Summary
Salmonella infections in humans are the cause of enteric fevers (Typhoid (TF) and Paratyphoid (PT)) and Non-Typhoidal Salmonellosis (NTS), which are major causes of morbidity and mortality in developing countries. There are no commercially available vaccines for PT and NTS and current vaccines for TF do not offer effective control of the disease. Thus a multi-valent vaccine that protects against all the different clinical manifestations remains an unmet public health need. Porins are outer membrane proteins from Salmonella enterica that generate immune responses in acute and convalescent TF and NTS patients. Purified porins have shown promise as a multivalent vaccine in preclinical testing and have also been shown to be safe and well tolerated in human volunteers. In addition, volunteers who received the porin-based vaccine carry circulating bactericidal antibodies up to 10 years after receiving one single immunisation. So far the porin-based vaccine formulation has been tested using conventional systemic delivery. In addition to being a non-invasive method for vaccine administration, mucosal delivery has the potential of eliciting protective immune responses at the pathogen site of entry. However, mucosal vaccination requires potent adjuvants and delivery systems to enhance immunogenicity, and to decrease the degradation rate. Particulate delivery systems such as micro/nano particles and liposomes have the ability to protect and carry subunit antigens to mucosal inductive sites. We aim to formulate particulate delivery systems for a porin multivalent vaccine for mucosal administration.
Project outcomes
Highly pure Salmonella porins were produced and formulated with liposomes and chitosan. Porins-Dimethyldioctadecylammonium (DDA) or trehalose 6,6-dibehenate (TDB). DDA:TDB liposomes were produced in either Tris buffer or two different concentrations of sucrose (2.5 and 20 %). After preparation, liposomes prepared in Tris buffer were 800-900 nm, whilst those prepared in sucrose were smaller in size (500 – 700 nm). All three formulations were highly cationic (zeta potential 50-60 mV), with no significant difference. The porins composed liposomes effectively retained porins within the liposomal adjuvants offering protection and enhanced delivery as shown by biodistribution studies. To enhance their shelf-life, a freeze-dried formulation was developed. After freeze-drying, high aggregation was noted with formulations prepared in Tris (with a 5-fold increase in size), whereas those prepared in 2.5% and 20% sucrose had no significant increase in size for up to 2 weeks at room temperature. These formulations produce strong antibody responses after intra-muscular administration.
Porins loaded chitosan micro- and nanoparticles were prepared with suitable particle size and distribution, surface charge, loading efficiency, in addition porins-chitosan gel was formulated. The particles and gel were shown to be stable, protecting porins integrity. Biocompatibility, cellular uptake and macrophage activation of these formulations were demonstrated in J774A.1 macrophage cell line. In BALB/c mice immunised with porins+chitosan nanoparticles or porins+microparticles showed an increased antibody titres when compared to porins alone. To analyse the protective response, BALB/c mice were immunised intraperitoneally on day 0 and boosted on day 14 with saline, or porins or porins-chitosan formulations, animals were challenged with Salmonella Typhi on day 30, and the reduction of bacterial numbers in liver and spleen was determined 24h after infection. We found that porins-chitosan formulation was more efficient than porins alone to reduce bacterial numbers in liver, whereas in the spleen, both formulations induced similar reduction of bacterial numbers.
Taken together, our results showed that porins could be efficiently formulated in liposomes and chitosan to form micro- and nano-particles, these formulations are immunogenic and suitable to induce protection against Salmonella challenge in mice. These formulations could be used to produce room temperature vaccines for non-invasive immunisations that improve its global distribution and facilitate its use.