Creating efficient vaccine candidates against Tuberculosis using mutant Mycobacterium smegmatis

One of the leading causes of mortality in low and middle income countries continues to be tuberculosis. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Recently, it has been shown that a nonpathogenic mycobacterium strain, Mycobacterium smegmatis (Msmeg), mutated in a protein secretion system called Esx-3 had superior protection abilities in a murine model of Mtb infection. In our proposed project we are investigating how mutant Msmeg can provide protection against Mtb infection. Our project is a «young scientist grant» project that in addition to excellent research also should deliver on career development for the principal investigator. The project has generated some papers and the project has also given the project leader the opportunity to be part of building a stronger research environment for Global Health research at NTNU and in Norway. In addition, we have been able to build a strong international network that has led to a successful application to the Joint programing intitiative in antimicrobial resistance (JPIAMR) that started in January 2015. In that project, we are work package leader and partner. Details regarding our results achievements: The project started in January 2013 and the first part of the project was used to develop tools for controllable gene expression in mycobacteria and tools to enable us to study Msmeg and mutants of Msmeg. This work has now been finished and we report results from these partial projects in one of our 2015 publications (PLOS One, September 2015). Another important part of our project is to understand the exact function of the protein secretion system Esx-3. Together with important collaborators at Harvard School of Public Health we published more on how ESX-3 function in protein secretion in the high impact open access journal mBio (mBio, April 2014). 2014 also saw the beginning of preparing strains for vaccine experiments. The work has centered on antigen selection and design of plasmid vectors for efficient expression of our selected antigens. We also started work on developing a tool for measuring antigen presentation from macrophages and dendritic cells. By the start of 2015 we adapted a tool for antigen presentation detection that use the well characterized protein ovalbumin as a reporter antigen for investigating our various vaccine vehicles. Ovalbumin has well characterized T-cell epitopes that are presented both from MHC-I and MHC-II, so both presentation ways can be investigated for our vectors. Our results from these experiments indicate that our vaccine vectors and vehicles will provide antigen presentation, and therefore have the potential of delivering a vaccine effect. We will continue these investigations in a mouse model of mycobacterial infection by the end of 2015. In 2014 and 2015 we continued investigating the role of ESX-3 in mycobacterial survival as well and based on findings related to the biological function of ESX-3 we were able to publish a paper in AAC (February 2015). We also published the development of a novel expression system for mycobacteria in PLOS One (September 2015). 2014 and 2015 has also shown the importance of international networking. The GLOBVAC project has enabled our group to build strong international networks in the field of mycobacterial research and such a network led to the successful proposal to the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR). Without the young scientist project from GLOVAC the connection to and continuing of such an important network would be much more difficult. In 2016 we have continued developing our vaccine vector and have initiated animal experiments to test our proposed vaccines. We have presented our data at the Conference on Molecular Mechanisms of Inflammation in Trondheim, Norway and the EMBO conference "Tuberculosis 2016: Interdisciplinary research on tuberculosis and pathogenic mycobacteria" in Paris, France. In 2017 we have investigated our vaccine candidates effect on mycobacterial infections in mouse models. Preliminary analysis has been carried out, and we observe interesting differences between our vaccine and the traditional BCG vaccine. We are currently re-testing the vaccine to confirm findings in the initial experiments. We also have been studying how mycobacteria change over the course of infection. In particular, we have focused on genetic changes that might influence virulence development. In 2018, the PhD-student associated with the project defended her thesis. We completed the study on mycobacterial genetic changes within a patient over the course of infection and have a manuscript accepted in the journal Infection & Immunity. We also managed to repeat the necessary animal experiments for the vaccination concept study and except that work to be published in 2019.

The project has resulted in closer collaboration between research groups at NTNU and Harvard School of Public health. In addition the project laied the foundation for a new project funded by the JPIAMR-program on antimicrobial strategies towards mycobacterial infection. A project with 5 European research partners.

One of the leading causes of mortality in low and middle income countries is tuberculosis. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Multidrug and extensively drug resistant Mtb strains are now common (6-10% of new cases) adding to the b urden of disease. To significantly reduce the burden of tuberculosis in low and middle income countries there is a need for improved health care systems, improved diagnostics, novel drugs and improved vaccines. Mtb are able to parasitize host phagocytes , including macrophages and dendritic cells. The establishment of this intracellular niche is known to be critical for the development of disease, but it also needed for the production of an effective innate and cell-mediated adaptive immune response. Recently, it has been shown that a nonpathogenic mycobacterium, Mycobacterium smegmatis (Msmeg) strain, mutated in the protein secretion system Esx-3 had superior protection abilities in a murine model of Mtb infection. In our proposed project we will i nvestigate how mutant Msmeg can provide protection against Mtb infection. By combining advanced proteomics technology with knowledge in mycobacterial genetics and cell biology we want to reveal changes in the mutant Msmeg, how the change affects killing o f the bacterium in macrophages and the antigen presenting machinery. The knowledge from understanding mutant Msmeg interaction with host will provide a scientific base for designing novel vaccine candidates. An additional important aspect of our proposal is the building of long term capacity for high quality research on infectious diseases through increased collaboration between three Norwegian groups working on tuberculosis (NTNU, Rikshospitalet and Gades) as well as collaboration with international gro ups on mycobacterial diseases (Harvard School of Public Health, Boston, USA and Tsinghua University, Beijing, China).