Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Detection of ESBL and AmpC producing Klebsiella pneumoniae ST11 and ST147 from urinary tract infections in Iran.

In the present study a total of 200 Klebsiella pneumoniae isolates were collected from patients with urinary tract infections (UTIs) in Tehran, Iran. Antibiotic resistance was determined by disk diffusion and broth dilution methods. Detection of extended-spectrum ß-lactamases (ESBLs) and AmpCs was performed using phenotypic tests. Polymerase chain reaction (PCR) was applied to detect the ESBL, AmpC, and integron genes. Analysis of AmpC and cassette arrays of integron genes was performed using DNA sequencing. Plasmids were analyzed by PCR-based replicon typing and conjugation. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were applied to explore the genomic relatedness among the isolates. The highest levels of resistance were observed against ampicillin (100%), followed by piperacillin (57.5%), ceftazidime (46%), trimethoprim/sulfamethoxazole (44%), ciprofloxacin (32.5%), and imipenem (19%). Approximately, 66.5% of isolates harbored at least one of the beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, and blaOXA-1). In addition, 22.5% of isolates carried at least one of the AmpC genes including blaDHA and blaCIT. Integron class I was the most prevalent integron among resistant isolates. According to the results of replicon typing, IncFII, IncL/M, and IncA/C were the most frequent replicons, respectively. All selected isolates were able to transfer blaCTX-M, also two isolates transferred the blaDHA-1 gene to Escherichia coli K12 through conjugation. Finally, 21 isolates were categorized into 4 pulsotypes and 11 unique clusters in PFGE. MLST identified ST147 and ST11 sequence types but ST147 was the most prevalent in the current study.

Investigation of the effects of antimicrobial and anti-biofilm peptide IDR1018 and chitosan nanoparticles on ciprofloxacin-resistant Escherichia coli.

Peptide IDR1018 and chitosan nanoparticles (CNs) showed antimicrobial and anti-biofilm activity against bacteria. In this study, the antimicrobial effects of peptide IDR1018 and CNs were evaluated on 50 clinical isolates of uropathogenic Escherichia coli (UPEC) resistant to ciprofloxacin. Ion gelation method was applied for CNs synthesis. Scanning electron microscope (SEM) and dynamic light scattering (DLS) were utilized to evaluate the nanoparticles. Antimicrobial and synergistic activity of peptide IDR1018 and CNs with ciprofloxacin were evaluated by microtiter broth dilution method. The checkerboard test was used to investigate the antimicrobial effects of IDR1018 and CNS in combination with ciprofloxacin. Anti-biofilm effect of ciprofloxacin, peptide IDR1018, and CNs was evaluated using crystal violet method. Fourteen (28%), 21 (42%), and 15 (30%) of clinical isolates produced strong, moderate, and weak biofilm, respectively. The CNs were spherical and uniform under electron microscopy with an average diameter of 246 nm. The minimum inhibitory concentration (MIC) values were 16-128, 20-40, and 375-750 (µg/ml) for ciprofloxacin, peptide IDR1018, and CNs, respectively. Fractional inhibitory concentration (FIC) analysis indicated a synergistic effect of ciprofloxacin in combination with peptide IDR1018, but in combination with CNs, this antibiotic showed an additive effect. Our results revealed that peptide IDR1018 and CNs have antimicrobial properties on UPEC isolates. Biofilm inhibition and biofilm eradication of clinical isolate were shown by peptide IDR1018 and CNs in a concentration-dependent manner. The antimicrobial agents alone and in combination decreased the number of viable bacteria in the biofilms. Therefore, these components seem to be a treating approach against biofilm-forming UPEC isolates.

The combination of CipA and PBP-7/8 proteins contribute to the survival of C57BL/6 mice from sepsis of Acinetobacter baumannii.

Due to the emergence of multi-drug resistant Acinetobacter baumannii strains, there is an urgent need to develop several new strategies to control this bacterium. In this context, vaccination may be the best approach to reduce the morbidity and mortality associated with MDR isolates in vulnerable groups. Serum resistance factors have a key role in the pathogenesis of A. baumannii and can be considered as potential vaccine candidates. This project aimed to evaluate the immunological reactivity of CipA and PBP-7/8 as two serum resistance factors in a combination form against sepsis infections of A. baumannii. Recombinant proteins were obtained and immunological evaluations were performed against sepsis infection in the C57BL/6 mouse model. The data showed a statistically significant increase in total IgG levels in all three immunization regimens (CipA, PBP-7/8, and CipA + PBP-7/8) compared to the control group. The ratios of IgG2c/IgG1 in the CipA, PBP-7/8, and CipA + PBP-7/8 schedules were 8.7, 46.50, and 33.29, respectively. It appears that the immunization schedules developed a strong polarized Th1 response. The cytokine profiles of the three plans showed that IFN-γ was highly concentrated in the combination plan. However, the highest concentration of IL-17 belonged to the PBP-7/8 plan. In conclusion, the data of total IgG, survival rates and splenic bacterial loads showed that the CipA + PBP-7/8 plan was more effective than each protein individually.

Evaluation of the immune response to a multi-epitope vaccine candidate in comparison with HlaH35L, MntC, and SACOL0723 proteins against MRSA infection.

Staphylococcus aureus is an important human opportunistic pathogen that can have a major influence on public health. Here, we aimed to evaluate different aspects of the immune response to a novel multi-epitope fusion protein (HMS) based on HlaH35L, MntC, and SACOL0723 proteins in comparison to the individual antigens. For this purpose, specific total IgG, IgG1, and IgG2a isotypes and the cytokines related to Th1, Th2, and Th17 were assessed. The Bio-efficiency of the fusion protein was evaluated by opsonic killing activity. The HMS fusion protein elicited a high specific IgG level and also induced a higher level of Th1, Th2, and Th17-related cytokines which were more polarized towards the Th1 and Th17 compared to individual antigens. The HMS-specific antisera also significantly promoted phagocytosis of S. aureus COL strain by mouse macrophages. In conclusion, the fusion protein might be an effective vaccine for potential protective immunity against a lethal infection of S. aureus in mice.

Surface display of uropathogenic Escherichia coli FimH in Lactococcus lactis: In vitro characterization of recombinant bacteria and its protectivity in animal model.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are very common, leading to high patient morbidity and substantial medical costs. The development of non-antibiotic strategies such as food-grade lactic acid bacterium can be recognized as an attractive and safe alternative way against UTI. Here, we report the construction of Lactococcus lactis (L. lactis) strain genetically modified to produce FimH virulence factor of UPEC on the cell surface. We showed the FimH inserted into the pT1NX vector is actively synthesized on L. lactis. The L. lactis-pT1NX-FimH exhibited an auto-aggregation phenotype in liquid cultures and formed robust biofilm on abiotic surface compared to vector-only bacteria. Then, we developed protective biofilms with L. lactis strains and examined their inhibitory effect for exclusion of uropathogenic biofilm formation. In the natural protective biofilm assays, L. lactis-pT1NX-FimH resulted in significant reduction in the pathogen load when compared to the L. lactis-pT1NX. Evaluation of the colonization ability in the bladder showed that L. lactis expressing FimH survived better in the mice bladder than L. lactis harboring vector. Protection assay against UPEC infection was investigated using a UTI mouse model. L. lactis-pT1NX-FimH displayed high effectiveness in the protection of the bladder as compared to the control group after UPEC challenge. The results suggest that genetically engineered L. lactis-pT1NX-FimH can be used as a safe alternative way for control of biofilm formation in UPEC. Furthermore, the possibility of using L. lactis-pT1NX-FimH as a new promising strategy against UTIs caused by UPEC strains is proposed.

Antibiotic resistance, virulence and genetic diversity of Klebsiella pneumoniae in community- and hospital-acquired urinary tract infections in Iran.

Klebsiella pneumoniae is among the most important causes of urinary tract infection (UTI). The aim of this study was to investigate the prevalence and correlation of antibiotic resistance with virulence characteristics and genetic diversity in K. pneumoniae isolated from UTIs in Iran. Phenotypic tests and antibiotic susceptibility were carried out on the isolates. Detection of the virulence and extended-spectrum ß-lactamase (ESBL) genes was performed by polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) was used for exploring the genomic relatedness. Hemolysin, biofilm, and hypermucoviscosity formation were observed in 87.1%, 86.4%, and 12.1% of isolates, respectively. The antibiotic resistance rate of K. pneumoniae isolates ranged from 12.1% for meropenem to 100% for amoxicillin. The prevalence of virulence genes ranged from 1.4% for cnf-1 to 100% for mrkD, fimH, kpn, and entB genes. In this study, 91.7%, 33.3%, and 4.2% of phenotypically ESBL-producers were positive for blaCTX-M, blaTEM, and blaSHV genes, respectively. An association was observed between the presence of traT, fyuA, or cnf-1 genes with antibiotic resistance. Two clone types were obtained by PFGE that indicate different K. pneumoniae clones in community- and hospital-acquired UTIs. The findings of this study are valuable in development of treatment strategies against UTIs in Iran.

Construction and evaluation of the immune protection of a recombinant divalent protein composed of the MrpA from MR/P fimbriae and flagellin of Proteus mirabilis strain against urinary tract infection.

Urinary tract infections (UTI) caused by Proteus mirabilis are prevalent among the catheterized patients. There is no effective vaccine to reduce the frequency of UTIs caused by P. mirabilis. In the present study, the immune responses and effectiveness of different combinations of MrpA and flagellin (FliC) of P. mirabilis were assessed intranasally in the mice model. The addition of FliC as adjuvant to MrpA in fusion form significantly raised the mucosal IgA and cellular (IFN-γ and IL-17) responses and maintained the serum IgG responses for 180 days after the first vaccination. Furthermore, MrpA in fusion form with FliC significantly increased the systemic, mucosal and IFN-γ responses of the FliC alone. In a bladder challenge assay with P. mirabilis, the fusion MrpA.FliC and the mixture of MrpA and FliC significantly decreased the colony count of the bacteria in the bladder and kidneys of mice in comparison to the control mice. It suggests a complex of the systemic, mucosal and cellular responses are needed for protection of the bladder and kidneys against P. mirabilis UTI. In our knowledge, the adjuvant property of the recombinant P. mirabilis flagellin was evaluated for the first time in a vaccine combination administered by an intranasal route. Our results suggest the recombinant flagellin of P. mirabilis could be used as an intranasal adjuvant in combination with other potential antigens against UTIs.

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.

Evaluation of prevalence, immunogenicity and efficacy of FyuA iron receptor in uropathogenic Escherichia coli isolates as a vaccine target against urinary tract infection.

Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.

Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.

Design and computational analysis of an effective multi-epitope vaccine candidate using subunit B of cholera toxin as a build-in adjuvant against urinary tract infections.

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 multi-epitope 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 protein-TLR2 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.

In silico and in vivo Investigations of the Immunoreactivity of Klebsiella pneumoniae OmpA Protein as a Vaccine Candidate.

Background: The growing threat of antibiotic resistance and Klebsiella pneumoniae infection in healthcare settings highlights the urgent need for innovative solutions, such as vaccines, to address these challenges. This study sought to assess the potential of using K. pneumoniae OmpA as a vaccine candidate through both in silico and in vivo analyses. Methods: The study examined the OmpA protein sequence for subcellular localization, antigenicity, allergenicity, similarity to the human proteome, physicochemical properties, B-cell epitopes, MHC binding sites, tertiary structure predictions, molecular docking, and immune response simulations. The ompA gene was cloned into the pET-28a (+) vector, expressed, purified and confirmed using Western blotting analysis. IgG levels in the serum of the immunized mice were measured using ELISA with dilutions ranging from 1:100 to 1:6400, targeting rOmpA and K. pneumoniae ATCC 13883. The sensitivity and specificity of the ELISA method were also assessed. Results: The bioinformatics analysis identified rOmpA as a promising vaccine candidate. The immunized group demonstrated significant production of specific total IgG antibodies against rOmpA and K. pneumoniae ATCC1 13883, as compared to the control group (p < 0.0001). The titers of antibodies produced in response to bacterial exposure did not show any significant difference when compared to the anti-rOmpA antibodies (p > 0.05). The ELISA test sensitivity was 1:3200, and the antibodies in the serum could accurately recognize K. pneumoniae cells. Conclusion: This study is a significant advancement in the development of a potential vaccine against K. pneumoniae that relies on OmpA. Nevertheless, additional experimental analyses are required.

Immunostimulatory chimeric protein encapsulated in gelatin nanoparticles elicits protective immunity against Pseudomonas aeruginosa respiratory tract infection.

This study presents the design and fabrication of an innovative vaccine candidate targeting Pseudomonas aeruginosa (P. aeruginosa). The vaccine consists of gelatin nanoparticles (GNPs) encapsulating a chimeric protein (CP) derived from the ExoS and OprI proteins from P. aeruginosa. The physicochemical properties of the GNPs were assessed using dynamic light scattering (DLS) and electron microscopy. The toxicity, encapsulation efficacy, release profile, and effectiveness of CP-encapsulated GNPs (CP-GNPs) in an animal model were investigated. The resulting nanovaccine demonstrated uniform spherical particles with an average size of 135 nm and an encapsulation efficiency of 85 %. The release assay revealed that 23 % of the antigen was released from the CP-GNPs after 20 days. The GNPs did not exhibit any toxic effects on L929 cells in vitro. The formulation induced both systemic and mucosal antibody responses. Additionally, CP-GNPs stimulated cytokine responses, including IFN-γ, IL-4, and IL-17, indicating the induction of both humoral (Th2) and cellular (Th1) responses. The CP-encapsulated GNPs formulation effectively protected the mice lungs against experimental respiratory tract infection, reducing colony count and inflammation. These findings suggest that CP-GNPs hold promise as a potential strategy for preventing respiratory tract infections caused by P. aeruginosa. Further research is needed to explore its clinical application.

Polydopamine-based nano adjuvant as a promising vaccine carrier induces significant immune responses against Acinetobacter baumannii-associated pneumonia.

The objective of this study was to assess the effectiveness of polydopamine nanoparticles (PDANPs) as a delivery system for intranasal antigen administration to prevent Acinetobacter baumannii (A. baumannii)-associated pneumonia. In the in vitro phase, the conserved outer membrane protein 22 (Omp22)-encoding gene of A. baumannii was cloned, expressed, and purified, resulting in the production of recombinant Omp22 (rOmp22), which was verified using western blot. PDANPs were synthesized using dopamine monomers and loaded with rOmp22 through physical adsorption. The rOmp22-loaded PDANPs were characterized in terms of size, size distribution, zeta potential, field emission scanning electron microscopy (FESEM), loading capacity, Fourier transform infrared spectroscopy (FTIR), release profile, and cytotoxicity. In the in vivo phase, the adjuvant effect of rOmp22-loaded PDANPs was evaluated in terms of eliciting immune responses, including humoral and cytokine levels (IL-4, IL-17, and IFN-γ), as well as protection challenge. The rOmp22-loaded PDANPs were spherical with a size of 205 nm, a zeta potential of -14 mV, and a loading capacity of approximately 35.7 %. The released rOmp22 from nontoxic rOmp22-loaded PDANPs over 20 days was approximately 41.5 %, with preserved rOmp22 integrity. The IgG2a/IgG1 ratio and IFN-γ levels were significantly higher in immunized mice with rOmp22-loaded-PDANPs than in rOmp22-alum, naive Omp22, and control groups. Furthermore, rOmp22-loaded PDANPs induced effective protection against infection in the experimental challenge and showed more normal structures in the lung histopathology assay. The results of this study suggest the potential of PDANPs as a nano-adjuvant for inducing strong immune responses to combat A. baumannii.

Antibiotic resistance and genetic diversity among Pseudomonas aeruginosa isolated from urinary tract infections in Iran.

Aims: To determine the antibiotic resistance and genetic diversity of Pseudomonas aeruginosa isolates. Methods: The antibiotic resistance, genetic diversity and the conjugate transformation among Pseudomonas aeruginosa collected from patients with urinary tract infection in Tehran, Iran, was investigated. Results: Antibiotic resistance against cefepime was seen in 51.74% of the isolates, followed by amikacin (47.76%). blaOXA-10 and blaVIM were the most prevalent extended-spectrum ß-lactamase and metallo-ß-lactamases genes, respectively. Five clusters (C1-C5) were obtained by pulse field gel electrophoresis and multilocus sequence typing revealed two strain types, ST235 and ST664. Conjugation detected blaOXA-48 and blaNDM genes were transferred to Escherichia coli K12. Conclusion: The resistance of P. aeruginosa to antibiotics is increasing, which highlights the need to determine the resistance patterns to design better treatment strategies.

Development of a multi-epitope vaccine candidate against Pseudomonas aeruginosa causing urinary tract infection and evaluation of its immunoreactivity in a rabbit model.

Pseudomonas aeruginosa is associated with different infections such as urinary tract infections (UTIs). The increased antibiotic resistance reaches the need to develop vaccine against the infections. In the present study, bioinformatics approaches were applied to design a novel multi-epitope of PcrV and OmpE from P. aeruginosa. The raised antibody against the multi-epitope was evaluated and challenge experiment was done to evaluate the efficacy of the multi-epitope. The results of epitope mapping of B-cells indicated 8 regions for PcrV and OmpE. The predicted 3D structure showed C-score = -1 and Z-score = -8.12. Molecular docking indicated high interaction between residues of Toll-like receptor 2 (TLR2) and TLR4 with the multi-epitope. The results of in silico simulation of the immune responses showed elevated levels of B-cell, T-cell, and memory cells. PcrV, OmpE, and the multi-epitope were expressed in pET28a-E. coli BL21 (DE3) and purified by Nickel columns. Our findings indicated that the sera collected from immunized rabbits with the multi-epitope reacted with the multi-epitope, PcrV, and OmpE in western blot. According to the ELISA results, the antibody developed against the multi-epitope showed cross-reactivity with individual proteins PcrV and OmpE. The level of antibody raised against the multi-epitope was significantly higher than the antibody reacted with PcrV or OmpE alone in ELISA. The challenge results confirmed that the load of bacteria was decreased in immunized rabbits as compared to the control. The results present the multi-epitope composed of PcrV and OmpE as a promising candidate against P. aeruginosa. Further evaluations are under investigation in animal model.Communicated by Ramaswamy H. Sarma.

Immunogenic Potential and Therapeutic Efficacy of Multi-Epitope Encapsulated Silk Fibroin Nanoparticles against Pseudomonas aeruginosa-Mediated Urinary Tract Infections.

Pseudomonas aeruginosa (P. aeruginosa) causing urinary tract infections (UTIs) are a major concern among hospital-acquired infections. The need for an effective vaccine that reduces the infections is imperative. This study aims to evaluate the efficacy of a multi-epitope vaccine encapsulated in silk fibroin nanoparticles (SFNPs) against P. aeruginosa-mediated UTIs. A multi-epitope is constructed from nine proteins of P. aeruginosa using immunoinformatic analysis, expressed, and purified in BL21 (DE3) cells. The encapsulation efficiency of the multi-epitope in SFNPs is 85% with a mean particle size of 130 nm and 24% of the encapsulated antigen is released after 35 days. The vaccine formulations adjuvanted with SFNPs or alum significantly improve systemic and mucosal humoral responses and the cytokine profile (IFN-γ, IL-4, and IL-17) in mice. Additionally, the longevity of the IgG response is maintained for at least 110 days in a steady state. In a bladder challenge, mice treated with the multi-epitope admixed with alum or encapsulated in SFNPs demonstrate significant protection of the bladder and kidneys against P. aeruginosa. This study highlights the promising therapeutic potential of a multi-epitope vaccine encapsulated in SFNPs or adjuvanted with alum against P. aeruginosa infections.

Design and fabrication of a vaccine candidate based on rOmpA from Klebsiella pneumoniae encapsulated in silk fibroin-sodium alginate nanoparticles against pneumonia infection.

The present study describes the design and fabrication of a novel vaccine candidate based on the outer membrane protein A (rOmpA) from Klebsiella pneumoniae (K. pneumoniae) encapsulated in silk fibroin-sodium alginate nanoparticles (SF-SANPs) against K. pneumoniae-mediated pneumonia. The physicochemical properties, toxicity, release profile, and in vivo potency of SF-SANPs encapsulated with rOmpA were evaluated. The spherical nano vaccine was created with an average particle size of 160 nm and an encapsulation efficiency of 80 %. Antigen release from SF-SANPs was 40 % after 22 days release assay. The SF-SANPs showed a zeta potential of -24.8 mV and had no toxic effect on the L929 cells in vitro. It was found that SF-SANPs in the vaccine formulation promoted systemic and mucosal antibodies and also stimulated cytokine responses, inducing both humoral (Th2) and cellular (Th1) immune responses, with a Th1-polarized response. The vaccine candidate was effective in protecting the mice lung against experimental pneumonia and reducing inflammation. These findings suggest that the rOmpA-based vaccine encapsulated in SF-SANPs could be a promising strategy for preventing pneumonia caused by K. pneumoniae.

Introduction of Novel Drug Targets against Staphylococcus aureus and Proposing Putative Inhibitors against Adenine N1 (m1A22)-tRNA Methyltransferase (TrmK) using Computer-aided Drug Discovery.

BACKGROUND: Nowadays, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains has dramatically restricted the treatment options against this microorganism. AIM: In this study, we aimed to discover new drug targets and inhibitors against S. aureus. METHODS: This study consists of two major sections. In the upstream evaluation, after a comprehensive coreproteome analysis, essential cytoplasmic proteins with no similarity to the human proteome were selected. Then the S. aureus metabolome-specific proteins were selected, and novel drug targets were identified using the DrugBank database. In the downstream analysis, a structure-based virtual screening approach was performed to reveal potential hit compounds against adenine N1 (m(m1A22)-tRNA methyltransferase (TrmK) using the StreptomeDB library and AutoDock Vina software. The compounds with a binding affinity > -9 kcal/mol were analyzed based on ADMET properties. Finally, the hit compounds were selected based on Lipinski's rule of five (RO5). RESULTS: Three proteins, including glycine glycosyltransferase (FemA), TrmK, and heptaprenyl pyrophosphate synthase subunit A (HepS1), were selected as feasible and promising drug targets based on PDB file availability and their essential role in the survival of the S. aureus. Finally, seven hit compounds, including Nocardioazine_ A, Geninthiocin_D, Citreamicin_delta, Quinaldopeptin, Rachelmycin, Di-AFN_A1 and Naphthomycin_ K were introduced against the binding cavity of TrmK, as a feasible drug target. CONCLUSION: The results of this study provided three feasible drug targets against S. aureus. In the following, seven hit compounds were introduced as potential inhibitors of TrmK, and Geninthiocin_D was identified as the most desirable agent. However, in vivo and in vitro investigations are needed to confirm the inhibitory effect of these agents on S. aureus.