A genetically modified nasopharyngeal commensal as a platform for human bacteriotherapy

Our long term objective is to invent a new type of medicine which consists of living bacteria which would be given as nose drops (akin to the Yacult drink that is commercially available and taken by mouth by people with bowel problems). We have recently published work in which we inoculated a small dose of a `friendly bacterium` - Neisseria lactamica - into the noses of participants and found that the bacterium was still present in their throats 6 months later, and caused no ill effects. Furthermore, the friendly bacterium stopped the paticipants from being infected with a related bacterium which can cause meningitis. This concept of `bacteriotherapy` is rapidly gaining credence as a way to treat bacterial infections - for example we now treat Clostridium difficile diarrhoea with bacteria derived from the stools of donors, and it works very well. We have discovered a way to genetically transform Neisseria lactamica with genes from a very wide range of living things, which means that we could potentially develop a range of bacterial medicines containing genes which exert specific desired effects in the recipients. For example, we could make a bacterial medicine which makes substances which kill harmful bacteria or viruses or which simply out-competes them. One of the problems with our idea is that when we inoculated students with the Neisseria lactamica, we found that 65% of them became colonised, but this was only 35% if we restricted the study to non-smokers. This would not be a very reliable bacteriotherapy and we would need to increase the likelihood of colonisation if this approach is to be of any use. So, in the proposed study, we will use our technology to make a strain of Neisseria lactamica which makes two proteins that are normally used by other bacteria to stick to cells. We will use for this purpose two proteins that are normally made by the related meningitis bacterium Neisseria meningitidis, to stick to stick to the inner surface of the nose when it colonises humans. We will show that the new genetically modified strain has all of the characteristics we expect including the ability to stick better to cells in the laboratory, and that is safe to release into the community (because it is not more resistant to the immune system, or to antibiotics, and does not have increased capacity to gradually change into something more dangerous). We will then apply to the appropriate regulators for permission to repeat our studies with human volunteers to see if the genetically modified strain that we have generated manages to colonise better than the wild type bacterium from which it is derived. This would be the subject of a follow-on study - if that were to be successful this strategy that we have devised would be a future therapy of relevance to any disease process involving colonisation of the nasopharynx, eg pneumonia, Chronic Bronchitis, sinusitis, ear infection, meningitis or MRSA colonisation and disease.

The commensal Neisseria lactamica (Nlac) will be genetically modified (GM-Nlac) as a live vehicle for expressing proteins in the human nasopharynx. We previously showed that induction of carriage by wild type Nlac is safe, and persists for >6 months, however at the inocula used, only 30-65% volunteers become colonised. GM-lac will therefore be modified to express Neisseria adhesins to enable enhanced colonisation of humans. With a view to using GM-Nlac in experimental human challenge, our cloning and mutation system avoids the use of antibiotic resistance markers, and instead utilises the Nlac-derived beta-galactosidase gene (lacZ) to discriminate colonies derived from successfully transformed bacteria by colour-change. Genes encoding NadA and Opc will be targeted to a 2.18 kbp intergenic region between NLY_27080 and NLY_27100. Insertion of heterologous antigen constructs into this site will minimize disruption of the existing genetic architecture. To facilitate future GMP Cell Bank manufacture all work will done with animal-free growth media. Surface expression of these antigens will be confirmed by flow cytometry and western blot. To establish functional modification, adherence assays using Chang cells which have previously been used to show functional enhancement by NadA and Opc will be done. GM-Nlac will then undergo assessment of complement-mediated killing using native and IgG-deleted human plasma and antimicrobial sensitivity testing, to ensure that GM-Nlac remains as susceptible as wild type, and that GM-Nlac remains sensitive to the antibiotics used to treat meningococcal disease. To investigate likelihood of GM-Nlac strains being transformed with exogenous DNA from the nasopharynx, we will assess the efficiency of transformation using both genomic DNA derived from a meningococcal mutant derivative, delta-siaD and an antibiotic resistance gene aphA3, and seek evidence of uptake of capsule biosynthesis machinery.

The major expected impact of this project will be a new delivery vehicle for antigens and DNA within a live bacterial vector into the precise site of bacterial colonisation of the upper respiratory tract. This will be used to prevent or treat antimicrobial resistant pathogens by (i) competitive bacteriotherapy, (ii) delivery of expressed proteins into the mucosa which could directly or indirectly (eg via microbiome effects) affect AMR pathogens, (iii) by providing a novel means of vaccination by expression of homologous or heterologous antigens.
Pharmaceutical Industry
At the point at which we test and prove that genetically modified organisms can be safely inoculated into humans and generate an anticipated effect (on colonisation efficiency and on immunogenicity) we will contact appropriate sectors of the Pharmaceutical Industry who are developing products that require prolonged expression within the mucosa, or by a bacterial vector likely to be processed by the mucosal immune system with delivery of antigen to local lymphoid cells. This will likely include SMEs with candidate products such as antimicrobial peptides, proteins that influence quorum sensing, bacteriocins or compounds that inhibit or promote biofilm. Even a simple GMO Neisseria lactamica that colonises at high efficiency even without further modification (such as we propose in this application) , will likely have profound competitive effects on other colonisers such as Neisseria meningitidis (as we have already shown - Deasy A, et al Clinical Infectious Diseases, 2015) and as such could be a highly effective prevention strategy for pathogens that are sensitive to colonisation by successful commensals. It will also provide a system that innovative small companies with new vaccine ideas might be able to accelerate the recognition of their new product. It is likely that we will be able to generate this new potential within 4 years of award of this grant. There is a groundswell of interest in probiotics for mucosal use - so far this has focussed on the gut (eg for Clostridium difficile disease) but the upper respiratory tract has a microbiome as well and colonisation of this mucosal surface is the first step in the pathogenesis of major diseases such as pneumonia, COPD, otitis media, sinusitis and meningitis.
Patients and Wider Public
The final objective is to create a general strategy for a range of bacterial medicines that likely would only require one single dose administered intranasally. As such the potential impact on the general public would be to improve greatly the simplicity and convenience of the medicine. Medicines of this type could play a role in preventing hospital infections, as well as treating established ones, and could for example be administered prior to a planned hospital visit or a course of chemotherapy, preventing considerable potential morbidity.
Government/Policy Makers
This development could increase the effectiveness of public services and policy. If this model is developed successfully, it could provide a suite of bacterial medicines that offer alternatives to vaccines for prevention of diseases that arise after colonisation of the upper respiratory tract.
Training/Development
It is essential that we train future leaders , and this project will train a researcher in a new and important multidisciplinary field involving microbiology, biochemistry and cellular and molecular biology, coupled with state-of-the-art technologies. The PDRA will have unique opportunities to interact with staff at Southampton, and will take part in the Southampton Network for Antimicrobial Resistance Action (NAMRA) 'Future Leaders in AMR Programme' and will include Engagement & Outreach, Gaining Funding, Communicating with the Public, 'Four star' publications, Impact and more.