors (CFs). CFA/I, a common CF, is composed of a polymeric stalk and a tip-localized minor adhesive subunit, CfaE. Vaccine delivery by the transcutaneous immunization of dscCfaE was safe but was poorly immunogenic in a phase 1 trial when administered to volunteers with LTR(192G) and mLT. To potentially enhance the immunogenicity of CfaE while still delivering via a cutaneous route, we evaluated the safety and immunogenicity of two CfaE constructs administered intradermally (ID) with or without mLT. Methods: CfaE was evaluated as a donor strand-complemented construct (dscCfaE) and as a chimeric construct (Chimera) in which dscCfaE replaces the A1 domain of the cholera toxin A subunit and assembles non-covalently with the pentamer of heat-labile toxin B (LTB). Subjects received three ID vaccinations three weeks apart with either dscCfaE (1, 5, and 25 µg) or Chimera (2.6 and 12.9 µg) with and without 0.1 µg of mLT. Subjects were monitored for local and systemic adverse events. Immunogenicity was evaluated by serum and antibody-secreting cell (ASC) responses. Results. The vaccine was well-tolerated with predominantly mild and moderate local vaccine site reactions characterized by erythema, induration and post-inflammatory hyperpigmentation. High rates of serologic and ASC responses were seen across study groups with the most robust responses observed in subjects receiving 25 µg of dscCfaE with 0.1 mcg of LT(R192G). Conclusion: Both ETEC adhesin vaccine prototypes were safe and immunogenic when co-administered with mLT by the ID route. The observed immune responses induced with the high dose of dscCfaE and mLT warrant further assessment in a controlled human infection model. Full article

Enterotoxigenic Escherichia coli (ETEC) are common causes of infectious diarrhea among young children of low-and middle-income countries (LMICs) and travelers to these regions. Despite their significant contributions to the morbidity and mortality associated with childhood and traveler’s diarrhea, no licensed vaccines are available. Current vaccine strategies may benefit from the inclusion of additional conserved antigens, which may contribute to broader coverage and enhanced efficacy, given their key roles in facilitating intestinal colonization and effective enterotoxin delivery. EatA and EtpA are widely conserved in diverse populations of ETEC, but their immunogenicity has only been studied in controlled human infection models and a population of children in Bangladesh. Here, we compared serologic responses to EatA, EtpA and heat-labile toxin in populations from endemic regions including Haitian children and subjects residing in Egypt, Cameroon, and Peru to US children and adults where ETEC infections are sporadic. We observed elevated IgG and IgA responses in individuals from endemic regions to each of the antigens studied. In a cohort of Haitian children, we observed increased immune responses following exposure to each of the profiled antigens. These findings reflect the wide distribution of ETEC infections across multiple endemic regions and support further evaluation of EatA and EtpA as candidate ETEC vaccine antigens. Full article

Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage. Full article

Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4′-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin–ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA–ETEC and formalin–ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA–ETEC. In addition, PUVA–ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin–ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models. Full article

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia. Full article

Japanese encephalitis virus (JEV) is an enveloped icosahedral capsid virus with a prime neutralizing epitope present in E protein domain III (EDIII). E dimers are rearranged into a five-fold symmetry of icosahedrons. Cholera toxin B (CTB) and heat-labile enterotoxin B (LTB) of AB₅-type toxin was used as the structural scaffold for emulating the pentameric axis of EDIII. We produced homo-pentameric EDIII through the genetic fusion of LTB or CTB in E. coli without recourse to additional refolding steps. Harnessing an RNA-mediated chaperone further enhanced the soluble expression and pentameric assembly of the chimeric antigen. The pentameric assembly was validated by size exclusion chromatography (SEC), non-reduced gel analysis, and a GM1 binding assay. CTB/LTB−EDIII chimeric antigen triggered high neutralizing antibodies against the JEV Nakayama strain after immunization in mice. Altogether, our proof-of-principle study creating a JEV-protective antigen via fusion with an AB₅-type toxin as both a pentameric scaffold and a built-in adjuvant posits the bacterially produced recombinant chimeric antigen as a cost-effective alternative to conventional inactivated vaccines against JEV. Full article

In this study, sixteen unique staphylococcal enterotoxin B (SEB)-reactive nanobodies (nbs), including ten monovalent and six bivalent nbs, were developed. All characterized nbs were highly specific for SEB and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb). The lowest limit of detection (LOD) reached 50 pg/mL in PBS. When applied to an ELISA to detect SEB-spiked milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the sixteen nbs, with a subset of nbs, SEB-5, SEB-9, and SEB-6², retaining activity even after exposure to 95 °C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat-labile properties. Several nbs demonstrated a long shelf-life, with one nb (SEB-9) retaining 93% of its activity after two weeks of storage at room temperature. In addition to their usage in toxin detection, eleven out of fifteen nbs were capable of neutralizing SEB’s super-antigenic activity, demonstrated by their inhibition on IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs are relatively small, thermally stable, and easy to produce, making them useful in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products. Full article