Two million individuals die each year from tuberculosis infection, which is an increasing problem because HIV/AIDS makes individuals highly susceptible and because antibiotic resistant strains of Mycobacterium tuberculosis are appearing. The present tuberculosis vaccine, BCG, is only partially effective, so development of a better vaccine is an urgent health care priority. Up to now, most new tuberculosis vaccines have been designed to be given after BCG, in order to boost the weak immunity provided by BCG. This is called prime boost immunisation. However, it is becoming clear that prime boost immunisation may not be sufficiently effective to control tuberculosis. In this project we will establish an alternative novel immunisation strategy called Simultaneous Immunisation (SIM). We have already shown that giving one tuberculosis vaccine by injection and simultaneously spraying another into the lungs is highly effective in mice. The lung vaccine establishes local immunity, which combats tuberculosis infection immediately after infection, and the injected vaccine has a slower effect, but the two work very effectively together. We now want to test several different SIM regimes in mice to find the most effective one and test its safety. We will also study how different white cells combine to protect the lungs against tuberculosis, in order to make even more effective vaccines in the future. At the same time we will study humans infected with tuberculosis to develop better tests to assess immunity to tuberculosis. This will help in testing new immunisation procedures, including SIM, in man. A better tuberculosis vaccine will have major health benefits for humans and can also be used to control bovine tuberculosis, which is currently widespread in the UK and causes considerable economic losses.

How can we improve the development of better new vaccines to protect poultry (and other livestock) against major disease threats such as bird flu? Genetic manipulation (GM) is having an increasing beneficial impact on our lives, particularly in human and veterinary health care; nowhere more so than in vaccines, where many commercial products have been licensed and released for use in livestock and companion animals. These new vaccines are based on 'vectors', which can be regarded as carriers for the target vaccine, and are generally based on well-understood vaccines, such as poxviruses, with a long history of safe use against important diseases. The best-known example is Vaccinia virus, used in the only successful global eradication of a virus disease, Smallpox, and as a recombinant in the elimination of feral fox rabies from Belgium and France. Fowlpox virus vaccination since the 1920s has effectively eliminated fowlpox from poultry in developed countries in temperate climates. Spread by biting insects, it remains a major problem in tropical and sub-tropical countries where vaccination of chicks in hatcheries is common and extensive. Using GM, we can incorporate into the 'genome' (or chromosome) of the vector, a gene from a different disease-causing virus (or pathogen), such as bird flu H5N1, making a 'recombinant vector'. When that gene carries the instructions to make a structural protein of the pathogen, vaccination with the recombinant vector will induce an immune response in the vaccinated animal against the pathogen (and vector). Recombinant poxviruses have been licensed for veterinary use against West Nile fever, canine distemper, feral rabies and equine influenza. The most extensively used is a commercial recombinant fowlpox vector incorporating the H5 surface spike of bird flu. Two billion doses have been used to vaccinate poultry against H5 bird flu in Mexico since '95. There, the lethal form of bird flu was eradicated but a less dangerous form remained in circulation. The recombinant vaccine reduces shedding and transmission of bird flu but does not completely prevent infection of birds, possibly driving evolution of the virus by random mutation. There, therefore, remains considerable scope for improvement, particularly in terms of immunity that will clear birds of infection. The vectors are not just inert delivery systems. Poxviruses activate the immune system and have to survive in the presence of the host's immune response. To do so, the vector deploys tens of different gene products. Some of these will reduce the effectiveness of the vector as a recombinant vaccine. To improve the response we can use GM to remove such genes from the vector but, with so many candidates, our problem is identifying those which should be removed. Currently the only way to see if the vaccine has been improved is to test it in animals. We propose to look in detail our panel of fowlpox virus mutants, each defective in just 1 of the 250 genes of the vector. When the vector enters a host cell, it turns up (or down) the production of protein from about 1000 of the host's 30000 genes. We will look to see how the different mutations affect the control of these host genes by the vector virus, using the microarray technique (performed in tissue culture dishes in the laboratory). In this study, we will also need to see how each mutation affects the ability of the recombinant vector to induce an immune response (against structural proteins of H5N1 in chickens). We will then look for correlation between improved immune responses to the recombinant vector and changes in control of the host genes by the vector. This should then give us a profile, or a fingerprint, of gene control that we can associate with improved vaccines. In future, we would look for this profile in the laboratory as a first step. This will give us a way of predicting which new vaccines are likely to be improved, before testing them in animals.

The project proposes a rapid response to the current monkeypox virus (MPXV) epidemic. It is led by the Pirbright Institute and the Centre for Virus Research - Glasgow - the two UKRI-funded institutes that lead on virus infections of animals and humans - and brings together relevant expertise from several other UK universities and institutions including the Universities of Cambridge, Oxford, Birmingham, Edinburgh and Surrey, Dstl, UKHSA, Guys and St Thomas NHS. Between April and 18th July 2022, there have been 2137 confirmed cases of human monkeypox (MPX) in UK and the WHO has reported infections in all 5 WHO regions and 50 member states. The current epidemic is the largest ever known for MPXV. An urgent response to this growing epidemic is needed. The consortium assembled proposes 6 inter-related work packages as follows. 1. Genomic characterisation of MPXV. 2. Examination of possible virus spillover from humans to UK animals 3. Study of the intrinsic and innate barriers to MPXV infection, and MPXV immune evasion strategies 4. Study of the immune response to MPXV infection and vaccination 5. Development of anti-viral drugs and monitoring for emergence of MPXV drug resistance 6. To develop point of care diagnostic tests for MPXV WP 1. This will undertake sequencing of MPXV genomes isolated from humans in UK and monitor virus evolution and adaptation to humans. The sequencing will pay particular attention to the acquisition of genome mutations that might affect virus replication, transmission, virulence or drug resistance and links to WP5. WP2. MPXV has a natural reservoir in rodents in parts of Africa and has a relatively broad host range that includes North American rodents, primates and humans. The widespread human infections provide a possible opportunity for human to animal transmission. This WP will evaluate this potential by examining the ability of MPXV to infect primary cells from a variety of UK animals. WP3. This WP will evaluate the host response to infection by measuring the transcriptomic and proteomic responses to infection of human cells and testing the roles of specific host proteins in protecting against MPXV infection. Further, the ability of MPXV to counteract these defences will be tested building on what has been learnt from studies of related orthopoxviruses. WP4. The immune response to MPXV infection of humans will be measured by determining the antibody and T cell responses. These will be compared with the responses to vaccination using the smallpox vaccine. A specific aim will be to identity signature T cell responses that are characteristic of MPXV infection. In addition to information vaccination programmes, the development of specific tests for immune monitoring will be undertaken. WP5. This WP is concerned with the development of anti-MPXV drugs and builds on the development of CRUSH (COVID-19 Drug Screening and Resistance Hub) at CVR-Glasgow. Currently, 2 drugs are licensed for use against MPXV and these each target a specific virus protein, but mutation of these proteins can lead to drug resistance. The WP proposes to screen additional FDA-approved drugs that have activity against VACV for activity against MPXV. Cyclosporin A and non-immunosuppressive derivatives will be included since these target a proviral cellular protein, cyclophilin A, and therefore emergence of virus resistance is difficult. WP 6. This will develop point of care (POC) diagnostic tests for MPXV. Currently, MPXV infection is confirmed by polymerase chain reaction (PCR), which is specific and sensitive but requires a specialist laboratory. A POC test (such as developed for SARS-CoV-2) would be of great benefit to speed diagnosis. Two approaches will be tried: a Lateral flow test (LAT) and a loop-mediated isothermal amplification (LAMP)-based assay.

Infectious bronchitis is an important endemic disease of chickens and caused by the avian coronavirus infectious bronchitis virus (IBV). Poultry meat is an important food source and during the course of a year approximately 40 x 109 chickens are reared globally. The increasing demand for poultry meat has lead to the introduction intensive farming methods but productivity is often limited by infectious diseases which spread rapidly through high density chicken populations. Importantly for this proposal, a report sponsored by the UK government published in 2005 (http://www.defra.gov.uk/science/Project_Data/Document Library/ZZ0102/ZZ0102 1215_FRP.doc) revealed that the number one cause of economic loss in the UK poultry industry resulting from infectious diseases of chickens was caused by IBV. The virus is not only responsible for respiratory disease, but also causes damage to the kidneys and to egg producing organs of hens, affecting both the production and quality of eggs. Despite the availability of live and inactivated vaccines, IBV continues to be a major problem. The virus causes high morbidity, is ubiquitous world wide, and endemic in the UK, and shows extensive antigenic variation and short lived immunity. These factors lead to high rates of infection and poor cross-protection following infection or vaccination. Most vaccines are given to poultry by spray or in drinking water. Both approaches are rather hit-and-miss. The 'holy grail' of vaccine developers is to have vaccines that can be given by robotic machine to chicks before they hatch. Unfortunately, no existing IB vaccine can be given in ovo because the viruses stop the chicks hatching. One means of controlling IBV is to have a systematic way of generating live attenuated vaccines that provide protection against virulent strains. The reverse genetics necessary for the modification or removal of genes associated with virulence from IBV has been developed by the coronavirus group at the IAH Compton. Together with DEFRA and Intervet International, a major commercial vaccine developer, the IAH coronavirus group are manipulating the genes of IBV to get an optimum balance between attenuation of virulence and capacity to induce immunity. To apply this technology to the rational design of live vaccines it is now necessary to identify genes that contribute to IBV virulence, and understand how they function. Importantly for this proposal, recent work on mammalian coronaviruses, and work on the avian IBV coronavirus at Compton, suggests that virulence may be determined by the way in which cells control virus replication. We have produced IBVs that do not make a series of small proteins called 3a, 3b, 5a and 5b. These viruses grow normally in cell culture and allow chicks to hatch after inoculation in ovo. This shows that we can attenuate IBV by removing non-essential genes, but this is an empirical process because we do not know how the 3(ab) and 5(ab) proteins function in the context of virulence. The purpose of the present grant application to BBSRC is to establish the science behind the empirical observations that we are making. Experiments underpinning this proposal have shown that virulence of IBV may be associated with proteins that control replication, and that these proteins associate with membranes in cells that have the potential to destroy the virus before it can leave the cell. We now want to understand how the replicase proteins avoid destruction, and in this way determine virulence. This will enable us to fine tune our mutants to make viruses that survive long enough to infect chicks 'in ovo', and induce an immune response, but are too weak to harm the chicks and prevent them from hatching.

Swine influenza attracts considerable attention because of the threat of zoonotic infections causing human pandemics. During the pandemic, a fear that viruses emerging from pigs may infect people resulted in the widespread destruction of animals in some countries and trade bans. Consequently, the insidious effects of this highly prevalent virus on the health and welfare of pig populations, estimated to increase the cost of production by £7 per finished pig, have not been given due regard. The primary disease caused by influenza virus in usually mild, but results in greater susceptibility to secondary infections. Vaccination will be a key control measure for influenza in pigs to improve general herd health. Through our studies we will develop a more detailed understanding of the dynamics of virus transmission and the consequences of transmission and vaccination in driving viral evolution. During these studies we will also define a range of parameters, for example local and systemic immune responses and sites of virus replication, which are associated with the onset and cessation of transmission. We need to know if current and proposed novel vaccines not only prevent clinical signs but also stop viruses being transmitted unnoticed. Furthermore, if viruses can be transmitted unnoticed are they changing because of the immune response in the population? To answer these questions we need to understand virus transmission in detail and how the viruses change when they pass between animals. We can then apply this new knowledge to population wide models of disease spread to predict the efficiency of any proposed control measures. This knowledge will also inform the design of novel vaccines. Vaccination against influenza in pigs is not routinely performed in Europe mainly for two reasons: the cost benefit of vaccination has not been clearly demonstrated and it is not clear that the available vaccines will protect against the strains currently circulating in the pig population. The most striking example of the latter is that current vaccines do not include pandemic H1N1 influenza virus antigen. These studies will provide essential evidence to design control programmes for influenza in pigs, most notably: i) finding out how efficient are the current prophylactic methods at controlling the spread of infection; ii) what level of immunity is required in a population to prevent the spread of infection and the evolution of new strains of virus and iii) determine whether new, broadly cross protective vaccines are more effective at controlling influenza infections in swine to enhance animal health and livestock production. Importantly, this type of information is not available for any natural mammalian hosts of influenza viruses, including humans and horses. Therefore, the results of our studies will have a broad impact on influenza control measures.