scholarly journals Immunogenicity Test of ESAT-6/CFP-10 Mycobacterium Tuberculosis (Indonesian Strain) Recombinant Protein Fusion: IFN-γ and CD8+ T Cells Expression in PBMC Culture

2019 ◽  
Vol 38 (4) ◽  
pp. 210-8
Author(s):  
Anung Sri Handayani ◽  
Tri Wahju Astuti ◽  
Teguh Rahayu Sartono ◽  
Maimun Zulhaidah Arthamin ◽  
Fransisca Srioetami Tanoerahardjo
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Healthy Subject ◽  
Cd8 T Cells ◽  
Vaccine Development ◽  
Laboratory Research ◽  
Protein Fusion ◽  
Pbmc Culture ◽  
Ifn Γ

Background: BCG vaccination is one way to control tuberculosis (TB) but still poor in efficacy thus new vaccine development is needed. Immunogenicity test is needed in developing new vaccine. The aim of this study was to understand whether the recombinant protein fusion of ESAT-6/CFP-10 Mycobacterium tuberculosis (M. tuberculosis) can stimulate cellular immune response, especially IFN-γ and CD8 + T cell expression in PBMC cultures. Methods: This study was an experimental laboratory research conducted on PBMC cultures of 3 groups of subjects (TB patients, latent TB patients and healthy subjects) at RSUD Dr. Saiful Anwar in April-July 2017. The sample of each groups was 8 subjects. Each groups induced by recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis as a standard protocol and to establish the immunogenicity status. CD8+ T cells IFN-γ expressed by C8+ were measured by flowcytometry. Result: Recombinant protein fusion ESAT-6/CFP-10 can stimulate CD8+ T cells and IFN-γ expressed by CD8+ T cells in all group. The highest stimulation of CD8+ percentage was found in healthy subject (37.533 ± 7.264) and IFN-γ expressed by CD8+ T cells was found in healthy subject (7.908 ± 4.457); There are increase significantly CD8+ T cells (p=0.001) and IFN-γ expressed by CD8+ T cells (p=0.217) not significantly in healthy subject compared in PPD and without antigen. Conclusion: Recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis can stimulate CD8+ T cells and IFN-γ expressed by CD8+ T cells in healthy subject. Recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis potential as a new vaccine candidate. (J Respir Indo. 2018;38: 210-8)

scholarly journals Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

2017 ◽  
Vol 24 (11) ◽  
Author(s):  
Ahreum Kim ◽  
Yun-Gyoung Hur ◽  
Sunwha Gu ◽  
Sang-Nae Cho
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Interleukin 2 ◽  
T Cell Epitopes ◽  
Content Type ◽  
Complete Form ◽  
Ifn Γ

ABSTRACT The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

Cytomegalovirus pp65 - Recombinant Protein: A New Antigen for Efficient Restimulation of CD4+ and CD8+ pp65-Specific T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3163-3163
Author(s):  
Anne Richter ◽  
Patricia Marschall ◽  
Marie Mohn ◽  
Uwe Odenthal ◽  
Silke Gösling ◽  
...  
Keyword(s):  
T Cells ◽  
Recombinant Protein ◽  
Cd8 T Cells ◽  
Matrix Protein ◽  
Peptide Pool ◽  
Effector Memory ◽  
Protein Antigens ◽  
Short Term ◽  
Ifn Γ

Abstract Short-term restimulation assays combined with the analysis of effector function, in particular the detection of cytokine production, are useful tools for the analysis and isolation of antigen-specific T cells. Until now, restimulations with soluble protein antigens failed to efficiently reactivate CD8+ T cells. We have developed a recombinant protein of the immunodominant cytomegalovirus (CMV) matrix protein pp65 for in vitro restimulation of pp65-specific CD4+ as well as CD8+ T cells. The efficiency of the CMV pp65 - Recombinant Protein to reactivate pp65-experienced CD4+ and CD8+ T cells and the specificity of the restimulated T cells were analysed. PBMC from CMV seropositive donors were restimulated with CMV pp65 - Recombinant Protein or a complete pool of overlapping pp65 peptides. Afterwards T cells were analysed for intracellular IFN-γ production by flow cytometry. Interestingly, we observed that stimulation with CMV pp65 - Recombinant Protein results in IFN-γ production in CD4+ as well as CD8+ T cells with frequencies comparable to that using the peptide pool as antigen (n=17). In contrast, upon stimulation of PBMC from CMV seronegative donors with CMV pp65 - Recombinant Protein neither IFN-γ nor TNF-α were detectable in T cells (n=6). Furthermore, we tested the specificity of CMV pp65 - Recombinant Protein-reactive CD4+ and CD8+ T cells. Therefore, IFN-γ-producing T cells were magnetically isolated after short-term stimulation with pp65 using the IFN-γ cytokine secretion assay and expanded for 7 days. Subsequently, the isolated and expanded CD4+ and CD8+ T cells were restimulated with pp65 peptide pool. More than 80 % of the CD4+ and CD8+ T cells produced IFN-γ and more than 80 % of the CD8+ T cells were positively stained with MHC class I/pp65 tetramers. These results demonstrate that CMV pp65 - Recombinant Protein efficiently and specifically reactivates pp65-experienced CD4+ as well as CD8+ T cells. Therefore, CMV pp65 - Recombinant Protein is a useful antigen for the detection and isolation of pp65-experienced CD4+ and CD8+ effector/memory T cells.

Age dependent increase in early resistance of mice to Mycobacterium tuberculosis is associated with an increase in CD8 T cells that are capable of antigen independent IFN-γ production

2006 ◽  
Vol 41 (11) ◽  
pp. 1185-1194 ◽  
Author(s):  
Bridget Vesosky ◽  
David K. Flaherty ◽  
Erin K. Rottinghaus ◽  
Gillian L. Beamer ◽  
Joanne Turner
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Cd8 T Cells ◽  
Age Dependent ◽  
Ifn Γ

scholarly journals Immunogenicity Test of ESAT-6/CFP-10 Mycobacterium tuberculosis (Indonesian Strain) Recombinant Protein Fusion: TNF-α, IL-17 and CD4+ T Cells Expression in PBMC Culture

2019 ◽  
Vol 39 (3) ◽  
pp. 160-168
Author(s):  
Eko Prasetyo ◽  
Triwahju Astuti ◽  
Nunuk Sri Muktiati ◽  
Maimun Z Arthamin
Keyword(s):  
T Cells ◽  
Recombinant Protein ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Vaccine Candidate ◽  
Interleukin 17 ◽  
Protein Fusion ◽  
Pbmc Culture ◽  
Tnf Α ◽  
Cell Expression

Background: Bascillus Calmette Guѐrin vaccination has not provided protection against TB in adults. ESAT-6/CFP-10 Mtb recombinant protein fusion as a vaccine candidate can stimulate the body's immune response. Interleukin-17, TNF-α and CD4+ play a major role against TB. This study aims to determine that the recombinant protein fusion ESAT-6/CFP-10 Mtb can stimulate TNF-α, IL-17 and CD4+ T cells expression in PBMC culture. Methods: Design of this study is experimental study. Number of research sample per group of 8 subjects. The subjects: healthy, TB contact and TB patients were taken their peripheral blood sample and treated with ESAT-6/CFP-10 Mtb recombinant protein fusion. TNF-α CD4+ T cells were measured by ELISA. Flow cytometry is used to measure IL-17 and CD4+ Tcells. As standard protocol research on tuberculosis vaccine, each subject also treated without antigen and with PPD. Results: There was no significant increase in the administration of ESAT-6/CFP-10 Mtb fusion compared without antigen on TNF-α expression of CD4+ (P=0.202), expression of IL-17 CD4+ (P=0.994) and percentage of CD4+ T cells (P=0.183). ESAT-6/CFP-10 Mtb Fusion was able to stimulate expression of TNF-α CD4+ in healthy subjects (29.91±1.23pg/ml), TB contact (32.21±4.02pg/ml) and TB patients (33.35±8.41 pg/ml). Expression IL-17 CD4+ in healthy subjects (33.24 ± 39.01%), TB contact (23.88 ± 21%) and TB patients (51.93 ± 36%). CD4+ T cell expression in healthy subjects 30.64 ± 7.63%, TB contact 24.58 ± 5.24% and TB patients (40.73±2.63%). Conclusions: ESAT-6/CFP-10 Mtb recombinant proteins fusion may stimulate the production of TNF-α, IL-17 CD4+ and CD4+ T cells in all subject. Expression of TNF-α, IL-17 CD4+ and CD4+ T cells in the healthy group, indicated that the ESAT-6/CFP-10 recombinant protein fusion has the potential as vaccine candidate. (J Respir Indo. 2019; 39(3):160-8)

scholarly journals Cytotoxicity and Secretion of Gamma Interferon Are Carried Out by Distinct CD8 T Cells during Mycobacterium tuberculosis Infection

2009 ◽  
Vol 77 (10) ◽  
pp. 4621-4630 ◽  
Author(s):  
Thorbjorg Einarsdottir ◽  
Euan Lockhart ◽  
JoAnne L. Flynn
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Cd8 T Cells ◽  
Gamma Interferon ◽  
Mycobacterium Tuberculosis Infection ◽  
Memory Cd8 T Cells ◽  
Ifn Γ ◽  
Sterilizing Immunity

ABSTRACT The host immune response is generally sufficient to contain Mycobacterium tuberculosis infection. It does not, however, efficiently prevent subsequent infection with M. tuberculosis or provide sterilizing immunity. While the understanding of the immune response generated against this pathogen is incomplete, improvements have been achieved due to advances in immunological tools. In this study, we analyzed the multifunctional nature of primary and memory CD8 T-cell responses generated during murine M. tuberculosis infection. We generated a recombinant M. tuberculosis strain expressing ovalbumin (OVA) epitopes in order to expand the peptides for the detection of CD8 T cells during M. tuberculosis infection and enable us to use OVA-specific reagents. Our results indicate that the majority of M. tuberculosis-specific CD8 T cells are limited to either cytotoxicity or the secretion of gamma interferon (IFN-γ), with cytotoxicity being far more prevalent than IFN-γ secretion. Memory CD8 T cells responded earlier and reached higher levels in the lungs than naïve CD8 T cells, as was expected. They were, however, less cytotoxic and secreted less IFN-γ than newly primed CD8 T cells, suggesting that one factor contributing to bacterial persistence and lack of sterilizing immunity may be the low quality of memory cells that are generated.

scholarly journals Induction of Mycobacterium tuberculosis-Specific Primary and Secondary T-Cell Responses in Interleukin-15-Deficient Mice

2005 ◽  
Vol 73 (5) ◽  
pp. 2910-2922 ◽  
Author(s):  
Vanja Lazarevic ◽  
David J. Yankura ◽  
Sherrie J. Divito ◽  
JoAnne L. Flynn
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Effector T Cells ◽  
Cell Responses ◽  
Interleukin 15 ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT Several studies have provided evidence that interleukin-15 (IL-15) can enhance protective immune responses against Mycobacterium tuberculosis infection. However, the effects of IL-15 deficiency on the functionality of M. tuberculosis-specific CD4 and CD8 T cells are unknown. In this study, we investigated the generation and maintenance of effector and memory T-cell responses following M. tuberculosis infection of IL-15−/− mice. IL-15−/− mice had slightly higher bacterial numbers during chronic infection, which were accompanied by an increase in gamma interferon (IFN-γ)-producing CD4 and CD8 T cells. There was no evidence of increased apoptosis or a defect in proliferation of CD8 effector T cells following M. tuberculosis infection. The induction of cytotoxic and IFN-γ CD8 T-cell responses was normal in the absence of IL-15 signaling. The infiltration of CD4 and CD8 T cells into the lungs of “immune” IL-15−/− mice was delayed in response to M. tuberculosis challenge. These findings demonstrate that efficient effector CD4 and CD8 T cells can be developed following M. tuberculosis infection in the absence of IL-15 but that recall T-cell responses may be impaired.

scholarly journals Th1 Cytokines Facilitate CD8-T-Cell-Mediated Early Resistance to Infection with Mycobacterium tuberculosis in Old Mice

2006 ◽  
Vol 74 (6) ◽  
pp. 3314-3324 ◽  
Author(s):  
Bridget Vesosky ◽  
David K. Flaherty ◽  
Joanne Turner
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Infectious Diseases ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Phenotype ◽  
Ifn Γ ◽  
Th1 Cytokines ◽  
Old Mice

ABSTRACT Numerous immunological defects begin to emerge as an individual ages, the consequence of which is heightened susceptibility to infectious diseases. Despite this decline in immune function, old mice display an early transient resistance to Mycobacterium tuberculosis infection in the lung, which is dependent on CD8 T cells and gamma interferon (IFN-γ) production. In this study, we investigated the mechanism of resistance by examining the CD8-T-cell phenotype and function in old naïve and M. tuberculosis-infected mice. Pulmonary CD8 T cells from naïve old mice expressed cell surface markers of memory in addition to receptors for several Th1 cytokines. Stimulation of lung cells from naïve old mice with a combination of Th1 cytokines (interleukin-2 [IL-2], IL-12, and IL-18) resulted in nonspecific production of IFN-γ by memory CD8 T cells. Following aerosol infection with M. tuberculosis, the lungs of old mice contained significantly more IL-12, IL-18, and IFN-γ than the lungs of young mice contained. Together, these data demonstrate that the increased and early production of Th1 cytokines in the lungs of M. tuberculosis-infected old mice, in combination with CD8 T cells that can nonspecifically produce IFN-γ, leads to transient control of M. tuberculosis growth in the lungs of old mice. Further characterization of this mechanism should provide essential information regarding the aging immune system and should contribute to the development of novel strategies to decrease the morbidity and mortality of the aging population associated with infectious diseases.

scholarly journals CD8 T Cells in Old Mice Contribute to the Innate Immune Response to Mycobacterium tuberculosis via Interleukin-12p70-Dependent and Antigen-Independent Production of Gamma Interferon

2009 ◽  
Vol 77 (8) ◽  
pp. 3355-3363 ◽  
Author(s):  
Bridget Vesosky ◽  
Erin K. Rottinghaus ◽  
Craig Davis ◽  
Joanne Turner
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Cd8 T Cells ◽  
The Elderly ◽  
Gamma Interferon ◽  
Ifn Γ ◽  
Aerosol Infection ◽  
Old Mice

ABSTRACT Elderly individuals have increased morbidity and mortality associated with infectious diseases due in part to the progressive age-associated decline in immune function. Despite this, the old mouse model of Mycobacterium tuberculosis infection has revealed a CD8- and gamma interferon (IFN-γ)-dependent early resistance to infection. In this study, we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to M. tuberculosis. Following a low-dose aerosol infection with M. tuberculosis, CD8 T cells were identified as being a dominant source of IFN-γ expression in the lungs of old mice early after infection, before the typical onset of antigen-specific immunity. In addition, M. tuberculosis-induced IFN-γ production by CD8 T cells isolated from naïve old mice was major histocompatibility complex class I independent but was dependent on interleukin-12p70, confirming an innate role of CD8 T cells during M. tuberculosis infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-γ levels in response to M. tuberculosis infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as being one possible mechanism by which CD8 T cells from old mice produce enhanced levels of IFN-γ. Additional characterizations of the signaling events that lead to enhanced innate IFN-γ production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.

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