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An increasing number of researchers are focusing on the influence of local peptide hormones such as angiotensin II (Ang II) and relaxin 2 (RLN2) in the regulation of inflammation and carcinogenesis. The interaction between the renin-angiotensin system (RAS) and relaxin family peptide system (RFPS) is known to influence the proliferation, adhesion and migration of normal and cancer prostate cell lines. The aim of the present study was to evaluate changes in the expression of nuclear factor-κB subunit 1 (NFKB1), nuclear factor-κB subunit 2 (NFKB2), REL proto-oncogene nuclear factor-κB p65 subunit (REL), RELA proto-oncogene nuclear factor-κB subunit (RELA) and RELB proto-oncogene nuclear factor-κB subunit (RELB) mRNA caused by Ang II and RLN2. The members of NF-kB family are involved in many processes associated with cancer development and metastasis. Reverse transcription-quantitative polymerase chain reaction analysis identified that both peptide hormones have an influence on the relative expression of nuclear factor-κB. Following treatment with either peptide, NFKB1 expression was downregulated in all prostate cancer cell lines (LNCaP, DU-145 and PC3), but not in normal epithelial cells (PNT1A). Conversely, RELB mRNA was enhanced only in non-cancerous prostate cells. RELA expression was strongly stimulated in the most aggressive cell line, whereas REL mRNA was unchanged. In many cases, the effect was strictly dependent on the cell line and/or the type of peptide: Ang II increased expression of both RELA and REL genes in the androgen-dependent cell line while RLN2 enhanced NFKB2 and RELA mRNA in androgen-independent cells (DU-145). Further research is needed to understand the regulation of NF-κB family members by key renin-angiotensin system and RFPS peptides in prostate cancer cells; however, prostate carcinogenesis appears to be influenced by the balance between the cross-regulation of nuclear factor-κB (NF-κB) and androgen receptor pathways by Ang II and relaxin 2.

Mucosal vaccination for COVID‐19 to boost preexisting though insufficient systemic and local/mucosal immunity remains an attractive prospect but there are currently no licensed mucosal vaccines against this infection. Here, using a plant expression system, we developed a novel mucosal vaccine platform for respiratory viruses and demonstrated its application in the context of SARS‐CoV‐2 infection. In addition to the antigen itself, the PCF (Platform CTB‐Fc) vaccine candidate incorporates two molecular adjuvants, the IgG‐Fc antibody fragment and the nontoxic cholera toxin B subunit (CTB), with the first targeting the vaccine to IgG receptors on antigen‐presenting cells, and the second providing local adjuvanticity by targeting cellular gangliosides in the mucosae. We demonstrated that this vaccine candidate is highly immunogenic in mice, inducing virus‐neutralising systemic and mucosal antibodies as well as tissue resident memory T cells in the lungs. We also demonstrated that SRBD‐PCF is recognised by immune cells from exposed or vaccinated individuals, and that circulating antibodies also bind to the antigen within the vaccine, forming immune complexes (IC). Finally, with a view of respiratory delivery, we demonstrated that the vaccine can be aerosolised without loss of material or biological activity, and that it is noncytotoxic and nonhaemolytic to human cells. Furthermore, we demonstrate that the plant expression system represents a suitable platform to produce these complex, multifunctional macromolecules capable of simultaneously binding to multiple targets. Our data strongly support the case for a safe, self‐adjuvanting mucosal COVID‐19 vaccine development, as means to boosting both systemic and mucosal immunity.

Proteus penneri ( P. penneri ) is a bacillus-shaped, gram-negative, facultative anaerobe bacterium that is primarily an invasive pathogen and the etiological agent of several hospital-associated infections. P. penneri strains are naturally resistant to macrolides, amoxicillin, oxacillin, penicillin G, and cephalosporins; in addition, no vaccines are available against these strains. This warrants efforts to propose a theoretical based multi-epitope vaccine construct to prevent pathogen infections . In this research, reverse vaccinology bioinformatics and immunoinformatics approaches were adopted for vaccine target identification and construction of a multi-epitope vaccine. In the first phase, a core proteome dataset of the targeted pathogen was obtained using the NCBI database and subjected to bacterial pan-genome analysis using bacterial pan-genome analysis (BPGA) to predict core protein sequences which were then used to find good vaccine target candidates. This identified two proteins, Hcp family type VI secretion system effector and superoxide dismutase family protein, as promising vaccine targets. Afterward using the IEDB database, different B-cell and T-cell epitopes were predicted. A set of four epitopes “KGSVNVQDRE, NTGKLTGTR, IIHSDSWNER, and KDGKPVPALK” were chosen for the development of a multi-epitope vaccine construct. A 183 amino acid long vaccine design was built along with “EAAAK” and “GPGPG” linkers and a cholera toxin B-subunit adjuvant. The designed vaccine model comprised immunodominant, non-toxic, non-allergenic, and physicochemical stable epitopes. The model vaccine was docked with MHC-I, MHC-II, and TLR-4 immune cell receptors using the Cluspro2.0 web server. The binding energy score of the vaccine was − 654.7 kcal/mol for MHC-I, − 738.4 kcal/mol for MHC-II, and − 695.0 kcal/mol for TLR-4. A molecular dynamic simulation was done using AMBER v20 package for dynamic behavior in nanoseconds. Additionally, MM-PBSA binding free energy analysis was done to test intermolecular binding interactions between docked molecules. The MM-GBSA net binding energy score was − 148.00 kcal/mol, − 118.00 kcal/mol, and − 127.00 kcal/mol for vaccine with TLR-4, MHC-I, and MHC-II, respectively. Overall, these in silico-based predictions indicated that the vaccine is highly promising in terms of developing protective immunity against P. penneri . However, additional experimental validation is required to unveil the real immune response to the designed vaccine.

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

•Development of an improved, thermostable dry formulation oral cholera vaccine (OCV).•Contains lyophilized formalin inactivated cholera bacteria and cholera toxin B subunit.•Single strain bacteria and high-yield CTB ensure low-cost manufacturing.•Proven stability for at least 26 months at 25 °C and 8 months at 40 °C.•Tolerability and immunogenicity equivalent to current, more complex, thermolabile liquid OCVs. The feared diarrheal disease cholera remains an important global health problem. Use of oral cholera vaccine (OCV) from a global stockpile against both epidemic and endemic cholera is a cornerstone in the World Health Organisations (WHOs) global program for “Ending cholera by 2030”. Three liquid inactivated whole-cell OCVs (Dukoral®, ShancholTM, and Euvichol-Plus®) are WHO prequalified and have proved to be safe and effective. However, their multicomponent composition and cold-chain requirement increase manufacturing, storage and transport costs. ShancholTM and Euvichol-Plus® OCVs used in WHOs global vaccine stockpile also lack the protective cholera toxin B-subunit (CTB) antigen present in Dukoral®, which results in suboptimal efficacy. WHOs Global Task Force on Cholera Control (GTFCC) has identified a thermostable, dry formulation vaccine as a priority for further OCV development. We describe here the development of such a vaccine, based on a lyophilized mixture of a single strain of formalin-killed Hikojima bacteria together with a low-cost, recombinantly produced CTB. The new vaccine, which is easy and inexpensive to manufacture, could be stored for at least 26 months at 25 °C and for at least 8 months at 40 °C with preservation of cell morphology and with no loss of protective Ogawa and Inaba lipopolysaccharides or CTB. It also proved to be well tolerated and to have equivalent oral immunogenicity in mice as ShancholTM and Dukoral® OCVs with regard to both serum and intestinal-mucosal antibody responses.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

Background Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. Results Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer’s patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels ( p  < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels ( p  < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. Conclusions The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24–59 months, 12–23 months, and 6–11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 1010 cells], quarter [2·5 × 1010 cells], or eighth [1·25 × 1010 cells] adult dose), with or without dmLT adjuvant (2·5 μg, 5·0 μg, or 10·0 μg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802. Between Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24–59 months, 100 aged 12–23 months, and 200 aged 6–11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24–59 months, 13 [13%] of 100 aged 12–23 months, and 29 [15%] of 200 aged 6–11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6–11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6–11 months. 78 (56%) of 139 infants aged 6–11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants. The encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas. PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.

G-protein coupled receptors help regulate cellular function and communication, and are targets of small molecule drug discovery efforts. Conventional techniques to probe these interactions require labels and large amounts of receptor to achieve satisfactory sensitivity. Here, we use frequency-locked optical microtoroids for label-free characterization of membrane interactions in vitro at zeptomolar concentrations for the kappa opioid receptor and its native agonist dynorphin A 1-13, as well as big dynorphin (dynorphin A and dynorphin B) using a supported biomimetic membrane. The measured affinity of the agonist dynorphin A 1-13 to the κ-opioid receptor was also measured and found to be 3.1 nM. Radioligand assays revealed a dissociation constant in agreement with this value (1.1 nM). The limit of detection for the κOR/DynA 1-13 was calculated as 180 zM. The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside was also monitored in real-time and an equilibrium dissociation constant of 1.53 nM was found. Our biosensing platform provides a method for highly sensitive real-time characterization of membrane embedded protein binding kinetics that is rapid and label-free, for drug discovery and toxin screening among other applications. The authors demonstrate a biosensing platform based on whispering gallery mode microtoroid resonators, which enables sensing at zeptomolar concentrations and real-time monitoring of molecular binding events

The occurrence of gastritis, gastric ulcers, distal gastric cancer, and gastric mucosal lymphoma in humans is strongly associated with Helicobacter pylori (H. pylori). Vaccination is an effective preventive measure due to the increasing prevalence of antibiotic resistance. Fusion vaccination is a potentially practical approach. A fusion vaccine was created in this study by combining the cholera toxin B subunit (CTB) with the antigenic H. pylori urease I subunit (CTB-UreI). The CTB-UreI DNA vaccine was chemically cloned into pIRES2-EGFP, and the success of the cloning was validated using PCR and restriction enzyme digestion. An investigation was conducted on the induction of CTB-UreI in Escherichia coli BL21(DE3). The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. The Western blot assay successfully identified the activation of CTB-UreI. In comparison, BALB/c mice receiving pIRES2-EGFP/CTB-UreI vaccination exhibited higher IgG, IgA, IFN-γ, IL-4, and IL-17 levels in their blood samples. In addition, there was a decrease in stomach injuries and bacterial loads. Furthermore, BALB/c mice inoculated with pIRES2-EGFP/CTB-UreI showed a high level of immunity (100%) against the H. pylori challenge. The pIRES2-EGFP/CTB-UreI elicited a combination of Th1/Th2/Th17 immune responses, possibly contributing to an effective defence mechanism. Our data suggests that using this fusion vaccine to prevent H. pylori infection is a promising option.