Search

Background Pertussis is a current contagious bacterial disease caused by Bordetella pertussis (Bp). Given the prevalence of pertussis, development of new vaccines is important. This study was attempted to evaluate the expression of main virulence factors (pertussis toxin [PTX], PRN [pertactin], and filamentous hemagglutinin [FHA]) from Bp predominant strains and also compare the expression of these factors in the outer membrane vesicles (OMVs) obtained from predominant circulating Bp isolate. Methods The physicochemical features of the prepared OMVs were analyzed by electron microscopy and SDS-PAGE. The presence of the mentioned virulence factors was confirmed by Western blotting. BALB/c mice (n = 21) immunized with characterized OMVs were challenged intranasally with sublethal doses of Bp, to examine their protective capacity. Results Electron microscopic examination of the OMVs indicated vesicles within the range of 40 to 200 nm. SDS-PAGE and Western blotting demonstrated the expression of all three main protective immunogens (PTX, PRN, and FHA), prevalent in the predominant, challenge, and vaccine strains, and OMVs of the predominant IR37 strain and BP134 vaccine strain. Significant differences were observed in lung bacterial counts between the immunized mice with OMV (30 CFU/lung) compared to the negative control group ((6 104 CFU/lung; p < 0.001). In mice immunized with OMVs (3 µg), the number of lungs recovered colonies after five days dropped at least five orders of magnitude compared to the control group. Conclusion OMVs obtained from circulating isolates with the predominant profile may constitute a highly promising vaccine quality. They also can be proposed as a potential basic material for the development of new pertussis vaccine candidate.

Background and Objectives: There are many pertussis outbreaks which is mainly due to the reduction in the immunity of acellular pertussis (aP) vaccines. Therefore, there is a crucial necessity to develop a new generation of pertussis vaccine. Pre- ceding researches have shown that Bordetella pertussis outer membrane vesicles (OMVs) have appropriate specifications, making them a suitable vaccine candidate against pertussis. Materials and Methods: The OMVs were separated by a new serial ultra centrifugation technique. Transmission electron microscopy (TEM) examination, SDS-PAGE, Western blotting and ELISA assay were used to characterize the OMVs. Results: TEM studies showed the size of the extracted OMVs at 40-200 nm. The presence of pertussis toxin, filamentous hemagglutinin, and pertactin was verified using Western blot and ELISA assay. Conclusion: The presented technique is a simple and effective way to obtain OMVs from Bordetella pertussis. So it can be utilized as an appropriate procedure in the development of an OMV-based vaccine against pertussis.

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough. Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX). Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering. Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91. Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.

In spite of high vaccination coverage in the Expanded Program of Immunization (EPI), pertussis has not been eradicated yet and the re-emergence of the disease is still reported worldwide. The genetic divergence study of circulating clinical strains of Bordetella pertussis among the population with high vaccination coverage is a useful tool to have an insight in the understanding of genetic patterns of this bacterium and deviation of them from vaccine strains. Different methods are accessible for studying of Bordetella pertussis that can perform appropriate assessment between populations. Strains used in this study were a collection of two pertussis vaccine strains used to create killed pertussis vaccine over years at Razi Vaccine and Serum Research Institute, 10 clinical and 2 reference strains (ATCC9797 and Tohama I) in Multilocus Sequence Typing (MLST), Pulsed-Field Gel Electrophoresis (PFGE), and serotyping. The genetic profiles of vaccine working and master seeds showed no important change(s) in frequencies of fingerprint types investigated in the vaccine strains and had homogeneity in PFGE method where the clinical isolates showed diversity in genetic profile. Serotyping method showed that all of 10 clinical strains expressing Fim 3. In MLST study, seven housekeeping genes including adk, pgm, fum C, tyr B, gly A, pep A and icd were analyzed which showed no changes in the sequence of clinical and vaccine strains with 100% homology. The genes that cause pathogenicity like ptxC, tcfA and fhaB were also evaluated and the results illustrated heterogeneity in the vaccine and circulating strains.

Background and Objectives: Leptospirosis is a widespread zoonotic disease that is transmitted directly or indirectly from animals to humans. Humans mainly acquire pathogenic leptospires through mucosal or percutaneous exposure to environment contaminated with urine from an infected animal. We aimed to identify pathogenic leptospiral serovars by detection of the ompL37 gene using polymerase chain reaction (PCR). Methods: Sixteen pathogenic leptospiral serovars and a saprophytic serovar, L. biflexa were cultured in modified semisolid Ellinghausen-McCullough-Johnson-Harris medium containing 5% rabbit serum. Genomic DNA extraction was done using the phenol-chlorophorm method. The ompL37 gene was amplified using specific primers. PCR products were analyzed by agarose gel electrophoresis. Results: The ompL37 gene was amplified only in the pathogenic leptospiral serovars. We detected no amplified fragment for the saprophytic serovar. Conclusion: Leptospirosis may be confused with other infectious diseases, and therefore, its early and accurate diagnosis is crucial. We showed that molecular detection of pathogenic leptospires based on the ompL37 gene could be used for laboratory diagnosis of leptospirosis. Keywords: Leptospirosis, PCR, ompl37 Gene, Pathogenic Leptospires.

Introduction: Pathogen adaptation is considered as one of the important reasons for the emergence of pertussis (whooping cough). Antigenic divergence between vaccine strains and clinical isolates of Bordetella pertussis has been occurred over the years. It is suggested that the predominant genomic profile of B. pertussis has an enough capacity to spread among the population. The aim of this study was to characterize a predominant B. pertussis strain isolated from Iranian patients during 2008-2015 period. Methods: Based on the epidemiologic results of B. pertussis circulating strains in Iran, a strain named BPIP91 with predominant genomic and virulence pattern was selected from Biobank of Pasteur Institute of Iran. The antibiotic susceptibility testing was done and the growth rate of this strain was analyzed. The lethal (LD50) and safety dose of infection of BPIP91 was also determined via mice intranasal infection. Results: Our results showed that BPIP91 was susceptible to erythromycin, azithromycin, clarithromycin, chloramphenicol, trimethoprim-sulfamethoxazole and rifampin antibiotics. The growth rate of BPIP91 was almost two-fold lower than the vaccine strain. In addition, the LD50 and infectious dose of BPIP91 strain were about 2 × 1010 and 4 × 106 colony forming units, respectively. Conclusion: In this study we obtained the growth curve, LD50 and intranasal infectious dose of a circulating strain with predominant genomic pattern in Iran. However, further examinations including determination of immunogenicity of this native strain in animal model is needed in order to evaluate its use as a vaccine strain candidate.

Introduction: In spite of high vaccination coverage, whooping cough (pertussis) is still a worldwide health problem. The main reason for pertussis outbreak is waning immunity of safer acellular vaccines which have replaced the more reactogenic cellular vaccines. A new generation of pertussis vaccines that is potent and safe is desperately needed to control the disease. Previous studies have indicated that outer membrane vesicles (OMVs) obtained from Bordetella pertussis have desirable characteristics which make them a good candidate for application as pertussis vaccine. They contain surface immunogens in a native structure, are self-adjuvant and are easily uptaken by the antigen presenting cells. Methods: B. pertussis Tohama strain was cultured at 35°C in Stainer-Scholte broth. The OMVs were isolated by a new sequential ultracentrifugation method. The extracted OMVs were characterized by electron microscopy, SDS-PAGE and ELISA assays. Results: The existence of pertussis toxin, filamentous haemagglutinin and a 69-kDa antigen in B. pertussis OMVs was verified using an ELISA assay. Electron microscopy showed the size of these OMV’s at 40-200 nm. The ELISA results indicated that the OMVs extracted using this protocol contain major immunogens. Conclusion: We report for the first time a simple protocol for the efficient extraction of B. pertussis OMVs. This protocol can be used in the process of making new generations of B. pertussis vaccines.

Background: Outer membrane vesicles (OMVs) release from Gram-negative bacteria and are interesting alternatives that can replace those vaccines that contain naturally incorporated bacterial surface antigens, lipopolysaccharides (LPS) and outer membrane proteins (OMPs). Nanoparticles can be used to encapsulate vesicles for slow release and prevent macromolecular degradation. Objective: Therefore, encapsulation of OMVs of B. pertussis into sodium alginate nanoparticles was the main aim of the current study. Methods: The OMVs of B. pertussis extracted and characterized by particle sizer, electron microscopy, SDSPAGE and Western blot assays. The extracted OMVs were encapsulated in sodium alginate nanoparticles (OMV-NP) using unique gelation process and injected into BALB/c mice. Immunogenicity indices such as different classes of antibodies and interleukins related to different T cell lines were evaluated in immunized mice by ELISA. The respiratory challenge was evaluated in the groups of mice. The existence of pertussis toxin (PTX), filamentous haemagglutinin (FHA) and PRN (pertactin) in B. pertussis OMVs was verified using SDSPAGE and Western blot analysis. Results: TEM electron microscopy showed the size of these OMVs to be around 20-80 nm. The OMVs with appropriate quality were encapsulated into sodium alginate nanoparticles (OMV-NP), and the final size was about 500 nm after encapsulation. Immunity indices were significantly higher in the OMV-NP receiving group. In challenge tests, the OMV-NP vaccine was able to show the highest rate of lung clearance compared to the control groups (OMV and wPV) at the lowest injection dose. Conclusion: The results indicate the potential of OMV-NP as a novel vaccine delivery system.

Background: Lately, the employment of nano-carriers has been known as an optimistic means of drug and vaccine delivery. Objective: Nano vaccines are a novel tier of vaccines that can develop humoral and cellular immune responses and can be introduced as a practical and secure nano vaccine candidate for the prevention of diseases. The purpose of this study was to accomplish and use biodegradable nano-carriers for the synthesis of pentavalent vaccine and immunogenicity evaluation in the animal models. The PLGA nanoparticle was prepared and modified with chitosan nanoparticles. Nano-carrier PLGA-chitosan was loaded by the DTP-HepB-Hib antigens and confirmed by DLS, SEM, TEM and FT-IR, then in vitro loading and release were evaluated. Toxicity was assessed by the MTT method in the Hec293 cells. Antigenicity evaluation and histopathological study were conducted by injection of new nano pentavalent vaccines in the BALB/c mice and the immune response was measured in the serum samples through an indirect ELISA method. Results: Conclusions drawn from the current study exhibited the plausible ability of nano-carrier to deliver the vaccine. A notable increase was shown in total IgG and IgM antibodies examined in mice vaccinated with new nano vaccines. The histopathological study in the treated mice showed no toxicity in the vital organs of mice. Conclusion: The engineered vaccine delivery system showed the ability to induce robust immune responses and also the suitability features of the PLGA-chitosan as a promising carrier to improve vaccine delivery.

Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M. haemolytica is surrounded by a polysaccharide capsule, and the degree of OmpA surface exposure is unknown. To investigate surface exposure and immune specificity of OmpA among bovine and ovine M. haemolytica isolates, recombinant proteins representing the transmembrane domain of OmpA from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were overexpressed, purified, and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. Immunogold electron microscopy and immunofluorescence techniques demonstrated that OmpA1 and OmpA2 are surface exposed, and are not masked by the polysaccharide capsule, in a selection of M. haemolytica isolates of various serotypes and grown under different growth conditions. To explore epitope specificity, anti-rOmpA1 and anti-rOmpA2 antibodies were cross-absorbed with the heterologous isolate to remove cross-reacting antibodies. These cross-absorbed antibodies were highly specific and recognized only the OmpA protein of the homologous isolate in Western blot assays. A wider examination of the binding specificities of these antibodies for M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognized OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognized OmpA2-type proteins but not OmpA1-type proteins. Our results demonstrate that OmpA1 and OmpA2 are surface exposed and could potentially bind to different receptors in cattle and sheep.

A protein designated Bap-5 (GenBank accession no. AF081494 ) or BapC (GenBank accession no. AJ277634 ) has been identified as a member of the Bordetella pertussis autotransporter family and the present work suggests that this protein, like the previously characterised BrkA, is a Bvg-regulated serum resistance factor and virulence determinant. B. pertussis bapC and brkA , bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA , bapC mutants, like the brkA mutant, were found to be more sensitive to the antimicrobial peptide cecropin P1 than the parent strains. In the genome sequence of B. pertussis strain Tohama, bapC is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5′ end of the gene which creates a truncated BapC protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms but it appeared that all had an ORF that would be able to produce BapC.