Development of biodegradable nanobeads as vaccines against tuberculosis.

Tuberculosis (TB), which is caused by infection with the bacterium Mycobacterium tuberculosis (M.tb), is a major burden globally and causes more than 1,3 million deaths every year. The only available vaccine against TB, the live vaccine Bacille Calmette Guerin (BCG), is however largely ineffective in protecting against TB in adults. Using nanotechnology, we were aiming to improve the foundation of the BCG vaccine by modifying live BCG bacteria with molecules able to stimulate the immune system (immunostimulatory molecules) either by coating them directly on the bacterial surface in a nanometer-sized layer or by adding them in form of nanometer-sized particles (nanoparticles). We were able to coat the surface of live BCG-bacteria with up to four layers of the immunostimulatory molecules poly(I:C) and chitosan without affecting the viability of the bacteria. Poly(I:C) is a potent inducer of so-called cell-mediated immune responses, which are typically very important in the protection against M.tb infection. Live poly(I:C)-coated BCG-bacteria strongly enhanced immune responses of macrophages, which is the primary cell type involved in fighting M.tb infection and in fact this enabled them to kill the BCG bacteria. Remarkably, the immune response to BCG was only enhanced by surface coated poly(I:C), but not by its soluble form. Together, this indicates that coating of the BCG vaccine with immunostimulatory molecules might be a valuable tool to improve the ability of the vaccine to trigger immune responses which are important in protecting against TB. In parallel, we have developed nanoparticles based on the same immunostimulatory molecules, which showed a similar ability to enhance the immune response to the BCG vaccine. This was found to be based on a hitherto undescribed powerful synergistic interaction of pathways activated by the BCG bacteria and by the poly(I:C) in the nanoparticles. These results provide a platform for testing these novel nanoparticles in future vaccination studies in mice. These discoveries have also led to collaborative work aiming to study the potential of nanoparticles in cancer immunotherapy and vaccination against fish viral diseases. The tumor immunology group at the Oslo University Hospital headed by Alexandre Corthay is exploring the use of these nanoparticles in combination with other activators of the immune system in immunotherapy against cancer. High densities of macrophages within solid tumor tissue are generally associated with a negative prognosis. However, when an inflammatory immune response is activated in these cells, they gain the ability to attack and kill the cancer cells. In this context, this nanoparticle formulation holds great promise for cancer immunotherapy. Our nanoparticles together with virus antigens have further proven to be an effective vaccine formulation against viral hemorrhagic septicemia (VHS), a viral disease in salmon and trout. Using a zebrafish model for virus infection, the group of Prof. Tor Gjøen at the school of pharmacy, UiO, has shown that this vaccine formulation is able to provide a high degree of protection against VHS virus infection. Finally, using the well-established zebrafish model for tuberculosis, we further studied the interaction of pathogenic TB-like mycobacteria (in this case M. marinum) with external surfaces of the body, in particular the gastro-intestinal tract, which is considered to be the main port of entry for M. marinum in fish. We were able to establish that M. marinum utilizes the uptake by a specialized cell type, also called antigen sampling cells, to cross the gastro-intestinal tract of adult zebrafish and to spread inside immune cells to the liver and spleen within a few hours. Similar patterns of uptake and transport were seen for different nanoparticles. Taken together, this model gives us more inside into the processes and mechanisms involved in mycobacterial infection across external barriers and how these might be targeted for nanoparticle-based vaccination. Our results show that our nanoparticle formulations are a novel and promising approach for improving the current TB-vaccine and for å developing vaccines against viral diseases in aquaculture with the prospect for further development to commercial products. The powerful synergistic interaction between nanoparticles and BCG in the activation of immune cells, which was discovered in line of this project, may further prove to be a promising starting point for developing an immune system-based therapeutic against cancer with negative prognosis.

One third of the world's population is infected with Mycobacterium tuberculosis (M.tb) and two million die annually of tuberculosis (TB). The standard therapy, using antibiotics, is increasingly ineffective due to multi-drug resistance. The only available , 80 year-old vaccine using M.bovis BCG has little therapeutic value, and no other vaccines have reached the market, despite enormous efforts. Here, we propose to develop technology to encapsulate whole mycobacteria inside biodegradable nano/microbeads to facilitate delivery of intact live or killed bacteria to vertebrate antigen-presenting cells (APC) to initiate the immune response. The Oslo nano-bead group of Nystrom will investigate different polymer materials that can encapsulate live and killed myco bacteria, focusing on the fish TB bacterium M.marinum, BCG and the mouse/human pathogen M.avium and Mycobacterium w, a promising anti-tuberculous vaccine candidate. The Oslo cell microbiology group of Griffiths has established a system using GFP-M.marinum lethal infection of transparent zebrafish. After first experiments with BCG- and M.avium-beads in macrophages in vitro we will search for the most effective GFP-bacteria-enclosing beads that when injected or fed to zebrafish cross mucosal barriers and in duce protection against subsequent infection with M.marinum. This rapid fish screening system will narrow down the promising polymer candidates/conditions for testing beads enclosing M.avium, Mw and later virulent M.tb, for their vaccine potential in mic e and guinea pigs against subsequent challenge with these pathogens by Verma and Khuller in India. Different oral, intra-nasal and subcutaneous vacination routes will be tested. A crucial advantage of nanobeads over freely-administered immunogens is thei r established ability to cross mucosal barriers intact. Our strategy ensures that the first location where mycobacteria become exposed to antigen digestion/ presentation systems is in the phago-lysosomes of APC's.