Mechanistic studies on transcutaneous vaccine delivery : microneedles, nanoparticles and adjuvants
Bal, S.M.
Citation
Bal, S. M. (2011, February 15). Mechanistic studies on transcutaneous vaccine delivery : microneedles, nanoparticles and adjuvants. Retrieved from
https://hdl.handle.net/1887/16485
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/16485
Chapter 1
Introduction, aim and outline of this
thesis
Chapter 1
3
Introduction
In recent years the search for alternatives for the classical manner of vaccination by injection into the muscle or subcutaneous tissue has increased tremendously. Improved safety, patient compliance and better efficacy are the most important reasons to develop novel vaccine delivery techniques [1, 2]. One of the alternative vaccination sites is the skin.
Previously regarded as an unconquerable barrier, the skin was first described as an attractive immunisation site by Glenn et al., who showed that it was possible to induce an immune response by topical application of cholera toxin (CT) or heat-labile enterotoxin (LT) on intact mice [3] and human skin [4]. Their research has led to the development of a vaccine patch against traveller’s diarrhoea currently being tested in a phase III study [5]
and one against influenza in a phase II study. These studies have boosted the research on novel techniques to apply a vaccine via the skin. An elegant example is the application of microneedle arrays, needles that pierce the stratum corneum while being short enough to avoid pain sensation. The first microneedle arrays introduced comprised solid microneedles that can be used to pre-treat the skin [6]. More recently a variety of types, including hollow, solid, coated and dissolvable microneedles, have been developed [7].
The research on efficient vaccination focuses not only on the delivery method and administration route, but also on the composition of vaccines. Safety issues with the traditional life-attenuated and inactivated vaccines have advanced the development of subunit vaccines, which are based on a purified protein, peptide or gene fragment of the pathogen and are less reactogenic than traditional vaccines. The main drawback of such subunits is their poor immunogenicity, which necessitates the addition of an adjuvant in order to yield a good immune response. An adjuvant is an additive that enhances the immunogenicity of an antigen. The adjuvant field is rapidly evolving. For a long time colloidal aluminium salts (alum) were the only approved adjuvants, but more recently squalene emulsions (MF59) and monophosphoryl lipid A have been licensed for usage in Europe. The more thorough understanding of the innate immune system has stimulated the research on developing new adjuvants. Another promising approach to increase the immunogenicity of subunit antigens is their formulation into (nano)particles [8, 9].
Particulates facilitate the uptake by the professional antigen presenting cells, such as dendritic cells, due to their similarity in size to pathogens. Furthermore, they can protect the antigen from enzymatic breakdown, allow sustained antigen release over time and offer the possibility of co-encapsulation of adjuvants. Knowledge on the effects of antigen formulation for transcutaneous vaccination, however, is sparse.
Chapter 1
Aim
The principal aim of the studies described in this thesis is to design subunit vaccine formulations that can be combined with microneedles for transcutaneous immunisation. In order to achieve this, understanding of the requirements of both the microneedles and the formulations needs to be acquired. Therefore several sub-aims needed to be formulated.
• The safety and efficacy of different microneedle arrays.
• The immunogenicity of the different vaccine formulations when used for vaccination via the skin. For this purpose the formulations are injected intradermally, to avoid the complicating factor of transport into the skin.
• The effectiveness of the formulations when applied transcutaneously with microneedle arrays.
Thesis outline
In chapter 2 the literature regarding transcutaneous immunisation is reviewed, with a strong focus on the immunological characteristics that makes the skin an excellent vaccination site and a critical view on the many different devices developed to deliver vaccines across the stratum corneum barrier. Adjuvants and particulate delivery systems currently used in (pre)clinical transcutaneous immunisation studies are also discussed.
The research described in the thesis is divided into three parts: the microneedle arrays used for transcutaneous vaccination (part I); the development and efficacy of several generations of N-trimethyl chitosan (TMC) based formulations (part II); and the usage of adjuvanted liposomal formulations for vaccination purposes (part III).
Part I: Safety and efficacy of microneedle pre-treatment on human volunteers
The ability of solid microneedle arrays differing in shape and length (200-550 µm) to disrupt the skin barrier is evaluated in chapter 3. The microneedles are applied with an electrical applicator onto the skin of human volunteers and the following parameters are studied: pain sensation, skin redness and blood flow as a measure of skin irritation and transepidermal water loss to indicate barrier disruption. These measurements are repeated in time to assess the closure time of the conduits.
This is followed by the visualisation of the microneedle conduits by confocal laser scanning microscopy in chapter 4. Two different solid microneedle arrays and the commercially available Dermastamp®, all three containing 300 µm long microneedles are applied onto the skin of human volunteers before or after the application of a fluorescent dye,
5
Part II: TMC-based formulations for intradermal and transcutaneous vaccination
This part focuses on preparing and testing different formulations based on the positively charged polymer N-trimethyl chitosan (TMC). This polymer has been successfully used preclinically as an adjuvant for mucosal vaccine delivery. In chapter 5 nanoparticles are prepared by ionic cross-linking of TMC with tripolyphosphate using either ovalbumin (OVA) or diphtheria toxoid (DT) as an antigen. These nanoparticles are physicochemically characterised and tested for their ability to enhance antigen uptake by dendritic cells (DCs) in vitro, DC maturation and T cell activation. The immunogenicity of the formulations is tested in Balb/c mice after intradermal injection. Antibody titres are measured to study the humoral immune response.In chapter 6 mice are immunised with DT-loaded TMC nanoparticles, a mixture of TMC and DT and non-adjuvanted DT by applying the formulations on microneedle pre-treated skin.
The antibody titres induced by vaccination via transcutaneous immunisation are compared to those after administration via the intradermal route. To obtain information on the efficiency of transport through the conduits, the localisation of TMC, as a solution and in nanoparticulate form, is visualised ex vivo.
Several parameters that can affect transcutaneous immunisation are optimised in chapter 7 by prolonging the application time of the formulations and using a smaller antigen- adjuvant entity, a TMC-OVA conjugate. To study the combined effect of diffusion through the conduits into the skin, transport to the draining lymph nodes and antigen uptake by DCs, the formulations are also applied by intradermal or intranodal injection. Besides the antibody titres, the amount of OVA+ DCs in the draining lymph nodes is quantified.
A second generation of OVA-loaded TMC nanoparticles is developed in chapter 8. A selection of adjuvants including Toll-like receptor ligands lipopolysaccharide (LPS), PAM3CSK4 (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and the GM1 ganglioside receptor ligand, cholera toxin B subunit are co-incorporated with the antigen into TMC nanoparticles. The immunogenicity of the formulations is assessed by determining the antibody response after nasal and intradermal vaccination.
Part III: Cationic liposomes to co-deliver antigen and adjuvant
The use of cationic liposomes, another type of nanoparticles, for intradermal vaccination is described in chapter 9. Two different Toll-like receptor ligands, PAM and CpG are encapsulated in OVA-containing liposomes. The ability of these ligands to interact with their receptors is studied in Toll like receptor (2 and 9) transfected HEK cells and their DC stimulating properties are investigated. Both humoral and cellular immune responses after intradermal immunisation are measured.
The formulation requirements for different administration routes are addressed in chapter 10. Liposomes containing OVA and CpG, as well as a mixture of soluble OVA and CpG, are
Chapter 1
administered via the transcutaneous, nasal, intradermal and intranodal route and the serum IgG and IgG subclass titres are measured. To further understand the working mechanism of the liposomes, the uptake of antigen and adjuvant by DCs, both in vitro and in vivo in the draining lymph nodes, is determined.
7
References
1. Giudice EL and Campbell JD, Needle-free vaccine delivery. Adv Drug Deliv Rev, 2006. 58(1): p. 68-89.
2. O'Hagan DT and Rappuoli R, Novel approaches to vaccine delivery. Pharm Res, 2004. 21(9): p. 1519- 30.
3. Glenn GM, Rao M, Matyas GR, and Alving CR, Skin immunization made possible by cholera toxin.
Nature, 1998. 391(6670): p. 851.
4. Glenn GM, Taylor DN, Li X, Frankel S, Montemarano A, and Alving CR, Transcutaneous immunization:
a human vaccine delivery strategy using a patch. Nat Med, 2000. 6(12): p. 1403-6.
5. Frech SA, DuPont HL, Bourgeois AL, McKenzie R, Belkind-Gerson J, Figueroa JF, Okhuysen PC, Guerrero NH, Martinez-Sandoval FG, Melendez-Romero JHM, Jiang ZD, Asturias EJ, Halpern J, Torres OR, Hoffman AS, Villar CP, Kassem RN, Flyer DC, Andersen BH, Kazempour K, Breisch SA, and Glenn GM, Use of a patch containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: A phase II, randomised, double-blind, placebo-controlled field trial. Lancet, 2008. 371(9629): p. 2019- 2025.
6. Henry S, McAllister DV, Allen MG, and Prausnitz MR, Microfabricated microneedles: A novel approach to transdermal drug delivery. J Pharm Sci, 1998. 87(8): p. 922-5.
7. Donnelly RF, Raj Singh TR, and Woolfson AD, Microneedle-based drug delivery systems:
Microfabrication, drug delivery, and safety. Drug Deliv, 2010. 17(4): p. 187-207.
8. Singh M, Chakrapani A, and O'Hagan D, Nanoparticles and microparticles as vaccine-delivery systems. Expert Rev Vaccines, 2007. 6(5): p. 797-808.
9. Rice-Ficht AC, Arenas-Gamboa AM, Kahl-McDonagh MM, and Ficht TA, Polymeric particles in vaccine delivery. Curr Opin Microbiol, 2010. 13(1): p. 106-12.
