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Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine
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Abstract Objectives: Inborn Errors of Immunity are characterized by infectious conditions and manifestations of immune dysregulation. The diversity of clinical phenotypes can make it difficult to direct the laboratory investigation. This article aims to update the investigation of immunological competence in the context of primary defects of the immune system. Source of data: Searches were carried out on Pubmed to review articles published in the last five years, in English, French or Spanish, using the terms "diagnosis" OR "investigation" AND "immunodeficiency" or "primary immunodeficiency" or "inborn errors of immunity" NOT "HIV". Recent textbook editions have also been consulted. Summary of findings: The immune system competence investigation should be started based on clinical phenotypes. Relevant data are: characterization of infectious conditions (location, recurrence, types of infectious agents, response to treatment), age during symptom onset and associated manifestations (growth impairment, allergy, autoimmunity, malignancies, fever and signs of inflammation without the identification of infection or autoimmunity) and family history. These data contribute to the selection of tests to be performed. Conclusions: The diagnostic investigation of Inborn Errors of Immunity should be guided by the clinical characterization of patients, aiming to optimize the use of complementary tests. Many diagnoses are attained only through genetic tests, which are not always available. However, the absence of a diagnosis of certainty should never delay the implementation of therapeutic measures that preserve patient life and health.
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Humans , Immunologic Deficiency Syndromes/diagnosis , Neoplasms , Phenotype , Recurrence , InflammationABSTRACT
Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.
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Objective:To build the DNA vaccine encoding hantavirus glycoprotein fused with lysosome-associated membrane protein (LAMP) and assess its immune response.Methods:BALB/c mice were immuned with the previous experimental expressing purified recombinant plasmids pVAX-Gn,pVAX-Gc,pVAX-LAMP/Gn,pVAX-LAMP/Gc,and inactivated vaccine.Indirect ELISA and neutralization test was used to detect specific antibody and neutralizing antibody in the serum of mice.The mice were tested to detect the protective effects in vivo.Results:Indirect ELISA results showed that the pVAX-LAMP/Gc group was the highest,followed by pVAX-Gc,pVAX-LAMP/Gn,and PVAX-Gn,and inactivated vaccine group.In neutralization test,there were significantly higher serum antibodies in LAMP group than those in conventional DNA group,which were higher than the inactivated vaccine group.The mice immuned had good protective effect in vivo without the specific antigen of hantavirus in vivo.Conclusion:Chimeric DNA vaccines induced higher levels of antibody in BALB/c mice and produced better protective efficacy,which illustrate the targeting strategy is expected to be an effective way to improve the DNA vaccine efficacy.
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Natural polymer like potato starch is a mixture of amylose and amylopectin, . Extraction of Potato starch was performed by rasping, centrifugation, refining and drying method. In our research method, we employed potato starch as a biodegradable polymer; It has a great impact on pharmaceutical applications due to its bioavailability, non toxic, high change density and biodegradability. In our research work, we have selected Potato starch polymer as a model of immunomodulatory effect of vaccine of tetanus antigen. According to WHO, Tetanus is a systemic infectious disease caused by genus Cl Tetanii. It has been estimated that the tetanus fever endemicity among large populations and global emergence of multidrug resistant strains to impose greater urgency on the evaluation of existing and new vaccines to prevent mortality of neonates and in pregnancy. Recently available recombinant vaccine was seems to be side effect and cost effective. The starch polymer in the form of microspheres was preferred in order to replace the alum to elicit sustained immune response because alum induces local granuloma and hypersensitivity reaction to some individual. We have employed microencapsulation technique by using 0.5% ml glutaraldehyde as a crosslinking agent. The particle size was analyzed as 40.23μm. Invitro studies was analyzed by SEM, stabilities studies Immunogenicity studies was carried out by incubating the sample, centrifuged and tested for an antigen and the Compatibility study was performed by Infrared Spectroscopy, the antigen integrity was studied by SDS PAGE and ELISA. Immunoglobulin titer values was found out ( IgG, IgA,IgM, IgE) to show the increase level of antibody response.
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Natural polymer like potato starch is a mixture of amylose and amylopectin, . Extraction of Potato starch was performed by rasping, centrifugation, refining and drying method. In our research method, we employed potato starch as a biodegradable polymer; It has a great impact on pharmaceutical applications due to its bioavailability, non toxic, high change density and biodegradability. In our research work, we have selected Potato starch polymer as a model of immunomodulatory effect of vaccine of tetanus antigen. According to WHO, Tetanus is a systemic infectious disease caused by genus Cl Tetanii. It has been estimated that the tetanus fever endemicity among large populations and global emergence of multidrug resistant strains to impose greater urgency on the evaluation of existing and new vaccines to prevent mortality of neonates and in pregnancy. Recently available recombinant vaccine was seems to be side effect and cost effective. The starch polymer in the form of microspheres was preferred in order to replace the alum to elicit sustained immune response because alum induces local granuloma and hypersensitivity reaction to some individual. We have employed microencapsulation technique by using 0.5% ml glutaraldehyde as a crosslinking agent. The particle size was analyzed as 40.23μm. Invitro studies was analyzed by SEM, stabilities studies Immunogenicity studies was carried out by incubating the sample, centrifuged and tested for an antigen and the Compatibility study was performed by Infrared Spectroscopy, the antigen integrity was studied by SDS PAGE and ELISA. Immunoglobulin titer values was found out ( IgG, IgA,IgM, IgE) to show the increase level of antibody response.