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
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Chapter 11
Summary and perspectives
Summary
Microneedle-based transcutaneous immunisation is an appealing alternative to the classical manner of injecting vaccines by intramuscular or subcutaneous route.
Importantly, as a consequence of the fact that the skin is in direct contact with the environment and should protect the body against pathogens, it contains more antigen presenting cells, such as dendritic cells (DCs) than the muscles or subcutaneous tissue and thereby offers the possibility to induce a more effective immune response. However, the perspective of the vaccinee, who generally prefer painless and safer vaccinations [1], is perhaps even more important. The combination of microneedles and adjuvanted subunit vaccines may offer effective vaccination whereas ensuring patient safety and vaccine application in a painless manner.
The principal aim of this thesis was to design subunit vaccine formulations that can be combined with microneedles for transcutaneous immunisation. The research focuses on both vaccine efficacy and safety as it starts with evaluating the safety and effectiveness of solid microneedles, followed by the development of adjuvanted formulations that can be delivered via the conduits made by the microneedles.
This thesis starts with a general introduction to the field of transcutaneous immunisation (chapter 2), giving an overview of the broad assortment of devices developed to deliver a vaccine transcutaneously. After successful transport into the skin, the vaccine should be taken up by the skin DCs. Pathogens are often particles. Formulating antigens into nanoparticles resembles the pathogens in terms of size, thereby promoting DC uptake.
Two types of nanoparticles are used in the studies described in this thesis: N-trimethyl chitosan (TMC) nanoparticles and cationic liposomes. Both have proven to be excellent vaccine delivery systems [2, 3], but their potential for vaccination via the skin remains to be elucidated.
Part I: Safety and efficacy of microneedle pre-treatment on human volunteers
In chapter 3 studies focusing on the safety of microneedle piercing in human volunteers, an often overlooked parameter, are described in terms of skin irritation (skin redness and blood flow) and pain sensation. Solid microneedles of different design and length (200-550 µm) were compared and, besides irritation, their ability to disrupt the stratum corneum was evaluated by the transepidermal water loss (TEWL). Longer microneedle length (400 µm) resulted in increased TEWL, redness and blood flow compared to 200 μm long microneedles. Needle design also had an effect. Of the two differently shaped microneedles, the ones with the sharpest tip induced higher TEWL values, while resulting
in less skin irritation. Most importantly, all microneedles resulted in minimal irritation which lasted less than 2 hours.
In a subsequent clinical study (chapter 4) for the first time the influence of microneedle geometry on the transport of fluorescein through the formed conduits was visualised with confocal laser scanning microscopy. Based on the fluorescence intensity a distinction was made between regions with high and low intensity fluorescence (HIF and LIF, respectively).
In most cases HIF areas were only present in the stratum corneum, while LIF areas were also present in the viable epidermis. After 15 minutes almost no HIF was observed anymore at the skin surface, whereas still LIF could be detected until a depth of 60 µm. All microneedle arrays were able to form conduits in the skin, but the geometry of the microneedles affected the shape and depth of the conduits. Microneedles with a sharp needle tip were able to penetrate the skin to a higher extent than microneedles with a blunter tip.
Part II: TMC-based formulations for intradermal and transcutaneous vaccination To prepare immunogenic subunit antigen formulations, the model antigen ovalbumin (OVA) and a relevant antigen, diphtheria toxoid (DT), were formulated into nanoparticles composed of TMC. As a comparison physical mixtures of TMC and the antigens were also prepared. The studies described in Chapter 5 revealed that nanoparticles with a size around 200 nm and a positive zetapotential could be prepared. A burst antigen release of 30% from these nanoparticles was observed in vitro, followed by no further antigen release over a time span of 8 days. In an in vitro human DC model we showed that TMC nanoparticles increased the uptake of OVA and that both nanoparticles and TMC/OVA mixtures were able to induce upregulation of maturation markers (MHC-II, CD83 and CD86) on these DCs. Co-cultures with T cells revealed production of cytokines of a Th2 biased profile. In vivo the humoral immune response was evaluated by measuring the total serum IgG antibodies and antibody subclasses after intradermal vaccination in mice. For both the OVA and DT vaccination studies, the TMC nanoparticles as well as the TMC/antigen mixture were able to increase the IgG titres compared to non-adjuvanted antigen and induced a Th2 biased immune response. Using DT-containing TMC formulations, IgG titres and toxin-neutralising antibody titres could match up to those obtained after subcutaneous injection of DT-alum.
The same formulations were used for transcutaneous immunisation using 300 µm long microneedles in the studies reported in chapter 6. Two different microneedle arrays were used and the formulations were applied before or after microneedle treatment.
Independent of the microneedle array used and the sequence of microneedle treatment and vaccine application, transcutaneous immunisation with the physical mixture of TMC and DT elicited 8-fold higher IgG titres compared to DT-loaded TMC nanoparticles or a DT
solution. Additional ex vivo confocal microscopy studies revealed that transport of the TMC nanoparticles across the microneedle conduits was limited compared to a TMC solution.
To optimise the transport of vaccine formulation across the conduits formed by microneedle pre-treatment the application time of the formulations was prolonged in chapter 7. An extension from 1 to 2 hours of transcutaneous application of an OVA solution resulted in a 30-fold increase of IgG titres. Besides the application time also the effect of antigen-adjuvant entity size and co-localisation was found to be of crucial importance. Superior IgG levels were induced by a TMC-OVA conjugate (28 nm) after the prime vaccination and this coincided with higher numbers of OVA positive DCs found in the lymph nodes. After the boost both the conjugate and the nanoparticles elevated the IgG titres compared to an OVA solution. The same formulations were also applied via intradermal and intranodal injection to study the aspect of delivery through the skin and to the lymph nodes. Intradermally TMC, irrespective of its physical form, was essential for increased antibody titres. These formulations formed a depot in the skin and prolonged OVA delivery to the lymph nodes. The prolonged delivery to lymph node resident DCs was also observed intranodally, but it did not correspond with elevated antibody titres. These findings emphasise that each delivery route has different requisites for the ideal vaccine formulation.
Chapter 8 describes a second generation of TMC nanoparticles containing 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. The effectiveness of these adjuvant loaded TMC particles was assessed after intradermal and nasal vaccination. After nasal vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG, IgG1 and sIgA levels than non adjuvanted TMC/OVA particles, whereas nanoparticles containing CTB, PAM or CpG did not. All nasally applied formulations induced only marginal IgG2a titres. After intradermal vaccination, the TMC/CpG/OVA and TMC/LPS/OVA nanoparticles provoked higher IgG titres than plain TMC/OVA particles. Additionally, the TMC/CpG/OVA nanoparticles were able to induce significant IgG2a levels. None of the intradermally applied vaccines induced measurable sIgA levels. These results confirm the conclusions of the previous chapter, i.e. the vaccine formulation, including the adjuvant, should be tailored to the needs of the route of administration.
Part III: Cationic liposomes to co-deliver antigen and adjuvant
In this part cationic liposomes, which in contrast to TMC nanoparticles do not possess intrinsic immunogenicity, were used to co-encapsulate an antigen (OVA) and an adjuvant (PAM or CpG).
In the studies described in chapter 9 we showed that, after liposomal encapsulation, both adjuvants retained the ability to activate TLR-transfected HEK cells, though PAM in liposomes only induced activation at elevated concentrations compared to a PAM solution.
In vitro DC maturation induced by the adjuvanted liposomes was superior compared to the free adjuvants. For intradermal immunisation, encapsulation of PAM and CpG in liposomes did not influence the total IgG titres compared to the antigen/adjuvant solution, but OVA/CpG liposomes shifted the IgG1/IgG2a balance more to the direction of IgG2a compared to non-encapsulated CpG. Moreover, only this formulation resulted in a cellular immune response as measured by IFN-γ production by restimulated splenocytes from immunised mice.
To obtain insight into the benefit of liposomes for various vaccination routes, we immunised mice via the intranodal, intradermal, transcutaneous (with microneedle pre- treatment) and nasal route with OVA/CpG liposomes and a mixture of OVA and CpG (chapter 10). OVA/CpG liposomes increased the IgG and IgG1 titres compared to OVA after intradermal and nasal administration. This effect could be attributed to the presence of CpG, as co-administration of OVA and CpG in solution induced similar (intradermal) or even higher (nasal) titres than OVA/CpG liposomes. After transcutaneous administration of an OVA and CpG solution, also elevated IgG titres were observed. Intranodally all formulations were equally potent. Although the serum IgG and IgG1 titres might suggest no added value of liposomes, for all routes, co-encapsulation resulted in the production of relatively more IgG2a than IgG1. Whereas after intradermal and intranodal vaccination with OVA/CpG liposomes the number of OVA+ and CpG+ DCs in the LNs increased, lower numbers were detected after transcutaneous and nasal vaccination compared to administration of a solution of OVA and CpG.
Conclusion
The approaches described in this thesis have generated new insights into the main requirements for transcutaneous immunisation. Microneedles definitively have the potential to be an excellent utensil for the delivery of vaccines into the skin. However, the skin is a very elastic organ and the actual conduits formed by microneedle pre-treatment will be considerably smaller than the diameter of the microneedles (chapter 4). These conduits can remain open for up to 2 hours (chapter 3) under non-occlusive conditions, but this time can be prolonged to 72 hours under occlusive conditions [4]. The conduit dimensions and lifetime should be considered when developing formulations to apply on microneedle pre-treated skin. As reported in chapter 7, extending the application time from 1 to 2 hours can increase the antibody titres by 30-fold, thereby revealing the beneficial effect of TMC nanoparticles for transcutaneous immunisation. Application times can be further prolonged if applied to humans. Mice need to be anaesthetised to prevent
them from grooming, which restricts the application time. However, a smaller antigen- adjuvant entity is preferable, as it will be transported more efficiently through the microneedle conduits. In this respect the TMC-OVA conjugate proved to be the best choice: a formulation which retains the co-delivery of antigen and adjuvant, while being as small as possible. This issue together with other perspectives are further discussed in the following section.
Perspectives
Ideal formulation for microneedle-based transcutaneous immunisation
The studies described in this thesis clearly define size of the adjuvant-antigen entity as the most important parameter to consider when designing a formulation to be applied transcutaneously after microneedle pre-treatment. This implies that the TMC-OVA conjugate (28 nm) offers better perspectives than the TMC/OVA nanoparticles and the OVA/CpG liposomes (200-300 nm). However, the preferred vaccine has additional requirements besides the size of adjuvant-antigen entity. The desired type of immune response and the adjuvant choice are also essential considerations. The CpG-containing TMC nanoparticles and liposomes were the only formulations that could reverse the bias of the induced immune response from Th2 to a more balanced Th1/Th2 response by inducing the production of IgG2a antibodies. After intradermal vaccination with the OVA/CpG liposomes a strong production of IFN-γ from restimulated splenocytes could be measured, indicating induction of a cellular Th1 response (chapter 9). Not all vaccines require the same immune response, but most of the currently approved adjuvants, such as alum, induce a Th2, rather than a Th1 response [5]. The induction of a strong cellular and Th1 response is indispensable for an appropriate response against viruses and intracellular pathogens. Consequently, the quality of the immune response, which can be steered by the vaccine formulation, depends on the disease against which the vaccine is developed. A formulation containing TMC-antigen and TMC-CpG conjugates may induce a more Th1 biased response. Another option is to conjugate the antigen to monophosphoryl lipid A (MPL), which was shown to promote Th1 responses [6]. Since intradermal immunisation with LPS-containing TMC nanoparticles induced superior antibody titres compared to non- adjuvanted TMC nanoparticles (chapter 8), conjugation of antigens to MPL (a derivative of LPS) as recently described by Tang et al. [7] might result in a potent Th1 skewing formulation for transcutaneous vaccination. These are just some options, and both the conjugation of antigens to adjuvants and the effectiveness of the formed conjugates remains to be studied. Co-localisation of antigen and adjuvant remains an important tool to enhance the immunogenicity of a subunit vaccine, but should not be established by
using a particulate delivery system. Antigen-adjuvant conjugates, due to their smaller size, may be more suitable for transcutaneous vaccination.
Besides the entity size and the adjuvant choice, also TMC itself could be further optimised.
TMC can be synthesised from chitosan using the classical synthesis as described by Sieval et al. [8], but more recently Verheul et al. developed an elegant and controlled method to synthesise TMC without O-methylation and with a tailorable degree of quarternisation [9].
This type of chitosan can be synthesised with more precise and reproducible characteristics, which may be an asset of approval for use in humans. A second important characteristic of TMC is that, irrespective of the synthesis method, it carries a positive charge. This might limit its transport across the microneedle conduits and was shown to induce depot formation after intradermal injection (chapter 7). The formulation could therefore benefit from for instance PEGylation to shield the positive charge. PEGylation of positively charged nanoparticles was shown to improve antigen expression after DNA vaccination via tattooing compared to unPEGylated nanoparticles [10]. It would be interesting to study the effect of PEGylation of the TMC nanoparticles on the transport through microneedle conduits and on the immune response generated. An important notion is that the positive charge of TMC is at least partly responsible for the interaction with DCs. The influence of PEGylation on the DC-stimulating properties of the TMC should therefore be investigated.
Transcutaneous vaccination: what is the target?
The type of immune response that is induced not only depends on the adjuvants used, but also relates to the type of DC that is targeted. An important consideration is whether transcutaneous immunisation should target the epidermis, the dermis, or both. Until now the main focus has been on the breaching of the stratum corneum barrier. Many effective devices have been developed to achieve this goal as described in chapter 2. The time has come to set our focus somewhat deeper in the skin. This, however, implicates that we have to make a distinction between mice and man. Murine skin is much thinner than human skin: the average epidermis thickness in mice is 10 µm, compared to 150 µm in humans [11, 12]. This makes applying a vaccine solely to the epidermis in mice an unfeasible challenge. Nevertheless, it has become clear that the epidermis and the dermis contain different types of DCs, with distinct, but not completely understood, immunological functions [13]. The separation between Langerhans cells in the epidermis and dermal DCs in the dermis is oversimplified, as there are now at least two types of dermal DCs described, both in human and murine skin. Next to the ‘classical’ dermal CD14+ DC, the human dermis contains a second type of DC that is CD1a+ and does not express DC- SIGN [14]. In mice, a new subset of dermal DCs has been found, the langerin+ CD103+ dermal DC [15]. Whether this subtype is the equivalent of the human CD1a+ dermal DC
remains to be investigated. It has been postulated that the CD14+ DCs are more linked to a humoral immune response, whereas the Langerhans cells preferably induce cellular responses [16]. However, further research is necessary to elucidate the role of the different DC subtypes. To study the effect of reaching only one of these subtypes in a mouse study, knock-out mouse models have to be developed or targeting ligands like langerin or DC-SIGN should be included in the vaccine. At the same time the differences and similarities of the skin immune system of mice and man needs to be thoroughly compared.
Are needles needless for vaccination?
Non-invasive vaccination is often mentioned as the ideal method of applying a vaccine.
Whereas this is possible by mucosal vaccination, applying a formulation onto the skin without disrupting the barrier will most probably not induce a potent immune response.
Although cholera toxin and heat-labile enterotoxin are exceptions that can induce an immune response if topically applied [17], even for these very potent antigens barrier disruption is preferred [18]. For instance, in the patch against travellers’ diarrhoea currently in phase III, heat-labile toxin is applied with a Skin Prep System to mildly abrade the skin [19, 20]. This system functions by pulling an abrasive strip over the skin in a force controlled manner, enabling a 10-fold reduction of the dose compared to intact skin.
Microneedles can exert the same function as this skin abrasion system, i.e. disruption of the skin barrier. Many different types of microneedles have been developed (solid, coated, dissolvable and hollow), but it is not yet clear what the perfect type is and this might depend on the selected antigen and adjuvant. Using solid microneedles for pre-treatment is a straightforward manner, but as only a small fraction of the applied amount of antigen will enter the skin, and even less will reach the lymph nodes (chapter 7 & 10), the dose applied will be relatively high compared to other vaccination strategies. Prolongation of the application time may make it possible to define the dose more precisely. By using hollow microneedles the exact dose can be injected into the skin, but trained physicians are needed to inject the vaccine. In particular, breakage and leakage are risks that should not be underestimated. Another important parameter is the microneedle material. Hollow and solid microneedles are often made of silicon or metal (stainless steel). Although silicon allows the fabrication of microneedles of many different shapes, it is an expensive process and requires clean room processing [21]. More importantly, it is not an FDA approved material, making the regulatory path considerably longer compared to microneedles made of stainless steel, which does have FDA approval. However, both materials are not biodegradable, leading to concerns about what happens if the needles break off in the skin. Alternatively, polymeric microneedles that have retained microneedle strength while
Coated or dissolvable microneedles are the most elegant devices to apply a vaccine into the skin, but developments are still in its infancy. By coating a vaccine onto the microneedles no additional patch or infusion solution is necessary, but only a small amount can be delivered into the skin and often stabilisers (e.g. trehalose) have to be added to preserve the immunogenicity of the vaccine. Dissolvable microneedles allow the possibility of controlled release of the antigen depending on the encapsulation technique used [22].
Recently it was shown that mice could be protected against a lethal influenza challenge by a single immunisation with polymer microneedles containing 6 µg of inactivated influenza vaccine [23]. Although this is only the first study to use dissolvable microneedles for vaccination purposes it shows that this type of microneedles offer great potential for the future of microneedle-based immunisation.
Besides the different manners of applying microneedles, factors that need to be taken into account are microneedle length, density and shape. These parameters vary greatly between the currently available microneedle arrays and studies comparing different arrays are sparse.
Perceived safety of vaccination – a role for microneedles?
In recent years vaccination has been a hot topic, both in the research community and in the general population. New vaccines developed against for instance the human papillomavirus (HPV) and the swine flu have raised questions in the society about the safety of vaccination in general and of these vaccines in particular. Entering the term “HPV vaccine” in a search engine results in a disturbing amount of sites advising people against getting the vaccination. Although this cannot directly be correlated to the perception of the general population, it is an indication that the current vaccination strategy is not ideal.
Besides people who refuse vaccination because of fear of needles or religious believes, the number of people doubting the vaccine safety has increased rapidly and the world wide usage of internet has boosted the influence of these ‘anti-vaccine’ groups [24]. Even though serious side effects are rare, the occurrence is usually broadly reported and this aids to the negative view on vaccination. It is therefore of crucial importance that health care professionals cooperate with the media to ensure people of the benefit of vaccinations, even if many diseases against which we are vaccinated are almost eradicated [25]. One example to properly address the fear of people, is the “Six common misconceptions about immunization” [26] published by the United States Center for Disease Control and Prevention together with the World Health Organization, discussing the most common objections to vaccination. This expression of distrust in vaccines shows that scientists’ view on vaccination probably differs significantly from that of at least part of the public. Scientists regard vaccines as valuable agents against many life-threatening diseases, which has boosted research on for instance effective adjuvants and the
investigation of therapeutic vaccines against cancer and HIV. However, it remains to be seen if these new vaccines will be accepted by the public, as concerns on the safety of even the most commonly used aluminium salt adjuvants is rising [27]. The development of safe and potent vaccines and delivery methods is essential in this respect.
Microneedles can function as an attractive alternative to the classical immunisation method as it allows for vaccination in a minimally invasive, pain-free manner. In this light, the study by Birchall et al. is of particular interest as it evaluates the opinion of the public and healthcare professionals on microneedles [28]. Both groups had a positive view on microneedle technology and believed that it would be a pain-free alternative for paediatric vaccinations, people with needlephobia and in the treatment of chronic diseases. The problems raised were of a practical kind. For instance, even though in the opinion of healthcare professionals self-administration is an important benefit, 75% of the people in the public focus groups rather had them applied by healthcare professionals. This underlines the fact that people tend to be suspicious of new technologies as most people preferred the hollow microneedles, which resemble most the current manner of vaccination. The most important concern raised was the uncertainty of delivering the appropriate dose. Both the public and professional groups mentioned the necessity of a feed-back mechanism, such as a colour indicator to ensure proper usage. This is valuable information for academic groups and industry working on the development of microneedle-based vaccines and should be taken into account in the development process.
This type of studies show that including the potential consumers in a relatively early stage may help to achieve public acceptance of the future product and to take away unnecessary objections against vaccinations in general, and microneedle-based vaccinations in particular. The development of microneedle-based vaccines may, provided that the technical issues will be solved, contribute to an improved perception about vaccination by the public and consequently, better vaccine coverage in the future.
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