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.

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.

Design peptide and multi-epitope protein vaccine candidates against monkeypox virus using reverse vaccinology approach: an in-silico study.

Monkeypox is a zoonotic virus that has recently affected different countries worldwide. On July 23, 2022, the WHO declared the outbreak of monkeypox as a public health emergency of international concern. Surveillance studies conducted in Central Africa in the 1980s and later during outbreaks in the same region showed smallpox vaccines to be clinically somewhat effective against Monkeypox virus. However, there is no specific vaccine against this virus. This research used bioinformatics techniques to establish a novel multi-epitope vaccine candidate against Monkeypox that can induce a strong immune response. Five well-known antigenic proteins (E8L, A30L, A35R, A29L, and B21R) of the virus were picked and assessed as possible immunogenic peptides. Two suitable peptide candidates were selected according to bio-informatics analysis. Based upon in silico evaluation, two multi-epitope vaccine candidates (ALALAR and ALAL) were built with rich-epitope domains consisting of high-ranking T and B-cell epitopes. After predicting and evaluating the 3D structure of the protein candidates, the most efficient 3D models were considered for docking studies with Toll-like receptor 4 (TLR4) and the HLA-A * 11:01, HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*07:02, HLA-A*15:01, HLA-A*30:01 receptors. Subsequently, molecular dynamics (MD) simulation of up to 150 nanoseconds was employed to assess the durability of the interaction of the vaccine candidates with immune receptors. MD studies showed that M5-HLA-A*11:01, ALAL-TLR4, and ALALAR-TLR4 complexes were stable during simulation. Analysis of the in silico outcomes indicates that the M5 peptide and ALAL and ALALAR proteins may be suitable vaccine candidates against the Monkeypox virus.Communicated by Ramaswamy H. Sarma.

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.

Evaluation of the dual effects of antiviral drugs on SARS-CoV-2 receptors and the ACE2 receptor using structure-based virtual screening and molecular dynamics simulation.

The use of US FDA-approved drugs is preferred due to the need for lower costs and less time. In in silico medicine, repurposing is a quick and accurate way to screen US FDA-approved medications to find a therapeutic option for COVID-19 infection. Dual inhibitors possess dual inhibitory activity, which may be due to the inhibition of two different enzymes, and are considered better than combination therapy from the developmental and clinical perspectives. In this study, a molecular docking simulation was performed to identify the interactions of antiviral drugs with the critical residues in the binding site of the main SARS-CoV-2 protease, spike glycoprotein, and papain-like protease receptors compared to the angiotensin-converting enzyme-related carboxypeptidase (ACE2) receptor of host cells. Each of the receptors was docked with 70 US FDA-approved antiviral drugs using AutoDock Vina. A molecular dynamics (MD) simulation study was also used for 100 ns to confirm the stability behaviour of the ligand receptor complexes. Among the drugs that had the strongest interaction with the SARS-CoV-2 main protease, spike glycoprotein and papain-like protease receptors, and host cell ACE2 receptors, Simeprevir, Maraviroc and Saquinavir had dual inhibitory effects. The MD simulation study confirmed the stability of the strongest interactions between the antiviral drugs and the main protease, ACE2, spike glycoprotein, and papain-like protease receptors to 100 ns. However the results of MMPBSA analysis showed that the bond between Saquinavir and the ACE2 receptor was weak. Simeprevir and Maraviroc drugs had acceptable binding energies with dual receptors, especially the Simeprevir.Communicated by Ramaswamy H. Sarma.

Genome-Wide Subtraction Analysis and Reverse Vaccinology to Detect Novel Drug Targets and Potential Vaccine Candidates Against Ehrlichia chaffeensis.

Human monocytotropic ehrlichiosis is an emerging tick-borne infection caused by the obligate intracellular pathogen, Ehrlichia chaffeensis. The non-specific symptoms can range from a self-limiting fever to a fatal septic-like syndrome and may be misdiagnosed. The limited treatment choices including doxycycline are effective only in the initiation phase of the infection. It seems that novel therapeutic targets and new vaccine strategies could be effective to control this pathogen. This study is comprised of two major phases. First, the common proteins retrieved through subtractive analysis and potential drug targets were evaluated by subcellular localization, homology prediction, metabolic pathways, druggability, essentiality, protein-protein interaction networks, and protein data bank availability. In the second phase, surface-exposed proteins were assessed based on antigenicity, allergenicity, physiochemical properties, B cell and T cell epitopes, conserved domains, and protein-protein interaction networks. A multi-epitope vaccine was designed and characterized using molecular dockings and immune simulation analysis. Six proteins including WP_011452818.1, WP_011452723.1, WP_006010413.1, WP_006010278.1, WP_011452938.1, and WP_006010644.1 were detected. They belong to unique metabolic pathways of E. chaffeensis that are considered as new essential drug targets. Based on the reverse vaccinology, WP_011452702.1, WP_044193405.1, WP_044170604.1, and WP_006010191.1 proteins were potential vaccine candidates. Finally, four B cell epitopes, including SINNQDRNC, FESVSSYNI, SGKKEISVQSN, and QSSAKRKST, were used to generate the multi-epitope vaccine based on LCL platform. The vaccine showed strong interactions with toll-like receptors and acceptable immune-reactivity by immune simulation analysis. The findings of this study may represent a turning point in developing an effective drug and vaccine against E. chaffeensis. However, further experimental analyses have remained.

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 Candidate Antigens to Achieving an Effective Vaccine against Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that causes various inflammatory local infections, from those of the skin to postinfectious glomerulonephritis. These infections could result in serious threats, putting the life of the patient in danger. Antibiotic-resistant S. aureus could lead to dramatic increases in human mortality. Antibiotic resistance would explicate the failure of current antibiotic therapies. So, it is obvious that an effective vaccine against S. aureus infections would significantly reduce costs related to care in hospitals. Bacterial vaccines have important impacts on morbidity and mortality caused by several common pathogens, however, a prophylactic vaccine against staphylococci has not yet been produced. During the last decades, the efforts to develop an S. aureus vaccine have faced two major failures in clinical trials. New strategies for vaccine development against S. aureus has supported the use of multiple antigens, the inclusion of adjuvants, and the focus on various virulence mechanisms. We aimed to present a compressive review of different antigens of S. aureus and also to introduce vaccine candidates undergoing clinical trials, from which can help us to choose a suitable and effective candidate for vaccine development against S. aureus.

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.

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.

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.

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.

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.