Development of liposome-based freeze-dried rods for vaginal vaccine delivery against HIV-1

The present investigation deals with development and characteriza- tion of the liposomes-based freeze-dried rods for the vaginal delivery of gp140 antigen in mice. Positively charged, negatively charged and neutral liposomes were prepared and characterized for various parameters e.g. morphology, size, polydispersity index, zeta potential and antigen encapsulation efficiency. To further improve the efficacy of vaccine delivery, antigen encapsulated liposomes were formulated as polymer gel-based freeze-dried rods, which were then characterized for moisture content. The redispersibility of the liposomes-based freeze- dried rods was determined in simulated vaginal fluid and liposome gel was investigated for mucoadhesion. The developed liposome-based freeze-dried rods systems could offer potential as stable and practical dosage form for the mucosal immunization against HIV-1 infection.

Dressing liposomal particles with chitosan and poly(vinylic alcohol) for oral vaccine delivery

Liposomes have been used as adjuvants since 1974. One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our aim was to combine liposomes that could encapsulate antigens (i.e., Dtxd, diphtheria toxoid) with chitosan, which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3: 1) can be sandwiched with chitosan (Chi) and stabilized by PVA (poly-vinylic alcohol), which are biodegradable, biocompatible polymers. Round, smooth-surfaced particles of REVs-Chi (reversed-phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on the REVs surface was accompanied by an increase of zeta-potential. In contrast, PVA adsorption on the REVs-Chi surface was accompanied by a decrease of zeta-potential. The presence of Dtxd increased the Chi surface-adsorption efficiency. The PVA affinity by mucine was 2,000 times higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new, stabilized particles were able to be adsorbed by intestinal surfaces, resisted degradation, and controlled antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant.

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

Needleless Vaccine Delivery Using Micro-Shock Waves

Shock waves are one of the most efficient mechanisms of energy dissipation observed in nature. In this study, utilizing the instantaneous mechanical impulse generated behind a micro-shock wave during a controlled explosion, a novel nonintrusive needleless vaccine delivery system has been developed. It is well-known that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle-free devices for epidermal delivery have inherent problems from the perspective of the safety and comfort of the patient. The penetration depth of less than 100 mu m in the skin can elicit higher immune response without any pain. Here we show the efficient utilization of our needleless device (that uses micro-shock waves) for vaccination. The production of liquid jet was confirmed by high-speed microscopy, and the penetration in acrylamide gel and mouse skin was observed by confocal microscopy. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model, and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. Vaccination using our device elicits better protection and an IgG response even at a lower vaccine dose (10-fold less) compared to other routes of vaccination. We anticipate that our novel method can be utilized for effective, cheap, and safe vaccination in the near future.

Fabrication of Poly (D,L-Lactic-Co-Glycolic Acid) Microparticles for Improved Human Papillomavirus Vaccine Delivery

Human papillomavirus (HPV) is the leading cause of cervical cancer and the most prevalent sexually transmitted disease worldwide. HPV vaccines require a multi-dose regimen to provide immunity, contributing to low patient compliance. We addressed this problem by formulating biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles and assessing their viability for use in controlled-release vaccines. We hypothesized that we could alter fabrication parameters to produce 1-10 μm microparticles in order to encapsulate ovalbumin (OVA) and HPV virus-like particles (VLPs). Microparticles were fabricated using a double emulsion method and used to elicit an immune response in JAWSII cells. Our results contribute to knowledge of vaccine delivery mechanisms and controlled-release technology, and could contribute to the creation of a viable controlled-release HPV vaccine.

Rheological characterization of thermoresponsive semisolids for vaginal HIV vaccine delivery

Purpose. To manufacture and characterize, through oscillatory rheology, thermoresponsive rheologically structured vehicles
(RSV’s) capable of enhanced retention times within the vagina for the purposes of HIV vaccine delivery.
Methods. Pluronics F127, F108 and F68 were investigated and RSV’s were prepared by dissolving sorbic acid (0.1% w/w)
and mucoadhesive component (Gantrez SBF97 or Noveon AA1, 3% w/w) in the required amount of H2O and NaOH.
Pluronic (10% w/w) was added via mixing in an ice-bath followed by hydroxyethylcellulose (5%) and subsequently
poly(vinylpyrollidone) (4%w/w). Oscillatory temperature sweeps between 10-38°C were preformed within the linear
viscoelastic region of the formulations on an AR2000 rheometer (T.A. Instruments, Surrey, England) with a 2cm diameter
parallel plate geometry and a plate gap of 1000µm at 1Hz.

Microneedle-mediated vaccine delivery:harnessing cutaneous immunobiology to improve efficacy

INTRODUCTION: Breaching the skin's stratum corneum barrier raises the possibility of the administration of vaccines, gene vectors, antibodies and even nanoparticles, all of which have at least their initial effect on populations of skin cells. AREAS COVERED: Intradermal vaccine delivery holds enormous potential for improved therapeutic outcomes for patients, particularly those in the developing world. Various vaccine-delivery strategies have been employed, which are discussed in this review. The importance of cutaneous immunobiology on the effect produced by microneedle-mediated intradermal vaccination is also discussed. EXPERT OPINION: Microneedle-mediated vaccines hold enormous potential for patient benefit. However, in order for microneedle vaccine strategies to fulfill their potential, the proportion of an immune response that is due to the local action of delivered vaccines on skin antigen-presenting cells, and what is due to a systemic effect from vaccines reaching the systemic circulation, must be determined. Moreover, industry will need to invest significantly in new equipment and instrumentation in order to mass-produce microneedle vaccines consistently. Finally, microneedles will need to demonstrate consistent dose delivery across patient groups and match this to reliable immune responses before they will replace tried-and-tested needle-and-syringe-based approaches.