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.

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.

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.

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 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.

Preparation and pre-clinical evaluation of flagellin-adjuvanted NOM vaccine candidate formulated with Spike protein against SARS-CoV-2 in mouse model.

From December 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was started as a cluster of pneumonia cases in Wuhan, Hubei Province, China. The disturbing statistics of SARS-CoV-2 promoted scientists to develop an effective vaccine against this infection. NOM protein is a multi-epitope protein that designed based on Nucleocapsid, ORF3a, and Membrane proteins of SARS-CoV-2. Flagellin is a structural protein that binds to the Toll-like receptor 5 and can enhance the immune response to a particular antigen. In this study, NOM protein as vaccine candidate was linked to the carboxyl and amino terminals of flagellin adjuvant derived from Salmonella enterica subsp. enterica serovar Dublin. Then, informatics evaluations were performed for both NOM protein and NOM protein linked to flagellin (FNOM). The interaction between the NOM and FNOM proteins with the TLR5 were assessed using docking analysis. The FNOM protein, which compared to the NOM protein, had a more suitable 3D structure and a stronger interaction with TLR5, was selected for experimental study. The FNOM and Spike (S) proteins expressed and then purified by Ni-NTA column as vaccine candidates. For analysis of immune response, anti-FNOM and anti-S proteins total IgG and IFN-γ, TNF-α, IL-6, IL-10, IL-22 and IL-17 cytokines were evaluated after vaccination of mice with vaccine candidates. The results indicated that the specific antisera (Total IgG) raised in mice that received FNOM protein formulated with S protein were higher than mice that received FNOM and S proteins alone. Also, IFN-γ and TNF-α levels after the spleen cells stimulation were significantly increased in mice that received the FNOM protein formulated with S protein compared to other groups. Immunogenic evaluations showed that, the FNOM chimeric protein could simultaneously elicit humoral and cell-mediated immune responses. Finally, it could be concluded that the FNOM protein formulated with S protein could be considered as potential vaccine candidate for protection against SARS-CoV-2 in the near future.

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.

Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Bioinformatics analyses for the designation of a hybrid protein against urinary tract infections caused by Pseudomonas aeruginosa and investigation of the presence of pre-existing antibodies in infected humans.

Pseudomonas aeruginosa is an important pathogen causing urinary tract infections (UTIs) and resistance to antibiotics has increased the need for a vaccine against this bacterium. P. aeruginosa V-antigen (PcrV), which is a component of the type III secretion system, delivers exoenzymes such as exoenzyme S (ExoS) into the host cells. In the present study, we aimed to design and express a hybrid protein composed of PcrV and ExoS from P. aeruginosa using bioinformatics. Finally, pre-existing antibodies were evaluated in sera collected from patients with UTI. The prediction results showed that the hybrid protein ExoS.PcrV had a C-score of -0.85 and Z-score of -5.55 versus C-score of -2.93 and Z-score of -2.65 for PcrV.ExoS. Based on BepiPred and ABCpred, 15 and 14 linear B-cell epitopes, as well as five conformational epitopes were identified in ExoS.PcrV. The interaction between the protein and immune receptor was validated in silico. Molecular docking indicated that the hybrid protein interacted strongly with Toll-like receptor 2. ExoS.PcrV was expressed in pET28a-BL21 and purified with a size of 57 kD by Nickel resins. The protein reacted with all sera collected from humans infected with P. aeruginosa following Western blot. The infected patients produced significantly higher IgG levels against the protein compared to the control as indicated by ELISA. The protein ExoS.PcrV was determined as a promising candidate against UTI caused by P. aeruginosa and the presence of pre-existing antibodies indicated the advantage of the hybrid protein. Evaluation of the efficacy of hybrid protein is ongoing in mice model. Communicated by Ramaswamy H. Sarma.

Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

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.

Immunization with recombinant protein Ag43::UpaH with alum and 1,25(OH)2D3 adjuvants significantly protects Balb/C mice against urinary tract infection caused by uropathogenic Escherichia coli.

The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Designing a vaccine will certainly reduce the occurrence of infection and antibiotic resistance of the isolates. Antigen 43 (Ag43) and autotransporter H (UpaH) have been associated with the virulence of UPEC. In the present study, the efficacy of different formulations of a hybrid protein composed of Ag43 and UpaH with and without alum and 1,25(OH)2D3 (Vitamin D3) adjuvants were evaluated in mice model. A significant increase in IgG and cellular responses was developed against Ag43::UpaH as compared to the control mice. The addition of alum or a mixture of alum and Vitamin D3 to the protein significantly enhanced the serum IgG responses and tended to remain in a steady state until 6 months. In addition, the mentioned formulations produced significant amounts of IgG1, IL-4, and IL-17 as compared to the fusion protein alone. In addition to the mentioned formulations, the combination of protein with Vitamin D3 also resulted in significantly higher serum IgA and IFN-γ levels as compared to the fusion protein alone. Mice immunized with fusion plus alum and formulation protein admixed with both alum and Vitamin D3 significantly reduced the bacterial load in the bladders and kidneys of mice as compared to the control. In this study, for the first time, the ability of a novel hybrid protein in combination with adjuvants alum and Vitamin D3 was evaluated against UPEC. Our results indicated that fusion Ag43::UpaH admixed with alum and Vitamin D3 could be a promising candidate against UTIs.

Protective multi-epitope candidate vaccine for urinary tract infection.

Urinary tract infections (UTIs) are induced by exogenous organisms including extraintestinal pathogenic such as Escherichia coli (ExPEC), Proteus mirabilis and Klebsiella pneumonia, which are closely related. These organisms can colonize in the urinary tract and cause UTIs. In this study, a cross-reactive multi-epitope vaccine was designed by two constructs to stimulate the immune system (CD8+ and CD4 + T cells) against ExPEC, Proteus mirabilis and Klebsiella pneumonia strains. Uropathogenic Escherichia coli (UPEC), Proteus mirabilis and Klebsiella pneumoniae are the main bacterial cause of UTI. They were used for designing experimental candidate vaccine, and their immunogenicity and protectivity were assessed. In this study, conserved antigens from their bacterial genomes were considered, and informatics-based immunological vaccine with cross-protective T and B-cells epitopes was designed and evaluated. The vaccine candidate was used as a broad immune system inducer, and its cross-protective immunity and protectivity were confirmed in in vivo experiments.

In silico analysis and in vivo assessment of a novel epitope-based vaccine candidate against uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are common pathogens in urinary tract infections (UTIs), which show resistance to antibiotics. Therefore, there is a need for a vaccine to reduce susceptibility to the infection. In the present study, bioinformatics approaches were employed to predict the best B and T-cell epitopes of UPEC virulence proteins to develop a multiepitope vaccine candidate against UPEC. Then, the efficacy of the candidate was studied with and without Freund adjuvant. Using bioinformatics methods, 3 epitope-rich domains of IutA and FimH antigens were selected to construct the fusion. Molecular docking and Molecular dynamics (MD) simulation were employed to investigate in silico interaction between designed vaccine and Toll-like receptor 4 (TLR4). Our results showed that the levels of IgG and IgA antibodies were improved in the serum and mucosal samples of the vaccinated mice, and the IgG responses were maintained for at least 6 months. The fusion protein was also able to enhance the level of cytokines IFN.γ (Th1), IL.4 (Th2), and IL.17. In challenge experiments, all vaccine combinations showed high potency in the protection of the urinary tract even after 6 months post first injection. The present study indicates that the designed candidate is able to evoke strong protective responses which warrant further studies.

A Synthetic Peptide 2Abz23S29 Reduces Bacterial Titer and Induces Pro-Inflammatory Cytokines in a Murine Model of Urinary Tract Infection.

INTRODUCTION: A urinary tract infection (UTI), which is often caused by uropathogenic E. coli (UPEC) strains, affects many people worldwide annually. UPEC causes the production of pro-inflammatory cytokines by the bladder epithelial cells; however, it has been proven that the UPEC can inhibit the early activation of the innate immune system. METHODS: This study aimed to examine the antibacterial and immunomodulatory effects of different doses of truncated alpha-defensins (human neutrophil peptide (HNP)-1) analog 2Abz23S29 on the mouse UTI model. Experimentally uropathogenic E. coli CFT073-infected mice were treated with low-dose 2Abz23S29 (250µg/mL), high-dose 2Abz23S29 (750µg/mL), ciprofloxacin (cip) (800µg/mL), or high-dose 2Abz23S29plus cip once a day 24 h post-infection. The 2Abz23S29 and cip treatment were given for two consecutive days. RESULTS: The in vivo results showed that fewer UPEC were recovered from the bladders of mice treated transurethrally with 2Abz23S29. Moreover, low-dose 2Abz23S29 significantly decreased the level of the interleukin-6 (IL-6), whereas high-dose 2Abz23S29 increased pro-inflammatory cytokines including IL-6, macrophage inflammatory protein/2 (MIP/2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in infected bladders of mice. Besides, the levels of cytokines IL-6 and MIP/2 in infected mice treated with a combination of high-dose 2Abz23S29 and cip were significantly higher than the untreated mice. In contrast, CFT073-infected mice treated with a combination of high-dose 2Abz23S29 and cip showed no changes in cytokines TNF-α and IL-1ß levels, indicating that ciprofloxacin may play an anti-inflammatory role. CONCLUSION: Collectively, apart from the direct antibacterial role of 2Abz23S29, our data illustrated that 2Abz23S29 modulates pro-inflammatory cytokine production of bladder in a dose-dependent manner, which has implications for the development of new anti-infective agents.

Pasteurella multocida Vaccine Candidates: A Systematic Review.

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.

Construction and assessment of the immunogenicity and bactericidal activity of fusion protein porin A from Neisseria meningitidis serogroups A and B admixed with OMV adjuvant as a novel vaccine candidate.

OBJECTIVES: The porins A and B and also outer membrane vesicles (OMVs) of Neisseria meningitidis are used for vaccine purposes. In the present study, we aimed to design a new vaccine candidate based on a fusion of PorA of serogroups A and B of N. meningitidis admixed with OMV and evaluate it in an animal model. MATERIALS AND METHODS: After bioinformatic studies, a fusion protein composed of porin A from both serogroups A and B of N. meningitidis was constructed, expressed, and purified by nickel resins. Extraction of OMV of N. meningitidis was performed using a chemical method. The mice were vaccinated subcutaneously in different groups with mixtures of PorA proteins, OMV, and Freund's adjuvants. Then, the immune responses were measured using the ELISA method. Finally, serum bactericidal activity (SBA) procedure was applied to assay the activity of the immune responses in mice. RESULTS: Mice received the PorA protein plus Freund's adjuvant. Mice vaccinated with PorA fusion of serogroups A+B plus Freund's adjuvant produced more IgG, IgG1, and IgG2a than combinations admixed with OMV. Furthermore, the vaccinated mice tended to direct the IgG responses toward IgG1. Sera of the mice that received PorA+Freund's and those that received PorA+OMV produced higher bactericidal activity than the controls. CONCLUSION: Fusion protein porin A could be a valuable target for developing vaccines against N. meningitidis. Although, Freund's adjuvant induced the strongest IgG responses, given that Freund's adjuvant has no human use, and OMV is a human adjuvant, OMV could be considered in vaccine design against N. meningitidis.

Inhibition and eradication activity of truncated α-defensin analogs against multidrug resistant uropathogenic Escherichia coli biofilm.

Today the development of antibiotic resistance, especially in the treatment of bacterial infections associated with biofilms, has led to increasing the importance of antimicrobial peptides (AMPs). In this work, antimicrobial and synergistic activity of three truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) with ß-lactam (amoxicillin and cefixime) and fluoroquinolones (ciprofloxacin and norfloxacin) antibiotics against multidrug-resistant (MDR) uropathogenic E. coli clinical isolates were evaluated. The anti-biofilm potential of peptides at different stages was also investigated. All peptides exhibited additive activity just with ß-lactam antibiotics in a checkerboard synergy assay. Inhibition and eradication of MDR uropathogenic E. coli biofilm were shown by all test peptides at different concentrations. Thus, truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) may have the potential for the treatment of urinary tract infections (UTIs) caused by biofilm-forming MDR uropathogenic E. coli.