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The hepatitis B virus is a dangerous infectious agent, with an estimated 300 million carriers worldwide. Therefore, developing effective vaccine production methods is essential. One such method involves the use of transgenic plants. Several factors influence foreign gene expression in transgenic plants, including the type of genes, their chromosomal location, and the promoter used. Based on these factors, various expression cassettes can be designed for transgenic plants. In this study, we designed an expression cassette for transgenic potato plants. Promoters play a crucial role, as some are tissue-specific while others are inducible. Additionally, certain elements can enhance or silence gene expression in transgenic plants. Enhancer sequences derived from plant viruses are also utilized. To increase the expression level of HBsAg in transgenic potato plants, the tobacco etch virus (TEV) 5' untranslated region and the Patatin promoter were employed. The results demonstrated that in the highest-producing transgenic plant, the expression level of HBsAg increased threefold when both patatin and TEV were used together, compared to plants containing only the patatin promoter without TEV. Furthermore, a comparison with pCaMV combined with TEV revealed that the use of patatin and TEV resulted in a 4-5 times higher expression level. [ABSTRACT FROM AUTHOR]

Introduction: Urinary tract infection (UTI) is one of the most common infections, usually caused by uropathogenic Escherichia coli (UPEC). However, antibiotics are a usual treatment for UTIs; because of increasing antibiotic-resistant strains, vaccination can be beneficial in controlling UTIs. Using immunoinformatics techniques is an effective and rapid way for vaccine development. Methods: Three conserved protective antigens (FdeC, Hma, and UpaB) were selected to develop a novel multiepitope vaccine consisting of subunit B of cholera toxin (CTB) as a mucosal build-in adjuvant to enhance the immune responses. Epitopes-predicted B and T cells and suitable linkers were used to separate them and effectively increase the vaccine's immunogenicity. The vaccine protein's primary, secondary, and tertiary structures were evaluated, and the best 3D model was selected. Since CTB is the TLR2 ligand, molecular docking was made between the vaccine protein and TLR2. Molecular dynamic (MD) simulation was employed to evaluate the stability of the vaccine proteinTLR2 complex. The vaccine construct was subjected to in silico cloning. Results: The designed vaccine protein has multiple properties in the analysis. The HADDOCK outcomes show an excellent interaction between vaccine protein and TLR2. The MD results confirm the stability of the vaccine protein- TLR2 complex during the simulation. In silico cloning verified the expression efficiency of our vaccine protein. Conclusion: The results of this study suggest that our designed vaccine protein could be a promising vaccine candidate against UTI, but further in vitro and in vivo studies are needed. [ABSTRACT FROM AUTHOR]

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway. [ABSTRACT FROM AUTHOR]

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin–streptavidin–biotin bridge. The utilization of the streptavidin–biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N‐acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents. [ABSTRACT FROM AUTHOR]

Objective(s): Formation of Staphylococcus aureus biofilm leads to persistent infection in tissue or on exter-nal and indwelling devices in patients. Cold atmospheric plasma (CAP) is used for eradication of bacterial biofilms and it has diverse applications in the healthcare system. However, there is not sufficient information on the behavior of biofilms during the CAP exposure period. Materials and Methods: Pre-established S. aureus biofilms were exposed to CAP for 0 to 360 sec, then subjected to washing steps and sonication. Subsequently, biomass, number of colonies, vitality of bacteria, structure of colonies, size of produced particles, and viability of bacteria were evaluated by different assays including crystal violet, colony-forming unit, MTT, scanning electron mi-croscopy, confocal laser scanning microscopy, and dynamic light scattering assays. Results: The results showed that the strength of biomass increased in the first 60 sec, then decreased to less than no-CAP treated controls. Moreover, short CAP exposure (<60 sec) ehances the fusion of the biofilm extracellular matrix and other components, which results in preservation of bacteria during ultra-sonication and washing steps compared with control biofilms. The S. aureus biofilm structure only breaks down following more CAP exposure (> 90 sec) and demolition. Interestingly, the 60 sec CAP exposure could cause the fusion of biofilm compo-nents, and large particles are detectable. Conclusion: According to this study, an inadequate CAP exposure period prevents absolute eradication of biofilm and enhances the preservation of bacteria in stronger biofilm compartments. [ABSTRACT FROM AUTHOR]

Background: The synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-γ) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model. Methods: Monocistronic and bicistronic cassettes, carrying genes encoding mIFN-γ and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5' leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-γ in different constructs and HBsAg was tested by ELISA. Murine IFN-γ was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow. Results: Extracts of agroinfiltrated leaves contained recombinant mIFN-γ and HBsAg proteins at about 14 unit/mg and 50 ng/mg of soluble protein, respectively. Subsequently, mIFN-γ was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence. Conclusion: The co-expression of recombinant HBsAg and mIFN-γ using TEV 5' leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-γ in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications. [ABSTRACT FROM AUTHOR]

Context: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. Objective: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. Materials and methods: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred intoAgrobacterium tumefaciensstrain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc–HBsAg was also evaluated by co-infiltration of a p19 expression vector. Results: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc–HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. Discussion and conclusion: The results indicated the possibility of expression of HCVpc–HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine. [ABSTRACT FROM AUTHOR]

Pasteurella multocida (P. multocida) is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by P. multocida include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome. [ABSTRACT FROM AUTHOR]

Background: Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. Methods: The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. Results: The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. Conclusion: This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting. [ABSTRACT FROM AUTHOR]

Background and Objectives: Antibiotic resistance is an indicator of the passively acquired and circulating resistance genes. Salmonella Gallinarum significantly affects the poultry food industry. The present study is the first study of the S. Gallinarum biofilm in Iran, which is focused on the characterization of the S. Gallinarum serovars and their acquired antibiotic resistance genes circulating in poultry fields in central and northwestern Iran., Materials and Methods: Sixty isolates of S . Gallinarum serovar were collected from feces of live poultry. The bacteria were isolated using biochemical tests and confirmed by Multiplex PCR. Biofilm formation ability and the antibacterial resistance were evaluated using both phenotypic and genotypic methods. The data were analyzed using SPSS software., Results: According to Multiplex PCR for ratA, SteB , and rhs genes, all 60 S . Gallinarum serovars were Gallinarum biovars. In our study, the antibiotic resistance rate among isolated strains was as follows: Penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). All isolates were susceptible to imipenem, ertapenem, ceftriaxone, ceftazidime, and ceftazidime+clavulanic acid. All sixty isolates did not express the resistance genes IMP, VIM, NDM, DHA , bla OXA48 , and qnrA . On the other hand, they expressed GES (85%), qnrB (75%), Fox M (70%), SHV (60%), CITM (20%), KPC (15%), FOX (10%), MOXM (5%), and qnrS (5%). All S. Gallinarum isolates formed biofilm and expressed sdiA gene., Conclusion: Considering that the presence of this bacteria is equal to the death penalty to the herd, the distribution of resistance genes could be a critical alarm for pathogen monitoring programs in the region. This study showed a positive correlation between biofilm formation and 50% of tested resistance genes. Also, it was found that the most common circulating S. gallinarum biovars are multidrug-resistant., (Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences.)

The human body produces two classes of antimicrobial peptides (AMPs), namely defensins and cathelicidins. In this study, a novel decapeptide (Catoid) and its dimer (Dicatoid) based on human cathelicidin (LL-37) have been designed by bioinformatics tools to be used in the treatment of bacterial keratitis. After the selection and synthesis of peptide sequences, their antimicrobial activities against the standard and resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus were evaluated. This test was performed with LL-37, gentamicin, ciprofloxacin, amikacin, and penicillin for a more accurate comparison. Furthermore, the cytotoxicity levels of the specified compounds on fibroblast cells and bovine corneal endothelial cells were investigated. The results demonstrated that the designed peptides had a superior antimicrobial activity on P. aeruginosa , compared to LL-37; however, Catoid had a better effect on the S. aureus strain. Additionally, a significant achievement is the very low toxicity level of Catoid and Dicatoid on the human skin fibroblast cell line and bovine corneal endothelial cells, compared to that of LL-37 as the initial design model., Competing Interests: Conflict of Interests: The authors have no conflict of interest to declare., (Copyright © 2022, Author(s).)

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope ( PlpE1 + 2 + 3 ) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli ), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.

Background: Pasteurella multocida is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate., Materials and Methods: In this study, the fusion protein ( PlpE1 + 2 + 3 ) and full PlpE genes ( PlpE-Total ) were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 Escherichia coli. Overlap polymerase chain reaction (PCR) using different primers for 5' and 3' end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning., Results: Cloning of both PlpE1 + 2 + 3 and PlpE-Total into the pET28a vector and their transform into the BL21 (DE3) E. coli host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration., Conclusion: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 E. coli have resulted in sufficient expression, which shows that Top10 E. coli may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Advanced Biomedical Research.)