Food-grade bacterial vectors as novel tuberculosis vaccines

Tuberculosis, caused by Mycobacterium tuberculosis, remains a major health threat, with nearly 10 million new cases and 1.4 million deaths each year. The current BCG vaccine has limited effectiveness against infection and transmission. Therefore, the highest current priority of TB research is to develop a more effective vaccine that will help to scale up immunization coverage in the poorest countries and help the global community to achieve the Millennium Development Goals. This is not an easy task and novel approaches are needed. This project aims at developing bacteria commonly found in food products such as yoghurt as delivery vehicle for a tuberculosis vaccine. The bacteria used, Lactobacillus, are food-grade, they thrive in the human digestive system, and they survive the harsh conditions in parts of this system (e.g. the stomach). This approach could in principle lead to vaccines that are easy-to-produce (e.g. yoghurt), easy to store, and easy to administer (no needles). Moreover, such vaccines could, if successful, induce mucosal immunity, meaning that they would have a unique and much sought after ability to prevent (rather than combat) infection and reduce transmission. The project builds on previous development of a genetic toolbox to engineer Lactobacillus strains so that they display vaccine components on their cell surface. We have the ability to display components in several ways, varying from sticking really out of the cells, so that they are "visible" to the immune system but also vulnerable for degradation, to being more hidden in the bacterial cell wall. We also have the option to equip the bacteria with signals that direct them to the most important immune cells. We collaborate with a European network on tuberculosis vaccine research, coordinated by the TuBerculosis Vaccine Initiative (www.tbvi.eu) and with the group of Rajko Reljic at St. George's University of London. After spending several years developing new tools to generate the best possible strains, the first vaccine candidates have now been tested in a mouse model. These are very large and expensive experiments, which require specialized expertise and infrastructure and we have performed such experiments at St. George's University of London, in the group of Dr Rajko Reljic. The Reljic group sees great potential in our work and therefore devotes significant own resources to our joint work. The experiments showed that several candidate strains protect mice against Mycobacterium tuberculosis, which is a very important and encouraging result. Alongside induced immunity, we also saw (desirable) activation of humoral and cellular immunity. Further steps in the development of vaccine strains include the use of different types of adjuvants (expressed on the bacterial surface), more advanced genetic modification (CRISPR / Cas), development of non-GMO alternatives, and other types of optimization of existing tools. In the very last phase of the project, we have focused on optimizing existing tools (including CRISPR / Cas), and we performed mouse tests to test the immunogenicity of new promising vaccine candidates (= newly developed Lactobacillus strains). We have completed systematic studies aimed at finding the Lactobacillus species that give the best effect when used as a Tb vaccine and we have compared several ways of "anchoring" the vaccine to the bacterial surface. Importantly, these experiments have shown that the candidate strains in many cases generate a desired mucosal immune response. To further test and develop the generic value of our technologies, we have run parallel projects, with Master students and guest researchers, where our protein secretion and anchoring tools are used to express other proteins, including other vaccines. Among other things, we look at cancer antigens. The project has been presented on national television (Schrödingers Katt, 2.10.2014, see https://tv.nrk.no/serie/schrodingers-katt/DMPV73002014/02-10-2014#t=16m48s; insert no. 5), in Aftenposten Viten in 2018 (https://www.aftenposten.no/viten/i/wERPwP/haap-for-ny-tuberkulosevaksine-norge-med-i-kapploepet), and, recently, at Forksning.no, with both text and video (https://forskning.no/nmbu-norges-miljo-og-biovitenskapelige-universitet-partner-vaksiner/nesespray-vaksine-mot-tuberkulose-viser-lovende-resultater-i-museforsok/1315644). Efforts are being made to build further on the very promising results of the project, primarily in collaboration with TBVI, and hopefully, among other things, in the form of a future EU project.

Outcome 1: A unique toolbox for anchoring proteins (e.g. antigens) to the surfaces of safe bacteria (lactobacilli) that occur in humans and food. Components of this toolbox are today used by researchers all over the world. The toolbox allows variation of the set-up: the Lactobacillus strain, the antigen, the anchoring method, the amount of antigen, the genetic technology (GMO, non-GMO). Outcome 2: A series of Lactobacillus strains with anchored tuberculosis antigens that induce favourable immune responses, including much sought after mucosal immune responses. Based on analysis of the many variables listed above, a picture of what set-ups work best is emerging. Outcome 3: Infection ("Challenge") trials with mice show that the most promising candidate Lactobacillus-based vaccine strains indeed reduce tuberculosis infections. Further steps to eventually reach clinical trials are coordinated with European research groups, through TBVI, The Tuberculosis Vaccine Initiative.

Tuberculosis (TB) is a re-emerging disease and is one of the leading causes of morbidity and mortality in humans, primarily in low-income countries. The current BCG vaccine has variable effectiveness and the need to develop novel vaccines with simplicity, safety and low cost is urgent. Furthermore, there is a need for vaccines that induce mucosal immunity, thus potentially blocking transmission. We will focus on innovative microbial delivery technologies based on food-grade Lactobacillus species, includin g further development of generic technologies for vaccine display on the surfaces of these unique commensal bacteria with interesting immunomodulatory properties. We will team up with the TuBerculosis Vaccine Initiative (TBVI), a top level international n etwork with partners from higher, middle and lower income countries. By doing so, we will combine the best of international tuberculosis vaccine research with advanced safe microbial delivery technologies, while at the same time ensuring an optimal pipeli ne for communication, implementation and knowledge transfer. The research comprises five interrelated activities: 1. Designing and engineering Lactobacillus strains, based on the applicant's generic technologies and with help of international experts for antigen and adjuvant selection. 2. Verification of immunological effects using cell cultures and basic mouse models, combined with a wide variety of response (immunological) analyses. 3. Pre-clinical efficacy tests in standardized TBVI-quality-controlled mouse models. 4. L. plantarum genome modification to increase the safety of promising strains. 5. Implementation, in particular in terms of designing and implementing product development trajectories, knowledge transfer, and initiating follow-up activitie s. Together with experts from the TBVI network, we will apply stringent criteria that need to be met for candidate strains to progress in this vaccine development and characterization pipeline.