N. Rabiei, S. A. Badi, F. E. Marvasti, T. N. Sattari, F. Vaziri, S. Siadat
Introduction: Extracellular vesicles (EVs) are spherical structures, naturally secreted by Gram-negative and Grampositive bacteria. EVs play a critical role in the modulation of immune responses, bioactive cargo delivery, and cellcell communication. The conventional method of EVs preparation involves the use of detergent (ultracentrifugation method). For the first time, we used a polyethylene glycol (PEG)-based method in our study to isolate EVs from prokaryotic cells, namely Faecalibacterium prausnitzii A2-165. We then compared various features of this method with those of the ultracentrifugation method. Methods: Extraction of EVs was performed via sequential deoxycholate ultracentrifugation and PEG-based methods. The physicochemical properties of the extracted EVs were compared via scanning electron microscopy (SEM), SDS-PAGE, and dynamic light scattering (DLS). Results: The protein content of the extracted EVs was 1.6 and 0.5 mg/mL, based on the ultracentrifugation and PEG-based methods, respectively. According to the SDS-PAGE analysis, vesicle-associated proteins were located at 20-150 kDa. The SEM analysis showed that the extracted EVs had a diameter of 50-200 nm in both methods. The results of DLS analysis showed 4 populations of approximately 50-8000 nm in the ultracentrifugation method and approximately 100-2000 nm in the PEG-based method. The EVs extracted by the ultracentrifugation method showed higher negative charge densities in contrast to EVs extracted by the PEG-based method. Conclusion: Our result showed that PEG-based extraction is a fast, simple, and cost-effective method and EVs purity was within the acceptable range. Further studies are needed to confirm the safety and the efficacy of EVs in clinical practices, especially as vaccine delivery vehicles.
{"title":"Comparison of two isolation methods for extracellular vesicles from Faecalibacterium prausnitzii A2-165","authors":"N. Rabiei, S. A. Badi, F. E. Marvasti, T. N. Sattari, F. Vaziri, S. Siadat","doi":"10.29252/VACRES.5.1.27","DOIUrl":"https://doi.org/10.29252/VACRES.5.1.27","url":null,"abstract":"Introduction: Extracellular vesicles (EVs) are spherical structures, naturally secreted by Gram-negative and Grampositive bacteria. EVs play a critical role in the modulation of immune responses, bioactive cargo delivery, and cellcell communication. The conventional method of EVs preparation involves the use of detergent (ultracentrifugation method). For the first time, we used a polyethylene glycol (PEG)-based method in our study to isolate EVs from prokaryotic cells, namely Faecalibacterium prausnitzii A2-165. We then compared various features of this method with those of the ultracentrifugation method. Methods: Extraction of EVs was performed via sequential deoxycholate ultracentrifugation and PEG-based methods. The physicochemical properties of the extracted EVs were compared via scanning electron microscopy (SEM), SDS-PAGE, and dynamic light scattering (DLS). Results: The protein content of the extracted EVs was 1.6 and 0.5 mg/mL, based on the ultracentrifugation and PEG-based methods, respectively. According to the SDS-PAGE analysis, vesicle-associated proteins were located at 20-150 kDa. The SEM analysis showed that the extracted EVs had a diameter of 50-200 nm in both methods. The results of DLS analysis showed 4 populations of approximately 50-8000 nm in the ultracentrifugation method and approximately 100-2000 nm in the PEG-based method. The EVs extracted by the ultracentrifugation method showed higher negative charge densities in contrast to EVs extracted by the PEG-based method. Conclusion: Our result showed that PEG-based extraction is a fast, simple, and cost-effective method and EVs purity was within the acceptable range. Further studies are needed to confirm the safety and the efficacy of EVs in clinical practices, especially as vaccine delivery vehicles.","PeriodicalId":52727,"journal":{"name":"Vaccine Research","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45075427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vaccination is a useful primary prevention against infectious diseases. In several countries, many infections are endemic and vaccines are usually provided free of charge by the local governments to the people. For vaccination, the assessment for the contraindication which is usually based on clinical history taking, is the primary requirement. Here, the experiences on a recent situation of free influenza vaccination is discussed. This review was based on the clinical history of 200 local patients who had attended a medical center in Bangkok, Thailand, during June 2018, asking for free influenza vaccination according to the local Thai public health policies. According to this report, it is observable that the clinical history taking is usually unreliable.
{"title":"Free vaccinations and clinical history takings of the vaccine recipients: reliable or not?","authors":"Wiwanitkit, S. Yasri","doi":"10.29252/VACRES.5.1.1","DOIUrl":"https://doi.org/10.29252/VACRES.5.1.1","url":null,"abstract":"Vaccination is a useful primary prevention against infectious diseases. In several countries, many infections are endemic and vaccines are usually provided free of charge by the local governments to the people. For vaccination, the assessment for the contraindication which is usually based on clinical history taking, is the primary requirement. Here, the experiences on a recent situation of free influenza vaccination is discussed. This review was based on the clinical history of 200 local patients who had attended a medical center in Bangkok, Thailand, during June 2018, asking for free influenza vaccination according to the local Thai public health policies. According to this report, it is observable that the clinical history taking is usually unreliable.","PeriodicalId":52727,"journal":{"name":"Vaccine Research","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42503151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Rashid, S. Shahsavandi, M. Ebrahimi, S. Soleimani
Introduction: Live and inactivated vaccines are wildly used against Newcastle disease (ND) which is a highly contagious and acute viral infection of domestic and wild birds. A higher and prolonged immune response is required to improve the control of the disease. The aim of this study was to evaluate the potential of the conserved TIR domain of an immune regulatory protein TLR7 (i.e. TIR-TLR7) as a biological adjuvant in enhancing cell-mediated immunity in vaccinated chickens against the inactivated ND virus (NDV) V4 strain antigen. Methods: NDV V4 strain was propagated in chicken embryonated SPF eggs, tittered and then inactivated by formalin. The amount of 10 μg of TIRTLR7 was mixed with the NDV antigen before intramuscular administration. Fifty SPF chickens were divided in A-E groups (n=10), consisted of negative control, TIR-TLR7, inactivated NDV antigen, TIR-TLR7/inactivated NDV antigen in prime, and the same regimen in boost platform. The blood samples were collected at week intervals up to 6 weeks post-vaccination. Humoral response was measured by detection of specific NDV antibody titer using the HI test. The cell-mediated immunity was evaluated by measuring lymphocyte proliferation in splenocytes cell culture using MTT. Results: All immunized chickens with TIR-TLR7/inactivated NDV antigen had significant (P < 0.05) cellmediated and HI responses to NDV compared to the control groups. No statistically-significant difference was observed between the prime and boost trials. Conclusion: The results indicated that the combination of TIR-TLR7 and inactivated NDV antigen gave a strong immune response at both the humoral and the cellular levels.
{"title":"Enhancement of cell-mediated immune response in chickens by combination of TIR-TLR7 with inactivated Newcastle disease vaccine","authors":"S. Rashid, S. Shahsavandi, M. Ebrahimi, S. Soleimani","doi":"10.29252/VACRES.5.1.19","DOIUrl":"https://doi.org/10.29252/VACRES.5.1.19","url":null,"abstract":"Introduction: Live and inactivated vaccines are wildly used against Newcastle disease (ND) which is a highly contagious and acute viral infection of domestic and wild birds. A higher and prolonged immune response is required to improve the control of the disease. The aim of this study was to evaluate the potential of the conserved TIR domain of an immune regulatory protein TLR7 (i.e. TIR-TLR7) as a biological adjuvant in enhancing cell-mediated immunity in vaccinated chickens against the inactivated ND virus (NDV) V4 strain antigen. Methods: NDV V4 strain was propagated in chicken embryonated SPF eggs, tittered and then inactivated by formalin. The amount of 10 μg of TIRTLR7 was mixed with the NDV antigen before intramuscular administration. Fifty SPF chickens were divided in A-E groups (n=10), consisted of negative control, TIR-TLR7, inactivated NDV antigen, TIR-TLR7/inactivated NDV antigen in prime, and the same regimen in boost platform. The blood samples were collected at week intervals up to 6 weeks post-vaccination. Humoral response was measured by detection of specific NDV antibody titer using the HI test. The cell-mediated immunity was evaluated by measuring lymphocyte proliferation in splenocytes cell culture using MTT. Results: All immunized chickens with TIR-TLR7/inactivated NDV antigen had significant (P < 0.05) cellmediated and HI responses to NDV compared to the control groups. No statistically-significant difference was observed between the prime and boost trials. Conclusion: The results indicated that the combination of TIR-TLR7 and inactivated NDV antigen gave a strong immune response at both the humoral and the cellular levels.","PeriodicalId":52727,"journal":{"name":"Vaccine Research","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44336718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Afrough, M. Vosogh, Asadi Karam, Ava Behrouzi, G. Mardani, S. Siadat
Introduction: As the causative agent of meningitis, Neisseria meningitidis has different serogroups. The purpose of this study was to investigate the molecular properties of N. meningitidis strains among Iranian cases. Methods: 450 samples were collected from children under 5 years of age. Detection of Neisseria genus was done by phenotypic and genotypic methods. Multiplex PCR was used to identify the serogroups of N. meningitides. The sequencing of variable regions of porA gene was performed for detection of the subserogroups. Results: From 137 (30.44%) Neisseria isolates, 4 isolates (0.88%) belonged to N. meningitidis and 133 isolates (29.55%) belonged to other species. Multiplex PCR results showed that one isolate belonged to serogroup A while 3 belonged to serogroup B. The analysis of amplified VR1 and VR2 variable regions of porA showed 100% identity of the serogroup A strain with strain BZ83N and the serogroup B strains with strain 528 of N. meningitidis . In accordance with other findings in Asia, serogroups A and B were the most prevalent serogroups of N. meningitidis. Sequencing of variable regions of porA could identify the subserogroups of the isolates. Conclusion: sequencing of porA could be a valuable method for identification of N. meningitidis strains to be used in epidemiological studies as well as improved vaccine designs.
{"title":"PorA typing of Neisseria meningitidis isolates from Iranian children for vaccine design","authors":"P. Afrough, M. Vosogh, Asadi Karam, Ava Behrouzi, G. Mardani, S. Siadat","doi":"10.29252/VACRES.5.1.4","DOIUrl":"https://doi.org/10.29252/VACRES.5.1.4","url":null,"abstract":"Introduction: As the causative agent of meningitis, Neisseria meningitidis has different serogroups. The purpose of this study was to investigate the molecular properties of N. meningitidis strains among Iranian cases. Methods: 450 samples were collected from children under 5 years of age. Detection of Neisseria genus was done by phenotypic and genotypic methods. Multiplex PCR was used to identify the serogroups of N. meningitides. The sequencing of variable regions of porA gene was performed for detection of the subserogroups. Results: From 137 (30.44%) Neisseria isolates, 4 isolates (0.88%) belonged to N. meningitidis and 133 isolates (29.55%) belonged to other species. Multiplex PCR results showed that one isolate belonged to serogroup A while 3 belonged to serogroup B. The analysis of amplified VR1 and VR2 variable regions of porA showed 100% identity of the serogroup A strain with strain BZ83N and the serogroup B strains with strain 528 of N. meningitidis . In accordance with other findings in Asia, serogroups A and B were the most prevalent serogroups of N. meningitidis. Sequencing of variable regions of porA could identify the subserogroups of the isolates. Conclusion: sequencing of porA could be a valuable method for identification of N. meningitidis strains to be used in epidemiological studies as well as improved vaccine designs.","PeriodicalId":52727,"journal":{"name":"Vaccine Research","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45971454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Sadeghi, M. Shahanaghi, Aghasadeghi, F. Motevalli, Amiran, S. M. Pargoo, M. Hamidi-Fard
Introduction: Hepatitis E virus (HEV) is a non-enveloped, single-stranded positive-sense RNA virus. It is one of the most important causes of liver failures and the mortality rate arising from HEV is more common in pregnant women. HEV is an entericallytransmitted virus and its outbreak is more common in the developing and poor-hygiene countries while vaccination against it can prevent its prevalence. The ORF2 is an immunogenic capsid protein of HEV with 660 amino acids that is being used in vaccine designs against HEV infection. ORF2 has been studied in a vast range of vectors and hosts, such as pRSET-C, pMAL and pSG vectors, as well as Escherichia coli BL21 and vaccinia virus hosts. A DNA vaccine expressing ORF2 has also been studied which has induced specific humoral and cellular immune responses in mice. This study was aimed to clone and express ORF2 as an immunogen protein in a eukaryotic host system. Methods: orf2 gene corresponding to 660 amino acids of ORF2 protein was subcloned from a pET21avector into pFastBac. The protein expression was achieved by transforming Sf9 insect cells with a pFastBac-orf2 construct. The over-expressed protein with ~72 kDa MW was assessed by SDS-PAGE. Results: The cloning was confirmed by PCR and restriction digestions. The expression of ORF2 with expected MW in Sf9 cells was confirmed by SDS-PAGE. Conclusion: ORF2 protein of HEV was successfully expressed in a baculovirus-based eukaryotic expression system as the first step for further studies on HEV vaccine designs, based on ORF2 protein.
{"title":"Cloning and expression of hepatitis E virus ORF2 as an immunogen protein in baculovirus expression system","authors":"S. Sadeghi, M. Shahanaghi, Aghasadeghi, F. Motevalli, Amiran, S. M. Pargoo, M. Hamidi-Fard","doi":"10.29252/VACRES.5.1.23","DOIUrl":"https://doi.org/10.29252/VACRES.5.1.23","url":null,"abstract":"Introduction: Hepatitis E virus (HEV) is a non-enveloped, single-stranded positive-sense RNA virus. It is one of the most important causes of liver failures and the mortality rate arising from HEV is more common in pregnant women. HEV is an entericallytransmitted virus and its outbreak is more common in the developing and poor-hygiene countries while vaccination against it can prevent its prevalence. The ORF2 is an immunogenic capsid protein of HEV with 660 amino acids that is being used in vaccine designs against HEV infection. ORF2 has been studied in a vast range of vectors and hosts, such as pRSET-C, pMAL and pSG vectors, as well as Escherichia coli BL21 and vaccinia virus hosts. A DNA vaccine expressing ORF2 has also been studied which has induced specific humoral and cellular immune responses in mice. This study was aimed to clone and express ORF2 as an immunogen protein in a eukaryotic host system. Methods: orf2 gene corresponding to 660 amino acids of ORF2 protein was subcloned from a pET21avector into pFastBac. The protein expression was achieved by transforming Sf9 insect cells with a pFastBac-orf2 construct. The over-expressed protein with ~72 kDa MW was assessed by SDS-PAGE. Results: The cloning was confirmed by PCR and restriction digestions. The expression of ORF2 with expected MW in Sf9 cells was confirmed by SDS-PAGE. Conclusion: ORF2 protein of HEV was successfully expressed in a baculovirus-based eukaryotic expression system as the first step for further studies on HEV vaccine designs, based on ORF2 protein.","PeriodicalId":52727,"journal":{"name":"Vaccine Research","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42604729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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