A reverse vaccinology approach to a bTB vaccine

Bovine tuberculosis, bTB, is the result of the infection of cattle by the bacterium Mycobacterium bovis. The bacterium is distributed widely in nature as it also infects many other wildlife species and as a result of this, wildlife infection acts as a reservoir for the bacterium which periodically get across into domestic cattle. The consequence of this is twofold. First, cattle that are bTB positive must be culled with a knock-on effect on the farmer and his ability to maintain a herd. Second, bTB is a threat for human infection, primarily via the consumption of contaminated milk. Today bTB infection of individuals is extremely rare, but the threat remains and for these reasons bTB infection is unwelcome and needs to be controlled or, preferably, eradicated. Since a historical review in the mid-1990s, the badger has been identified as one of the major routes of transmission of bTB to domestic cattle and this, in turn, has led to attempts to break this transmission route via badger culling. These have met with only limited success and immense public concern leaving the physical breaking of the transmission route mostly unaltered. An alternative approach is to accept that bTB circulates widely in the environment but to prevent cattle infection by previous vaccination. At the present time however an effective vaccine for bTB in cattle is not available and new methods to develop such a vaccine are urgently needed. It is generally accepted that vaccines function by generating an antibody response in the target animal which prevents the bacterium from establishing the initial infection. These antibodies, which are a normal product of the immune system of all mammals, generally bind to the outside of the bacterium and so prevent it from binding to cattle cells, usually the epithelial cells of the lung. It follows that the protective components of bTB, that is, the components that will generate the antibody response that is protective, are to be found on the outside of the bacterium surface. Bacteria have many components on their surface and any, or perhaps a combination of many, of these components, proteins encoded by the bacterium genome, could be essential for the development of effective immunity. However exactly which are required is currently unknown.

In this research, we propose to produce all of the surface components of bTB and to test them in batches for their ability to induce an effective immune response. We propose to do this work in cattle so that the response measured to our test vaccines is typical of what will be found if an eventual vaccine is used in typical herds. Our work breaks down into three related components. Firstly, we will identify all of those proteins from the M. bovis bacterium that are to be found on the surface of the organism and produce each of them in a safe and efficient manner. Our initial calculations suggest that several hundred such proteins may be required in order to find the few that are necessary for effective immunity. Secondly, we will use our surface proteins as test vaccines in cattle and to make this process efficient we will carry out this work with mixes of proteins so that the least number of cattle has to be used. Following the immunizations we will take blood samples from the cattle and test them for the ability to prevent M. bovis infection. Finally, we will examine the mechanism of protection and how the individual components so we have identified work together to provide the cattle with an effective barrier of immunity. Our approach is exhaustive but it has the potential to draw a line under the vaccine discovery program for bTB and to identify the best mix of candidates for eventual effective vaccine production.

We will identify vaccine candidates for bTB through the use "reverse vaccinology" wherein the open reading frames (ORFs) representing the surface proteins of M.bovis are identified through bioinformatics and cloned and expressed using previously developed high throughput technologies. These proteins represent the maximum complexity of any bTB vaccine and within them will be individual components which together represent a protective vaccine cocktail. Such protective antigens will be identified through a tiered screening approach once the initial catalogue of all surface proteins is assembled. Several hundred proteins are likely to complete this screening process. Proteins will be used to immunise cattle in formats designed to induce antibodies and, separately, T-cells . The former will use classical immunisation with purified proteins and adjuvant while the latter will use a newer "RNA vaccine" approach. Sampling of the cattle following vaccination by each route, i.e. protein or RNA, will derive blood samples which will be tested for seroconversion and for the induction of T-cells to the proteins used. Blood samples will also be tested directly for their ability to inhibit the growth of M. bovis in established blood culture techniques. Vaccine candidates that emerge from this screen will be assessed further, individually and together, by subsequent rounds of vaccination using the relevant delivery route and by using mixed routes (protein+RNA). M.bovis ORFs identified as containing epitopes of vaccine candidates will be subject to structural study in combination with epitope mapping in order to deduce their immunogenic surface. Antibodies will be immortalised by cloning of immunoglobulin genes directly from activated B cells followed by expression and isolation as purified recombinant proteins. Antibody structures will be derived, as will antibody-antigen complexes, to understand the structural basis of protection and to inform the design of 2nd generation vaccines

Bovine TB in the UK is an emotive issue in which the direct effects of infection, the indirect effects of infection and the effects that might occur as a consequence of preventative actions become muddled. As a result, the number of groups of individuals ultimately affected by the issues around bTB is considerable. The primary beneficiaries of a successful vaccine candidate which will lower the pathogenic burden of bTB infection in commercial cattle herds are farmers and those immediately dependant on their success. A second direct beneficiary group would include those who wish to farm or those who wish to develop dairy or beef herds but are currently prevented from doing so by the fact that they occupy land where there is a high endemicity of bTB infection. A third group of direct beneficiaries would include those charged with the current monitoring and culling policies and there time would be freed to spend on other areas of farm biosecurity. The broader beneficiaries include land users of all forms, be it productive use or leisure use. Conservationists currently concerned about the badger culling policy would also benefit from a knowledge that this route of infection control would no longer be required. The public at large, those who do not fall into one of the previously described categories, would benefit from a safe dairy industry in which the threat of bovine TB transmission to man, albeit extremely small, would be reduced even further.

Further benefits from a focused research plan to develop an effective bovine TB vaccine include the possibility of vaccinating species other than cattle, for example pigs and deer, that represent other farming stocks. Vaccination of wildlife is also a possibility although the logistics of this and the lead organisation involved would clearly be very different to the vaccination of any farm species. The basic research data obtained from the proposed study could feed into the development of vaccines for other complex diseases and of course the identity of the proteins responsible for the generation of a level of protective immunity against M.bovis inform the improvement of a vaccine designed for TB in man. It has recently been found that BCG, the attenuated form of M.bovis used as a vaccine in humans, lacks a considerable number of genes when compared to the parental strain or indeed to mycobacterium tuberculosis. A potential outcome from our research programme would be the reintroduction of the genes encoding beneficial proteins into BCG such that it becomes a more effective universal vaccine than is currently the case. This would benefit Pharmaceutical manufacturers, who would be producing a more efficacious product, and the general health of society, in the UK and worldwide. An improved BCG would certainly be easier to adopt by current manufacturing regimens when compared to a wholly different form of vaccine, which may be more expensive and difficult to administer.

Overall, a positive outcome to this work would benefit many groups, not only those immediately affected by bTB positivity.