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This paper aims to investigate the preparation and characterisation of the alginate nanoparticles (NPs) as antigen delivery system loaded by diphtheria toxoid (DT). For this purpose, both the loading capacity (LC) and Loading efficiency (LE) of the alginate NPs burdened by DT are evaluated. Moreover, the effects of different concentrations of sodium alginate and calcium chloride on the NPs physicochemical characteristics are surveyed in addition to other physical conditions such as homogenization time and rate. To do so, the NPs are characterised using particle size and distribution, zeta potential, scanning electron microscopy, encapsulation efficiency, in vitro release study and FT-IR spectroscopy. Subsequently, the effects of homogenization time and rate on the NPs are assessed. At the meantime, the NPs LC and efficiency in several DT concentrations are estimated. The average size of the NPs was 400.7 and 276.6 nm for unloaded and DT loaded, respectively. According to the obtained results, the zeta potential of the blank and DT loaded NPs are estimated as -23.7 mV and -21.2 mV, respectively. Whereas, the LC and LE were80% and90%, in that order. Furthermore, 95% of the releasing DT loaded NPs occurs at 140 h in the sustained mode without any bursting release. It can be concluded that the features of NPs such as morphology and particle size are strongly depended on the calcium chloride, sodium alginate concentrations and physicochemical conditions in the NPs formation process. In addition, appropriate concentrations of the sodium alginate and calcium ions would lead to obtaining the desirable NPs formation associated with the advantageous LE, LC (over 80%) and sustained in vitro release profile. Ultimately, the proposed NPs can be employed in vaccine formulation for the targeted delivery, controlled and slow antigen release associated with the improved antigen stability.

Background: Pertussis is still one of the major public health problems. The increase of the disease emerged in recent decades due to the replacement of the reactogenic whole cell vaccine with the safer acellular vaccine and the genetic diversity of the bacterium. As outer membrane vesicles (OMVs) obtained from Bordetella pertussis contains surface immunogenic antigen in its structure, it has an acceptable characteristic that could be considered as a good candidate for pertussis vaccine. Materials & Methods: Vaccinal strain BP134 strain of B. pertussis was cultured under standard conditions and OMVs were isolated by modifying the method without the use of ultracentrifuge. The isolated vesicles were examined by transmission electron microscopy and protein content was measured by the Bradford method. The expression of virulence factors was confirmed by SDS-PAGE and protein expression was confirmed by Western immunoblot. Pyrogenicity test and abnormal toxicity test were performed on extracted vesicles. Results: The morphology of the vesicles was confirmed in the range of 40 to 200 nm. The protein concentration of the extracted vesicles was determined as 600 μg. Expression analysis by SDS-PAGE and western blot confirmed the presence of virulence factors, pertussis toxin, filamentous hemagglutinin, and pertactin using monoclonal antibodies in OMVs of the vaccinal strain. Pyrogenicity test and abnormal toxicity test were negative. Conclusion: The proposed method is a simple and efficient method for isolation of the B. pertussis OMVs. The OMVs extracted from B. pertussis could be a candidate for a new generation of pertussis vaccine alone or in combination with adjuvants to design future acellular vaccines.

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: 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.

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.

Background and Objectives: The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis. Materials and Methods: The genetic diversity and evolutionary characteristics of circulating Iranian isolates of Bordetella pertussis during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subsequently ptxA, ptxP and fim3 alleles were characterized. The next generation genome sequencing was then used to compare the genomics of ptxP1 and ptxP3 of selected isolates from PFGE dendrogram. Results: PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015–2018. The predominant B. pertussis genotype harbored pertussis toxin promoter allele, ptxP3 and the expansion of ptxA1 isolates, were also observed in our population. Conclusion: No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the ptxP3 and ptxP1 isolates indicate that changes in ptxP3 genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the ptxP3 clone. We compared ptxP3 and ptxP1 isolates in pathogenicity-associated genes and found five of them were specific for the ptxP3 isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.

Background and Objectives: The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis. Materials and Methods: The genetic diversity and evolutionary characteristics of circulating Iranian isolates of Bordetella pertussis during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subse- quently ptxA, ptxP and fim3 alleles were characterized. The next generation genome sequencing was then used to compare the genomics of ptxP1 and ptxP3 of selected isolates from PFGE dendrogram. Results: PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015-2018. The predominant B. pertussis genotype harbored pertussis toxin promoter allele, ptxP3 and the expansion of ptxA1 isolates, were also observed in our population. Conclusion: No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the ptxP3 and ptxP1 isolates indicate that changes in ptxP3 genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the ptxP3 clone. We compared ptxP3 and ptxP1 isolates in pathogenicity-associated genes and found five of them were specific for the ptxP3 isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.

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: 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.

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.