Development of a rapid and facile platform for testing viral escape-resistance of therapeutic antibodies & vaccines & determining escape mutations

Vaccines can be extremely effective for preventing people becoming ill from viruses, and anti-viral medicines can help those who do become ill to recover. However, viruses evolve and can acquire mutations leading to the appearance of some viral variants that can partially or fully by-pass these preventions and treatments. These so-called escape variants are dangerous as they can cause even vaccinated people to become ill, and in the worst case leave us without protection against the virus. It is very difficult to predict what mutations can occur in a virus that will lead to loss of effectiveness of medicines and vaccines. This project aims to develop a new method for very quickly and easily testing which new drugs and vaccines would still work best against future viral variants, and for revealing the mutations the virus could acquire that would lead to escape.

This new method does not use viruses. Instead, it uses the protein that the vaccines and drugs normally target in the virus, and tests whether they are still effective against a whole range of possible mutant versions of this target protein. It also reveals the different variant forms of this protein that the virus could produce. This new method could be used during the development of vaccines and drugs to enable those that will be most effective against any new variants to be selected and developed, even before the variants appear. In addition, if escape-resistance is not possible, it reveals how the virus would be able to escape, allowing us to develop the appropriate follow-up booster vaccines and drugs that would block these escape routes and be most effective at protecting the population.

Mutational escape of viruses from therapeutics and vaccines is a major challenge to controlling the spread and impact of viral diseases. Development of the most effective therapeutics and vaccines requires methods for testing resistance to viral escape. However, current methods are technically demanding and time consuming.

This project aims to optimise and validate a rapid and facile new method for testing escape resistance of therapeutic monoclonal antibodies and vaccines that target viral binding to host cell receptors, and for revealing mutational escape routes. The methodology entails expression of the target receptor binding domain (RBD), or full-length spike, of the virus in a eukaryotic cell surface display and in-cell mutagenesis system. Libraries of cells expressing mutant variants of viral protein are produced by simply expanding the cells, and escape variants are selected by fluorescence activated cell sorting. Iterative cycles of selection and expansion accumulate mutations to enable emergence of escape routes that require mutational combinations.

The methodology will be optimized and validated using SARS-CoV-2 as an example target virus, and tested for its ability to reveal mutations leading to escape of CoV-2 RBD and full-length spike protein from a series of monoclonal antibodies, and post-vaccination plasma. The functional significance of the mutations will be confirmed by in vitro binding and pseudotyped virus entry assays. To validate the method against current approaches, the numbers and identities of the escape mutations found will be compared to those already identified by current yeast surface display and replication-competent pseudotyped virus methods, for the same monoclonal antibodies and similar post-vaccination plasma.