TY  - CONF
AU  - Panić, Marko
AU  - Prijić, Ivana
AU  - Simić, Mihajlo
AU  - Lukić, Ivana
AU  - Petrušić, Marija
AU  - Živković, Irena
AU  - Kojić, Milan
PY  - 2024
UR  - http://intor.torlakinstitut.com/handle/123456789/876
AB  - Tetanus toxin, a highly potent neurotoxin produced by Clostridium tetani, is the primary agent responsible for causing tetanus. This serious, potentially fatal disease can be effectively prevented through vaccination. Thanks to successful vaccination campaigns, tetanus has become exceedingly rare in both developed and most developing countries. However, the widespread presence of C. tetani spores in the environment means that tetanus cannot be completely eradicated, underscoring the ongoing need for vaccination. Traditionally, tetanus vaccines are produced by cultivating C. tetani, extracting a crude form of the tetanus toxin, and then chemically inactivating it for use in immunization. This method has proven clinically effective and is in widespread use. A challenge with this approach, however, is that the vaccine contains hundreds of various C. tetani proteins, with the active component making up only a variable and small fraction of the overall vaccine mass. To improve the current tetanus vaccine, there is potential in the recombinant production of a genetically inactivated tetanus vaccine. Prior studies have demonstrated the feasibility of engineering the full-length tetanus toxin in E. coli, and our current work builds on this foundation. We have successfully cloned the complete tetanus toxin open reading frame into the pMAL expression vector. This step was followed by the creation of a genetically inactivated protein, achieved through standard site-directed mutagenesis which altered 8 critical amino acid residues. These mutations have been confirmed via sequencing, ensuring that the toxin is genetically inactivated and thus does not require chemical inactivation for vaccine production. Our present focus is on optimizing the expression of this protein in E. coli. Following this, we intend to conduct thorough assessments of the biochemical and immunological properties of the recombinant tetanus toxin. This research represents a promising avenue towards enhancing the efficacy and specificity of tetanus vaccines, potentially improving global health outcomes.
PB  - Serbian Society for Microbiology
C3  - XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april
T1  - Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development
EP  - 113
SP  - 113
UR  - https://hdl.handle.net/21.15107/rcub_intor_876
ER  - 

@conference{
author = "Panić, Marko and Prijić, Ivana and Simić, Mihajlo and Lukić, Ivana and Petrušić, Marija and Živković, Irena and Kojić, Milan",
year = "2024",
abstract = "Tetanus toxin, a highly potent neurotoxin produced by Clostridium tetani, is the primary agent responsible for causing tetanus. This serious, potentially fatal disease can be effectively prevented through vaccination. Thanks to successful vaccination campaigns, tetanus has become exceedingly rare in both developed and most developing countries. However, the widespread presence of C. tetani spores in the environment means that tetanus cannot be completely eradicated, underscoring the ongoing need for vaccination. Traditionally, tetanus vaccines are produced by cultivating C. tetani, extracting a crude form of the tetanus toxin, and then chemically inactivating it for use in immunization. This method has proven clinically effective and is in widespread use. A challenge with this approach, however, is that the vaccine contains hundreds of various C. tetani proteins, with the active component making up only a variable and small fraction of the overall vaccine mass. To improve the current tetanus vaccine, there is potential in the recombinant production of a genetically inactivated tetanus vaccine. Prior studies have demonstrated the feasibility of engineering the full-length tetanus toxin in E. coli, and our current work builds on this foundation. We have successfully cloned the complete tetanus toxin open reading frame into the pMAL expression vector. This step was followed by the creation of a genetically inactivated protein, achieved through standard site-directed mutagenesis which altered 8 critical amino acid residues. These mutations have been confirmed via sequencing, ensuring that the toxin is genetically inactivated and thus does not require chemical inactivation for vaccine production. Our present focus is on optimizing the expression of this protein in E. coli. Following this, we intend to conduct thorough assessments of the biochemical and immunological properties of the recombinant tetanus toxin. This research represents a promising avenue towards enhancing the efficacy and specificity of tetanus vaccines, potentially improving global health outcomes.",
publisher = "Serbian Society for Microbiology",
journal = "XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april",
title = "Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development",
pages = "113-113",
url = "https://hdl.handle.net/21.15107/rcub_intor_876"
}

Panić, M., Prijić, I., Simić, M., Lukić, I., Petrušić, M., Živković, I.,& Kojić, M.. (2024). Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development. in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april
Serbian Society for Microbiology., 113-113.
https://hdl.handle.net/21.15107/rcub_intor_876

Panić M, Prijić I, Simić M, Lukić I, Petrušić M, Živković I, Kojić M. Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development. in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april. 2024;:113-113.
https://hdl.handle.net/21.15107/rcub_intor_876 .

Panić, Marko, Prijić, Ivana, Simić, Mihajlo, Lukić, Ivana, Petrušić, Marija, Živković, Irena, Kojić, Milan, "Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development" in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april (2024):113-113,
https://hdl.handle.net/21.15107/rcub_intor_876 .

TY  - JOUR
AU  - Petrušić, Marija
AU  - Stojić-Vukanić, Zorica
AU  - Pilipović, Ivan
AU  - Kosec, Duško
AU  - Prijić, Ivana
AU  - Leposavić, Gordana
PY  - 2023
UR  - http://intor.torlakinstitut.com/handle/123456789/633
AB  - The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of “inflammescent” cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy.
PB  - Elsevier
T2  - Experimental Gerontology
T1  - Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development
SP  - 112009
VL  - 171
DO  - 10.1016/j.exger.2022.112009
ER  - 

@article{
author = "Petrušić, Marija and Stojić-Vukanić, Zorica and Pilipović, Ivan and Kosec, Duško and Prijić, Ivana and Leposavić, Gordana",
year = "2023",
abstract = "The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of “inflammescent” cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy.",
publisher = "Elsevier",
journal = "Experimental Gerontology",
title = "Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development",
pages = "112009",
volume = "171",
doi = "10.1016/j.exger.2022.112009"
}

Petrušić, M., Stojić-Vukanić, Z., Pilipović, I., Kosec, D., Prijić, I.,& Leposavić, G.. (2023). Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development. in Experimental Gerontology
Elsevier., 171, 112009.
https://doi.org/10.1016/j.exger.2022.112009

Petrušić M, Stojić-Vukanić Z, Pilipović I, Kosec D, Prijić I, Leposavić G. Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development. in Experimental Gerontology. 2023;171:112009.
doi:10.1016/j.exger.2022.112009 .

Petrušić, Marija, Stojić-Vukanić, Zorica, Pilipović, Ivan, Kosec, Duško, Prijić, Ivana, Leposavić, Gordana, "Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development" in Experimental Gerontology, 171 (2023):112009,
https://doi.org/10.1016/j.exger.2022.112009 . .