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.
Introduction: Serum bactericidal assay is the gold standard index of protection against Vibrio cholerae. The outer membrane vesicles (OMVs) which are released during the bacterial growth have intrinsic immune stimulatory properties, based on their nature and composition. In this study, the induction of serum bactericidal activities in immunized BALB/c mice was determined using different vaccine regimens, using V. cholerae O1 (El Tor biotype) OMVs. Methods: A single clone of V. cholerae O1 (El Tor biotype) isolated during the 2005 outbreak in Iran, was used. A detergent-centrifugation procedure was used for OMVs production. Various vaccination regimens were inoculated into female mice via an oral route. The vaccine formulas included V. cholerae OMVs, killed whole cells of V. cholerae (WC), combination of WC-OMV and licensed cholera vaccine (Dukoral).The serum vibriocidal activity of mice sera was determined by measuring the complement-mediated lysis. Results: Electron microscopy of the purified OMVs from the isolated V. cholerae revealed the spherical-shaped vesicles of the size range 20-300 nm. In vitro reactivity of mice sera bactericidal capability against regimens of vaccination showed a significant immune response of antibody titers in comparison with negative control groups. Also, there was a significant increase in serum bactericidal titer of WC-OMV obtained from wild-type V. cholerae which had a satisfactory reactivity as Dukoral cholera vaccine. Conclusion: The results indicated that the combination of WC-OMV from the local strain is able to induce a high level of bactericidal antibody responses and it can be useful in optimization of the vaccine formula.
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 The secretion of extracellular vesicles (EVs) has been neglected in Gram-positive bacteria due to the absence of an outer membrane and the difficulties of proper visualization. Here we aimed to prove that lactobacillus casei can secrete extracellular vesicles. Methods: EVs were extracted from Lactobacillus casei, cultured in De Man, Rogosa and Sharpe broth, using a polyethylene glycol (PEG) solution. The characteristics of the EVs were analyzed by electron microscopy, Dynamic Light Scattering (DLS) and SDS-PAGE. Results: The electron microscopy showed rounded vesicles with average diameter of 300 nm. The protein content of this nanostructure was 2.5 mg/ml with a protein pattern within the range of 10-200 kDa. DLS result showed populations of approximately 300 nm while the extracted EVs had a negative zeta potential. Conclusion: A new method of producing functional molecules from probiotic bacteria was presented. Our results indicated EVs purity with acceptable conformation. Further investigations are necessary to elucidate the efficacy, practicality and mechanism of action of such EVs in clinical practices, especially for development of bio-compounds and vaccine delivery vehicles.
Introduction: Leishmania infantum is the causative agent of visceral leishmaniasis (VL). Cell-mediated immunity (CMI) is required to control leishmaniases. Therefore, simple tests that can evaluate the cellular immunity of the target populations can help to understand the immune status of the human subjects, their immunity to the re-infection and the evaluation of the effectiveness of the potential vaccines. Here, we compared antigens based on single clones of L. infantum promastigotes and axenic amastigotes by in vitro and in vivo tests. Methods: Using serial dilutions, L. infantum promastigotes were selected as single clones (PSC) or were grown under axenic conditions with succinate-tris to prepare amastigote-like single clones (ASC). Antigens prepared from PSC and ASC were then compared with typical Leishmania major and L. infantum amastigotes by SDS-PAGE, Western-blotting and proliferation tests as well as an in vivo delayed-type hypersensitivity test on guinea pigs. Results Both PSC and ASC exhibited a distinctive ~50-kDa band could be detected by Western-blotting. The proliferation tests results indicated that both PSC and ASC could cause higher lymphocyte proliferation compared to typical L. infantum and L. major promastigotes; however the differences were not significant. Moreover, both PSC and ASC had an ability to induce comparable DTH and hence CMI. Conclusion: Similar proliferation or delayed-type hypersensitivity could be caused with antigens based on PSC, ASC or the typical promastigotes and any of these reagents could potentially be used for in vivo detection of CMI in VL epidemiological or vaccine studies.