Microfluidic manufacturing and development of a liposomal vaccine for Clostridium difficile

Student thesis: Doctoral Thesis

Abstract

Oral vaccines offer significant social and economic advantages over vaccines delivered by parenteral routes including higher patient compliance and ease of administration, allowing vaccines to be given by health workers without medical training. Furthermore, oral immunisation can generate both systemic and localised immune responses, making it ideal for generating effective protection against many enteric pathogens such as the gram-positive spore forming bacterium Clostridium difficile (C. difficile). However, design of an effective vaccine for oral administration remains a significant challenge. In order to reach the relevant sites for the induction of immunological responses, the vaccine must first traverse the hostile environment of the gastrointestinal tract (GIT).For subunit antigens, the acidic pH, degradative enzymes and bile salts present in the GIT can adversely impact their structural stability. This can limit the effectiveness of the immune response. In addition, the mucosal surface within the gut can impair the transport of the vaccine antigen and limit the accessibility to the underlying antigen presenting cells. In order to circumvent these barriers, delivery systems can be employed. Lipid-based delivery systems are commonly used to deliver therapeutics via the oral route of administration. Of the lipid based systems, liposomes have been widely investigated but tend to be used for parenteral delivery.Therefore, the aim of the work in this thesis was to explore the use of liposomes for the oral delivery of a C.difficile vaccine. Liposomes generally exhibit low toxicity, enhance interactions with biological membranes and can be incredibly versatile in regards to what compounds they can incorporate (whether they are encapsulated within the aqueous core, within the lipid bilayer or surface associated). However, the manufacture of liposomes has been limited to batch scale production which has inherent risk associated. By employing microfluidics as a manufacturing platform, in conjunction with tangential flow filtration for purification, scalable production can be achieved.Therefore, this method was employed for the formulation and manufacture of the liposomal vaccine systems. Key production parameters were identified and formulations were selected based upon their physicochemical characteristics and protein loading efficiency. Selected formulations were screened for immunological effectiveness on THP-1 macrophage-like cells regarding antigen processing ability and activation marker expression. Finally, a range of formulations were then administered orally to mice to determine in vivo efficacy.Enhanced antibody responses (IgG) could be observed when antigen was administered encapsulated within liposomal formulation DSPC:Chol (alongside the potent adjuvant cholera toxin) when compared to free antigen alone; however, poor localised IgA responses were observed for all liposomal formulations tested. The work within this thesis presents a platform for the rapid and scalable manufacture, purification and at-line analysis of liposomal formulations incorporating protein, de-risking the translation of liposomal based protein products from bench to large scale production.
Date of Award11 Nov 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsEPSRC (Engineering and Physical Sciences Research Council)
SupervisorYvonne Perrie (Supervisor) & Paul Hoskisson (Supervisor)

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