Optimisation of DNA vector activity and delivery for improved vaccination

DNA-based vaccines represent a powerful technology that could be well-suited to the quick development and rapid, large-scale production needed for effective response to rapidly emerging or mutating pathogens. However, DNA vaccines have not yet been widely accepted or utilised as a vaccine technology due to the lack of immunogenicity observed. Intramuscular injection is the most commonly used vaccination dosing route but the skin represents an immune-rich environment that has been shown to enable significant dose-sparing and broader immunogenicity advantages. However, traditional needle and syringe methods are not adept at accessing this potentially powerful vaccination site. Cavitation has been shown to be able to permeabilise the stratum corneum and hence, provides a method for accessing the epidermis in a reliable and pain-free way. In this thesis, cavitation-mediated delivery of DNA vaccines into the dermal compartment is explored as a strategy for improved vaccination.
This thesis begins by exploring the formulation and ultrasound parameters of needle-free, cavitation- mediated delivery of DNA into the skin. Data shows that relatively low quantities of DNA are delivered into the skin compared to direct injection but that cavitation can be a mechanism to drive increased delivery into the skin. It is also demonstrated that, once cavitation is achieved, exposure of skin to higher ultrasound pressure amplitudes provides no additional benefit. It is also proposed that cavitation-mediated delivery targets a different cell population compared to intradermal injection, based on the observation that the balance of anti-transgene antibody isotypes is altered. Specifically, for cavitation mediated delivery an IgG1-dominant IgG profile is produced compared to a balanced IgG1/IgG2a response observed upon injection. An initial exploration of improved DNA vectors for vaccination, showcases the scope for improvement. Hence, subsequent chapters are devoted to three avenues of optimisation.
The potential of protein cavitation nuclei (p-CaN) is demonstrated, both as cavitation nuclei and as an immune-stimulatory particle. It is shown p-CaN can match delivery of DNA compared to polymeric cavitation nuclei produced to GMP standard, as well as induce an immune response against the protein used to formulate the p-CaN. A number of p-CaN iterations are validated in this work, including p-CaN produced by the scalable technology of hydrodynamic shearing.
The second optimisation explored was an improved ultrasound set-up (V2). Crucially, the ultrasound transducer was inverted to enable easier handling and the scruff of the mouse is treated to prevent accidental ultrasound exposure of internal structures. Optimisation of delivery parameters using this improved set-up are determined and validated through reporter gene expression, anti-transgene antibody assays, as well as qPCR for DNA delivery quantification.
The final optimisation explored is in terms of DNA vectors used. In particular, perforin-encoding DNA was identified as a powerful adjuvant for cavitation-mediated delivery and shown to improve antibody titres and avidity compared to a non-adjuvanted DNA vaccine. A self-amplifying vector was also explored with the transgene expression profile defined and the immunogenic potential characterised. This showed that the self-amplifying vector has a marked increase in protein production after 5 days and can produce more reproducible antibody titres, higher avidity and a more IgG2a-skewed immune response compared to a non-amplifying vector.
In the final chapter, the potential of the self-amplifying vector and the perforin-encoding DNA vector are tested in a challenge study, which shows that the self-amplifying vector improves survival, and reduces symptomatic disease in a mouse model.

Keywords: vaccine
DNA
ultrasound
synthetic biology
immunology
perforin
p-CaN
cavitation
influenza
Advisor: Carlisle, R