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Expert Review of Vaccines
ISSN: 1476-0584 (Print) 1744-8395 (Online) Journal homepage: http://www.tandfonline.com/loi/ierv20
Needle-free epidermal powder immunization
Dexiang Chen, Yuh-Fun Maa & Joel R Haynes
To cite this article: Dexiang Chen, Yuh-Fun Maa & Joel R Haynes (2002) Needle-free epidermalpowder immunization, Expert Review of Vaccines, 1:3, 265-276
To link to this article: http://dx.doi.org/10.1586/14760584.1.3.265
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Technology Report
© Future Drugs Ltd. All rights reserved. ISSN 1476-0584 265
CONTENTS
Why skin is a good target for vaccination
Needle-free skin immunization technologies
Epidermal powder immunization
Particle-mediated DNA vaccination
Advantage of needle-free skin immunization
Conclusions & key issues
Five-year view & expert opinion
Information resources
References
Affiliations
www.future-drugs.com
Needle-free epidermal powder immunizationDexiang Chen†, Yuh-Fun Maa and Joel R Haynes
Due to the presence of a network of antigen-presenting cells and other cells with innate and adaptive immune functions, the skin is both a sensitive immune organ and a practical target site for vaccine administration. A handful of needle-free immunization technologies have emerged in recent years that aim to take advantage of these characteristics. Skin delivery technologies provide potentially safer alternatives to needle injection and promises increased efficacy in the prevention and/or therapy of infectious diseases, allergic disorders and cancer. In this review, we will cover advances in needle-free skin vaccination technologies and their potential applications to disease prevention and therapy. Emphasis will be placed on epidermal powder immunization and particle-mediated (‘gene gun’) DNA immunization, which use similar mechanical devices to deliver protein and DNA vaccines, respectively, into the viable epidermis.
Expert Rev. Vaccines 1(3), 265–276 (2002)
†Author for correspondencePowderJect Vaccines Inc., 585 Science Drive, Madison, WI 53711, USATel.: +1 608 231 3150Fax: +1 608 231 [email protected]
KEYWORDS: adjuvant, dendritic cells, DNA vaccine, epidermal powder immunization, gene gun, Langerhans cells, vaccine
Vaccination is the most cost-effective means ofpreventing infectious diseases and had an enor-mous impact on public health in the last cen-tury. With few exceptions, most current vac-cines are administered by injection using aneedle and syringe. Each year, an estimated 1billion injections are administered throughnational immunization programs and up to30% of the injections are thought to be unsafe[1]. Needle-stick injuries not only put the pub-lic and health workers at risk of infection withHepatitis B, C, HIV and other viruses but alsoincrease the economic burden on healthcaresystems [1]. Due to these alarming statistics,needle-free vaccination alternatives are beingsought in numerous academic and industrialinstitutions.
The search for alternatives to parenterally-inoculated vaccines is a priority of the GlobalAlliance for Vaccines and Immunization(GAVI). In addition to developing needle-freevaccine delivery technologies, GAVI has iden-tified the development of cold chain-inde-pendent vaccine formulations as a critical fac-tor for vaccination programs, particularly indeveloping countries. One of the most feasibleapproaches to achieving room-temperaturestability is the development of solid-state or
dry vaccine formulations. As a result of newformulations, the development of needle-freeskin vaccination technologies may requirenovel delivery strategies and devices.
Why skin is a good target for vaccinationHuman skin can be subdivided into three lay-ers: the stratum corneum (10–20 µm in-depth), the viable epidermis (50–100 µm) andthe dermis (1–3 mm) (FIGURE 1). The stratumcorneum is composed of multiple layers ofclosely packed, flattened, dead keratinocytes,known as squames [2,3]. The intercellularspaces between the squames are filled with athick layer of lipid cement forming a semiper-meable barrier that prevents microbes andlarge biomolecules from penetrating the skin.The underlying viable epidermis, the targettissue for needle-free immunization, is com-posed of cells, such as keratinocytes, melano-cytes and Langerhans cells (LCs). The epider-mis generally lacks blood vessels and sensorynerve endings, important characteristics in atarget site for needle-free delivery alternatives.The underlying dermis consists of an amplesupply of blood vessels, lymph vessels, nerveendings, hair follicles, dendritic cells (DCs),collagen fibers and sweat glands. Successful
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skin immunization requires that the vaccine formulation beeffectively delivered past the stratum corneum and into eitherthe epidermis or dermis. Based on the above-mentioned con-stituents, there is a clear advantage in delivery technologiesthat effectively target the epidermis.
Skin is not only a physical barrier that shields the body fromexternal hazards, but is also an integral part of the immune sys-tem. The immune function of the skin arises from a collectionof residential cellular and humoral constituents of the viableepidermis and dermis with both innate and acquired immunefunctions, collectively referred to as the skin immune system[4]. One of the most important components of the skinimmune system is the LCs, which are specialized antigen-pre-senting cells (APCs) found in the viable epidermis. The den-sity of LCs in most areas of the human skin, with the excep-tion of sole and palm, is approximately 500–1000 cells/mm2
(an estimated total of 109 LCs for an adult) [5–7]. LCs form asemicontinuous network in the viable epidermis due to theextensive branching of their dendrites between the surround-ing cells. The normal function of LCs is to detect, capture andpresent antigens to evoke an immune response to invadingpathogens [8]. LCs perform this function by internalizing epi-cutaneous antigens, trafficking to regional skin-draininglymph nodes and presenting processed antigens to T-cells (FIG-
URE 1). LCs are capable of presenting antigen to both naive andantigen-specific T-cells of CD4+ and CD8+ phenotypes toinduce or stimulate antibody and cellular immune responses.In addition to LCs, dermal DCs play a similar role to APCsthat internalize, process and present antigens entering the der-mis. Other cells, such as keratinocytes, are important sourcesof proinflammatory cytokines that may augment the immuneresponses to antigens introduced via the skin route [9].
The effectiveness of the skin immune system is responsible forthe success and safety of vaccination strategies that have beentargeted to the skin. Vaccination with a live attenuated smallpoxvaccine by skin scarification has successfully led to global eradi-cation of the deadly smallpox disease [10]. Intradermal injectionusing 1/5 to 1/10 of the standard intramuscular doses of variousvaccines has been effective in inducing immune responses to anumber of vaccines, while a low-dose rabies vaccine has beencommercially licensed for intradermal application [11,12].Although skin scarification and intradermal injection have beensuccessfully applied to specific vaccines, a wave of new needle-free immunization technologies are currently in developmentand may see broad application in the future.
Needle-free skin immunization technologiesA number of vaccine delivery technologies are under develop-ment to exploit the skin immune system. In broad terms, theycan be classified into noninvasive and invasive approaches andare reviewed in the sections below.
Noninvasive immunizationNoninvasive skin immunization involves application of vaccinesto the surface of intact skin in the form of a liquid solution,cream, or patch. Due to the safety and practicality of such anapproach, successfully developed noninvasive strategies shouldachieve widespread appeal. The success of noninvasive skin vac-cination immunization technologies is dependent on the identi-fication of mechanisms to deliver antigens across the semiper-meable stratum corneum barrier, into the viable epidermis.Large biomolecules, when applied to intact skin, normally fail topass through the stratum corneum and are unable to evoke animmune response. However, there are a few exceptions. Cholera
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Figure 1. A schematic representation of the skin structure and role of Langerhan’s cells (LCs) in immune responses to antigens introduced by the skin route. The human skin has three layers of structure, stratum corneum, viable epidermis and dermis. Only the dermis has blood vessels and nerve endings. LCs are derived from bone marrow progenitor cells and are localized in the viable epidermis. LCs take up antigens introduced via the skin route, migrate to the draining lymph nodes and present processed antigen to T-cells to induce an immune response.
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toxin (CT) and the heat-labile enterotoxin of E. coli (LT) areable to penetrate the stratum corneum of mice and humans insufficient quantities to elicit immune responses [13–15]. In thiscase, large antigen doses are required for evoking responses inhumans [15], suggesting the limited penetration of these andother coadministered macromolecules [16–18].
In addition to protein vaccines, noninvasive immunizationwith DNA and live vectored vaccines has induced immuneresponses in mice [16–18]. In these studies, removal of a portionof the stratum corneum with a depilatory agent appears to ben-efit the efficacy, but may not be mandatory for immunogenic-ity in small animals. Data on the effectiveness of this approachin larger animals are not available. The main challenge for thisand other noninvasive immunization technologies is the lowefficiency of antigen uptake. Limited success has been demon-strated with simple antigens, often at an extremely high doseand not with common vaccine formulations in use today thatmay contain antigens adsorbed onto an insoluble adjuvant,such as aluminum compounds.
Invasive immunizationA number of new technologies are under development thatemploy needle-free mechanisms to break or perforate thestratum corneum barrier to facilitate vaccine administrationto the skin, particularly the epidermis. Treatment of the skinby tape striping with an adhesive tape or brushing withsandpaper is well known to remove or break the stratum cor-neum barrier and expose the viable epidermis [19,20]. Addi-tional methods of perforating the skin include electropora-tion, ablation by laser or heat and microneedle injection.The latter method combines skin perforation and vaccinedelivery into a patch containing an array of microprojec-tions of 100–300 µm in length. Vaccines can be eithercoated on the surface of the microprojections (e.g., Mac-roflux® Skin Interface Technology, Alza Corp.) or containedin the hollow interior spaces of the projections (microfabri-cated arrays). Application of the patch to the skin results in theperforation of the stratum corneum and delivery of vaccineantigens to the viable epidermis [21,22].
While not limited to the epidermis, low-volume liquid jetinjectors have been used to deliver small volumes of DNAsolutions intradermally in mice, rabbits and monkeys [23–25].Extracellular delivery of DNA to the skin using this methoddoes not achieve gene expression levels as high as can beobtained by particle-mediated DNA delivery to the epider-mis, but achieves significant antibody and cellular immuneresponses nonetheless.
Invasive skin immunization strategies demonstrate theadvantages of technologies that can breach the stratum cor-neum barrier, facilitating a more efficient uptake of vaccineantigens by immune cells of the skin. Preclinical studies withprotein or DNA vaccines by microneedle and jet injectionhave demonstrated good efficacy relative to deeper tissueinjection.
Epidermal powder immunizationEpidermal powder immunization (EPI) delivers powder-formvaccines to the LC-rich epidermis using a needle-free powderdelivery device powered by a small volume of compressedhelium gas (5 ml or less). EPI has its roots in ‘gene gun’ tech-nology that was developed in the 1980s for plant genetic engi-neering purposes and later adapted for DNA immunization ofanimals and humans in the 1990s [26–28]. The term ‘EPI’ gen-erally refers to the epidermal delivery of powdered vaccineformulations composed of proteins, polysaccharides, inacti-vated pathogens and is reviewed in sections: EPI delivery sys-tem to Potential applications of EPI in disease control. Theparticle-mediated delivery of DNA vaccines to the epidermisusing what is commonly referred to as a ‘gene gun’ device isreviewed in the Particle-mediated DNA vaccination section.
EPI delivery systemThe delivery systems for EPI are closely related to devicesemployed for particle-mediated DNA vaccine delivery thathave been described under such names as gene gun, Accell®
(Agracetus Inc.), Helios (BioRad), PowderJect XR® andND devices (PowderJect Vaccines Inc.) [29,30]. While thesedelivery systems have different outward appearances, theirdelivery mechanisms are similar in that a powdered formula-tion is delivered into the LC-rich viable epidermis using themotive force of a burst of helium gas. While most of thesedevices employ an external helium gas source, the Powder-Ject ND device is configured as a disposable device with aself-contained, single-use helium gas canister [31,32]. The sin-gle-use, disposable device is ideal for clinical use since itobviates problems of patient-to-patient contamination andpossible degradation in device performance through routineor excessive use.
The single-use device is approximately 15 cm in length andcomposed of a gas cylinder (5 ml volume), rupture chamber, avaccine containing cassette, a nozzle and a silencer [31]. The gascylinder is filled with medical grade helium gas to a nominalpressure of 30–50 bar. The cassette (11 mm OD, 6 mm ID and4 mm height) is constructed of a ethylene vinyl acetate washerwith rupture membranes heat sealed to either side within whichthe powder sample is housed. The rupture membranes are thinfilms (10–20 µm) made of semitransparent polycarbonate.Upon actuation, the helium gas is released from the gas cylin-der and causes pressure build-up in the rupture chamber. Theescaping gas ruptures the membranes of the trilaminate cassetteand accelerates the vaccine powders through the stratum cor-neum and into the viable epidermis. The helium gas is reflectedoff the skin and exhausted through the silencer.
Powder formulationsFor effective delivery and optimal immunogenicity, powder for-mulations for EPI must possess certain particle characteristics(size, density, flowability, particle integrity, shape/morphologyetc.) and long-term physical and chemical stability. Two differ-ent formulations based on either precipitation of antigens onto
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2 µm gold particles or formulation of antigens into larger parti-cles using mono- and disaccharride excipients have been devel-oped and both exhibit good performance in preclinical andclinical studies.
Gold microparticles
Gold microparticles were initially used as carriers to achieveintracellular delivery of DNA vaccines and have also proven use-ful for protein vaccine delivery [33,34]. Due to the extremely highdensity of gold (18 g/ml), particles as small as 1–2 µm can reacha sufficient momentum to penetrate intact stratum corneum andlodge within the cytoplasm and nuclei of viable epidermal cells.Intracellular delivery of protein antigens precipitated onto goldparticles results in MHC class I antigen presentation and theinduction of cell-mediated immunity, which is not normallyaccomplished by traditional needle injection [34].
Sugar excipient formulations
The sugar excipient formulations are designed to formulateprotein (or polysaccharide) antigens for delivery by EPI. Sincethe majority of pharmaceutical excipients, including sugars,have a density of ≤1.5 g/ml, the low density of the sugar for-mulation relative to gold is compensated by larger particlesizes (20–70 µm) to achieve effective delivery. Delivery ofsugar formulations to the epidermis usually results in the dis-ruption of cells at the particle deposition site due to the largesize of the particles. Antigens that are released upon particledissolution are taken up by APCs and presented via the MHCclass II antigens to elicit mainly humoral immune responses[33]. Thus, sugar formulations are appropriate for vaccinesagainst those pathogens in which protection is mediatedlargely by antibody responses.
Several powder formulation methods, including spray-freeze-drying (SFD), supercritical methods, fluid-bed spray-coating and spray-drying, are suitable for the preparation ofpowdered formulations for EPI [35–37]. Of these, SFD wasfound to be most suitable because of its scalability (both smalland large scale). SFD is a two-step process in which a liquidvaccine formulation containing antigen and excipients isatomized through a high-frequency ultrasonic nozzle to gen-erate fine liquid droplets that are flash frozen in liquid nitro-gen. Lyophilization of the frozen particles produces a vaccinepowder with appropriate characteristics for EPI (size, densityand moisture content).
The composition of the sugar formulation prepared by SFDplays an important role in achieving the desirable particle char-acteristics and long-term physical and chemical stability of thepowdered vaccine. After evaluating a range of excipients, wefound that the combination of trehalose, mannitol and a poly-mer, such as dextran, polyvinylpyrrolidone, or polyethyleneglycol, produced powders with the most desirable properties.Importantly, these excipients are highly water soluble, allowingfor the use of excipient concentrations in the liquid stage tohigh enough to achieve the appropriate particle density in thepowdered stage.
A number of vaccine powder formulations, including thosecontaining influenza and hepatitis B antigens, have been pre-pared by SFD. FIGURE 2 illustrates the stability of both vaccineswhen stored at 40oC for up to 16 weeks. The potency of theinfluenza vaccine was measured using a single radial-immunod-iffusion assay (SRID) that demonstrated no potency loss for upto 4 months. Accelerated stability studies of this nature lead toan estimated room temperature stability (25oC) of over12 months, obviating the need for an intact cold chain.
Evidence for targeting antigens to LCs by EPIHistological data show that EPI delivers powdered vaccinesinto the LC-rich viable epidermis. Antigen targeting of LCs inthe target tissue following EPI is dependent on whether goldor sugar formulations are employed. EPI with gold formula-tions result in intracellular deposition of gold and antigen inepidermal cells. Since LCs comprise about 3% of epidermalcells, only a fraction of the particles are delivered directly tothese APCs. Typical EPI target sizes are approximately110 mm2, which comprises approximately 1 x 105 LCs [34].Fluorescence microscopy has revealed that the delivery of0.5 mg of antigen-coated gold results in up to 50% antigen-positive LCs at the vaccination site (FIGURE 3A). This is a signif-icant number of antigen-loaded LCs and is consistent withthe observed levels of immunogenicity.
EPI with sugar formulations results in a different antigendistribution pattern than that observed using gold formula-tions. The footprint of the sugar particle target site can bemarked by codelivery of a tissue-marking dye that binds tocells with minimal diffusion after particle dissolution [33]. Dis-crete dark spots that arise following particle delivery to the tar-get site are readily visible under a light microscope. Followingsugar particle dissolution, Texas Red-labeled ovalbumin (TR-OVA) clearly diffuses into the surrounding tissue and appearsto be taken up by LCs. Nearly all LCs in the target tissueappear to contain fluorescent antigen (FIGURE 3B). Antigen and
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Figure 2. Stability of powdered influenza vaccine. Trivalent influenza vaccine (Fluvirin®, PowderJect Pharmaceuticals Plc, Oxford, UK) was formulated with trehalose, mannitol and dextran and spray-freeze-dried. Powder samples were stored in sealed glass vials at 40°C/75%RH. Data are HA content per dose determined by a single radial-immunodiffusion assay.
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antigen-carrying LCs are present at the site of immunizationfor up to 5 days, but the concentrations are markedly reducedafter the first 48 h (Chen D, unpublished).
EPI inherently activates LCs in the target site, as evidenced byenlarged cell morphology and brighter staining with an I-Ad-spe-cific fluorescent antibody due to enhanced expression of class IIantigens [33]. LC activation is likely mediated by cytokines releasedby epidermal cells when triggered by microscopic tissue damage asa result of particle penetration. In vitro culture of epidermal sheetsprepared from immunization sites results in the migration of LCsinto the culture medium. In vivo, antigen-positive LCs can beobserved in the regional draining lymph nodes as early as 20 h fol-lowing immunization and can be detected for several days(FIGURE 3C–F) [34]. Recent adoptive transfer studies have shown thatthe transplantation of LCs collected from the vaccine target sites ofimmunized mice to naive mice results in the induction of antigen-specific antibody responses confirming the role of antigen-positivemigratory LCs following EPI (Chen D, Unpublished).
Breadth of the immune responses induced by EPISerum antibody responses
EPI elicits strong serum antibody responses in mice to a variety ofconventional vaccines, including inactivated influenza viruses,diphtheria toxoid, hepatitis B surface antigen (HBsAg) and HIV-1 gp120 [31,33,34,38]. Using the same vaccine, EPI elicits signifi-cantly stronger serum titers than subcutaneous, intramuscular, orintraperitoneal inoculations [31]. Dose-range studies indicate thatEPI with an influenza vaccine may require a smaller antigen dose
than needle injection to achieve maximalimmunity and protection (FIGURE 4) [31].Serum antibody titers elicited by EPI are asdurable as those elicited by needle injection[38]. More recently, we determined that EPIelicits strong serum antibody titers to influ-enza HA and HBsAg in monkeys demon-strating the effect of this technology inlarger animals (Chen D, unpublished). Theefficacy of EPI is being assessed in humansusing an influenza vaccine.
Mucosal antibodies
Since the majority of infectious agentsgain entry through mucosal epithelialsites, secretory mucosal antibodyresponses play a vital role in early protec-tion from infectious disease. The immunesystem of humans and other higher ani-mals is compartmentalized in such a waythat mucosal antibodies are generallyinduced only by the direct application ofvaccines to mucosal sites. Vaccination vianonmucosal routes normally does notelicit mucosal immunity. However, recentdata has shown that this dogma may nothold true for vaccines administered to the
skin. Following EPI of mice with an inactivated influenza vac-cine, antigen-specific SIgA antibodies were detected in mucosallavages of the small intestine, trachea and vaginal tract [39]. Thelocal origin of the SIgA antibodies was demonstrated by meas-uring antibodies released from cultured tracheal and smallintestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays.Local antibody responses of this nature are likely to be impor-tant for protection against infectious challenge. Interestingly,the induction of mucosal immunity via EPI appears to bedependent on the use of adjuvants. CT, LT, CpG-containingoligodeoxyribonucleoties and other adjuvants were found toenhance the mucosal antibody responses elicited to antigensdelivered by EPI [39].
Cytotoxic T-lymphocyte responses
Cytotoxic T-lymphocytes (CTLs) play a vital role in hostdefense against viral and intracellular bacterial infections. How-ever, nonreplicating vaccines administered by intramuscularinjection using a needle and syringe elicit predominantlyhumoral responses and little, if any, CTL activity. MHC class I-restricted antigen presentation is required for the induction ofCTLs and is facilitated by the presence of protein antigens inthe cytosol of APCs. EPI using gold particle formulations deliv-ers proteins to the cytosol of LCs and other epidermal cells andwould be expected to elicit antigen-specific CTL responses [34].Not surprisingly, gold formulation EPI studies using HBsAg,HIV-1 gp120 and an influenza virus nucleoprotein peptide
A C E
B D F
Figure 3. Epidermal powder immunization (EPI) targets antigens to Langerhan’s cells (LCs). LCs in the skin site of EPI using Texas red-labeled ovalbumin (TR-OVA) coated gold formulation (A) or sugar formulation (B). Immediately after EPI, epidermal sheet from the site of immunization was prepared and
LCs were stained with Mab to I-Ad antigen conjugated to FITC. The TR-OVA inside the green-colored LCs is yellow, whereas the TR-OVA in nonfluorescent cells (keratinocytes) is red (60x). LCs from the site of EPI migrate to the draining lymph node (C: gold formulation, D: sugar formulation). The vaccination sites were treated with FITC by topical application (C only) 2 hrs prior to EPI with TR-OVA. Fluorescent cells in the draining lymph nodes were examined 20 hrs later by fluorescent microscopy under an UV light. The LCs that contain TR-OVA originating from the site of EPI (240x). E and F are the same fields as C and D, respectively, examined under a bright light.
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resulted in significant CTL responses that were similar in mag-nitude to those elicited via DNA and live attenuated vaccines[34]. In contrast, sugar formulation EPI does not routinely elicitCTL responses and is consistent with the extracellular deliveryof the antigens that results in endocytic uptake mechanismsthat often bypass the MHC class I presentation pathway.
T-helper responses & immunomodulation with adjuvants
The quality of T-helper cell responses (Th1 versus Th2) followingEPI have been examined using a number of antigens. Not sur-prisingly, T-helper cell responses are dependent on the presenceor absence of an adjuvant. In the absence of adjuvant, both diph-theria toxoid and HBsAg induce Th2 responses as reflected bythe prevalence of interleukin (IL)-4 versus interferon (IFN)-γ pro-duction and the IgG1 to IgG2a subclass ratio. These responsescan be shifted towards a Th1 phenotype, or more strongly in theTh2 by the use of adjuvants. For example, CpG-containingDNA, QS-21 and LT adjuvants induce responses that are moreTh1-like, while aluminum adjuvants and a synthetic polymeradjuvant, poly[di(carboxylatophenoxy)phosphazene], promoteTh2 responses [38].
Potential applications of EPI in disease controlVaccination against infectious diseases
EPI can elicit high levels of serum antibodies that are importantfor prophylactic immunization against infection by extracellu-lar bacteria and many viruses. Mucosal antibody responses areparticularly important because they represent a first line ofdefense and may prevent pathogens from gaining entry to thedeeper tissue. EPI may also have immunotherapeutic potentialfor chronic infectious disease caused by viruses and intracellularbacteria, since antigen-coated gold particle formulations can beused to elicit CTL responses. It is also important to note thatEPI, since it targets the skin, may allow for the use of new andmore potent adjuvants that may otherwise be unsafe or poorlytolerated when administered parenterally. Adjuvants adminis-tered by EPI appear to have low systemic bioavailability, mark-edly reducing the risks of systemic toxicity while maintainingtheir adjuvant properties [40]. Moreover, the continual slough-ing and regeneration of the epidermis ensures that much of anydelivered adjuvant will be lost from the local target site and intothe environment.
Cancer immunotherapy
Immunotherapeutic immunization with whole tumor cells,tumor-cell lysates and with tumor-associated antigens are yield-ing promising results in animal models and the clinic [42]. Sinceantigen presentation is a critical regulatory element for theinduction of humoral and cellular immune responses to tumors,DCs are being exploited for their antigen-presenting capabilitiesin tumor immunotherapy. DC-based cancer immunotherapyoften involves the ex vivo cultivation of autologous DCs fromthe peripheral blood of patients, followed by in vitro antigenloading and the reinfusion of loaded DCs back to the patient.Alternatively, EPI using gold formulations represents an in vivo
Figure 4. Enhanced antibody responses and protection by EPI with A/Aichi/68 influenza vaccine in the murine influenza challenge model. Mice (8 per group) were immunized with 25, 5, or 1 mg (total protein) of inactivated virus by either EPI or subcutaneous (sc.) needle injection. Sera werecollected 28 days after the immunization and assayed in an ELISA. The data are IgG titer (A) of individual animals (¨) and the mean titer of 8 mice (-). p was <0.001 between the EPI and sc. control for each dose (t-test). Mice were challenged 32 days postimmunization with a mouse-adapted A/Aichi virus. Allmice receiving the EPI (q ) of 25, 5, or 1 mg of vaccine survived the 21-day monitoring period (B). The p values were 0.14, 0.06, and <0.01 between the EPand sc. control for the 25, 5, and 1 mg doses, respectively (JMP Survival Analysis). Weight-loss data are the mean percent body weight relative to that prior to challenge (C). The "*" marks indicate fewer than 50% of the mice werealive at that time-point.
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approach that could achieve similar DC activation and loadingeffects without the need for ex vivo culture. EPI is compatiblewith the delivery of tumor cell lysates, purified antigens andother immunostimulating agents (e.g., granulocyte-macrophagecolony-stimulating factor [GM-CSF]) and adjuvants.
Allergy immunotherapy
Allergic diseases, including asthma and hay fever, are commonlytreated with corticosteroids and antihistamine drugs. Immuno-therapy by subcutaneous injection of the allergen over a pro-longed period has been used to treat several types of allergieswith variable clinical effects. This appraoch is effective in induc-ing prolonged remission of insect venom anaphylaxis, but isonly moderately effective against hay fever and even less effectiveagainst asthma. Although the mechanism of immunotherapy inhumans is not fully understood, studies using animal modelssuggest that immunization strategies promoting a Th1 responsecan decrease the formation of IgE antibodies and eosinophilia[42]. Alternatively, formation of IgG antibodies will neutralizethe allergens before being captured by the IgE antibodies. EPIusing antigen and an appropriate adjuvant (CpG DNA,saponin) promotes strong Th1 responses in animal models indi-cating that it may be possible to reprogram the immune systemof sensitized individuals and offers a more effective means ofallergy immunotherapy. Given that EPI has been shown to elicitmucosal responses, allergy immunotherapy treatment of the skinmay result in therapeutic benefits systemically, as well as in theskin and at mucosal sites.
Particle-mediated DNA vaccinationParticle-mediated DNA vaccination involves the precipitationof plasmid DNA molecules encoding one or more antigensonto a preparation of approximately 2 µm gold particles, fol-lowed by the intracellular delivery of these particles into viablecells of the epidermis using a particle delivery (‘gene gun’)device. Successful intracellular delivery of the plasmid vectorresults in transient antigen production in both keratinocytesand LCs and the induction of humoral and cellular responses tothe specified antigen. This technology has advanced from ani-mal studies to human clinical trials and is the only DNA vac-cine delivery system to date that has elicited significanthumoral and cellular responses in humans [43]. Potential appli-cations of this technology include prophylactic and therapeuticvaccines for infectious diseases, as well as therapeutic vaccinesfor cancer and allergies.
Brief history of particle-mediated DNA vaccinationParticle-mediated DNA vaccination has its roots in the devel-opment of the original ‘gene gun’ concept by John Sanford thatwas instrumental in the successful genetic engineering of severalplant species. This novel gene delivery system employed a phys-ical delivery process to propel DNA-coated tungsten particlesdirectly into the interior of cells of whole plants or plant tissues[27,28,44]. Due to the physical, rather than biological nature ofthe delivery system, this technology was shown to function
equally well for the transient delivery of DNA into mammaliancells, either in tissue culture or in living animals using DNA-coated gold particles [26,29,45]. While transient gene expressioncan be induced in a variety of tissues, the most practical tissuetarget is the skin, due to accessibility in the absence of surgicalintervention. The first demonstrations of the induction of anti-gen-specific immune responses following particle-mediatedDNA delivery and expression in the skin took place in the early1990s using delivery devices employing the motive force ofeither a controlled electric discharge [46,47] or compressedhelium [48]. While these devices differed markedly in design,they employed similar delivery mechanisms that involved therapid acceleration of a flat membrane, carrying a DNA/goldpreparation into a porous barrier. Rapid deceleration of themembrane upon impact with the porous barrier allowed theDNA/gold particles to carry on through the barrier and intothe adjacent skin tissue with the depth of delivery being a func-tion of the gold particle size and the amount of motive forceapplied. The impracticality of a large electric discharge deviceand the use of fragile gold-coated membranes led to the devel-opment of a completely new hand-held, helium-powdereddevice design employing the formulation of DNA/gold insmall, open-ended, tubular cartridges (FIGURE 5). Actuation ofthe PowderJect XR-1 powder delivery device (formerly Accell®
system) involves the release of a defined volume of pressurizedhelium that is directed through the cartridge, entraining theDNA/gold formulation in the flow and accelerating the parti-cles into the target skin. This device has been used to induceantigen-specific humoral and cellular responses and protectionagainst pathogenic challenge in a variety of small and large ani-mals and was employed in the first human DNA vaccine clini-cal trial that resulted in the induction of significant humoraland cellular responses (see below). A similar device (Helios,Bio-Rad) is commercially available for research purposes andhas been used successfully for DNA vaccination in a number ofanimal models.
SolenoidSnap lock Disposable
nozzle
DNA cartridge
Trigger
Helium inlet
Power cord
Figure 5. PowderJect XR-1 DNA vaccine delivery system. Actuation of this device results in the release of a bolus of pressurized helium gas that travels through a single use disposable DNA/gold cartridge and disposable nozzle. The DNA/gold formulation is entrained in the gas flow and is directed into the epidermis in the skin target site.
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The evolution of delivery devices for DNA vaccines is con-tinuing with the development of completely disposable, single-use devices very similar to those described in the section on EPIdelivery system. Although they are yet to be tested in the clinicfor DNA vaccination, animal studies to date demonstrateequivalence with the reusable XR-1 system in terms of DNAvaccine efficacy (unpublished). For clinical purposes, single-usedisposable devices will offer convenience and consistency ofdelivery and avoid the complications associated with the moni-toring of reusable device performance.
Induction of humoral & cellular responses in small animalsNumerous published reports have demonstrated the induction ofhumoral, cellular and protective immune responses in small ani-mals using both the original carrier membrane devices and thesecond generation XR-1 and Helios devices [46,47,49–57]. Thecommon thread among these reports is the induction of vigorousimmune responses using only microgram levels of DNA vaccinevectors delivered intracellularly to the viable epidermis. In caseswhere the relative performance of particle-mediated andparenterally-inoculated DNA vaccines have been compared, itwas observed that particle-mediated DNA vaccination results instronger responses with much less DNA than is required byparenteral DNA inoculation [30,46,51,58,59]. The dramatic dose–response differences are almost certainly due to the requirementfor intracellular uptake mechanisms associated with needle andsyringe injection of DNA. While uptake and expression of DNAoccurs in some muscle cells following intramuscular injection,evidence for expression in APCs is lacking [60]. On the otherhand, particle-mediated DNA delivery to the skin results indirect transfection to APCs due to the considerable concentrationof LCs in the viable epidermis [61,62]. Directly transfected migra-tory cells in the skin were shown to be responsible for the estab-lishment of immunological memory and cellular responses in aseries of skin ablation and transplantation experiments in miceemploying particle-mediated DNA vaccination [63].
Another distinguishing feature of particle-mediated DNAvaccines is the lack of evidence demonstrating that bacterialCpG motifs in DNA vaccine vectors contribute to immuno-genicity following particle delivery. Although ample evidencesupports the adjuvant properties of CpG motifs in parenterally-inoculated DNA vaccines, CpG motifs may contribute little tothe vigorous responses elicited following particle-mediatedDNA vaccination [64,65]. This observation is consistent with thetheoretical mechanism of CpG adjuvanticity in that endocyticuptake of extracellular DNA is followed by DNA degradationand CpG engagement of Toll-like receptor 9 in the endocyticpathway [66]. By circumventing the endocytic uptake mecha-nism, direct intracellular delivery of DNA vaccines appears toinduce immune responses in a CpG-independent fashion.
Particle-mediated DNA vaccine efficacy in large animals & humansDue to a number of experiments in mice, a popular myth in theDNA vaccine field is that particle-mediated DNA vaccination
elicits predominantly Th2 responses. While this phenomenonmay hold true for certain DNA vaccine vectors in Th2-proneBalb/c mice, it is apparent that the quality of responses elicitedby this technology is influenced by a number of factors, includ-ing the identity of the antigen and the species being vaccinated[51,67]. By evaluating particle-mediated DNA vaccines in largeranimals and humans, it is clear that particle delivery technologyis equally effective in eliciting humoral, cellular and protectiveimmune responses in larger, nonrodent species, such as ferrets[58], pigs [50,68], monkeys [69–71] and humans [43]. More impor-tantly, studies in larger animals have shown the absence of theTh2 tendency, particularly in monkeys [69] and humans [43],where significant Th1-like cellular responses were documented.Moreover, SIV-specific Th1 responses in rhesus monkeys wereassociated with protection against heterologous pathogenicmucosal challenge [69]. Accumulated data in large animals andhumans clearly demonstrate the important role particle-medi-ated DNA vaccination is likely to play in the clinic. Moreover,the ability to elicit Th1-like cellular responses suggests thepotential this technology may have for successful therapeuticDNA vaccination.
Adjuvantation of particle-mediated DNA vaccinesInsofar as CpG motifs appear to play little part in the immuno-genicity of particle-mediated DNA vaccines (see above), thisdoes not imply that adjuvantation will not be effective with thistechnology platform. A number of studies in animal modelshave shown the potential to augment or modulate immuneresponses by the codelivery of vectors encoding cytokines andchemokines using both parenterally-inoculated and particle-mediated DNA vaccines. While GM-CSF- and IL-12-encod-ing vectors have stood out as potential cytokine adjuvant vec-tors with the most promise for particle-mediated DNA vaccines[72–77], the strongest adjuvant effects to date have been obtainedwith vectors encoding CT and LT [78]. The delivery of CT andLT adjuvants in a DNA vector form results in dramatic aug-mentation of cellular immune responses as well as protectionagainst viral challenge (Haynes J, unpublished), while eliminat-ing toxicity that is typically associated with these molecules.The incorporation of powerful, yet well-tolerated adjuvantswith an effective delivery system is likely to markedly improvethe performance of particle-mediated DNA vaccines in largeanimals and humans.
Advantage of needle-free skin immunizationNeedle-free skin immunization holds a number of advantageover parenteral needle injection. First, effective skin immuniza-tion may induce stronger serum antibody responses using thesame dose of vaccine or induce similar immune responses usingsmaller antigen doses. This has been demonstrated in animalstudies by EPI using a number of antigens and recently bymicroneedle injection with ovalbumin. The improved immuneresponses and reduced antigen dose requirement are probablyrelated to targeted antigen delivery to APCs in the skin. Sec-ond, effective skin immunization may induce mucosal antibody
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responses and offer improved protection against pathogens thatinfect via mucosal routes and may be applicable to both DNA-and protein-based vaccines. Third, induction of cell-mediatedimmunity by DNA vaccines or intracellular delivery of protein-based vaccines by EPI opens the door for developing therapeu-tic vaccines against diseases, such as cancer and chronic infec-tious diseases. Fourth, needle-free immunization is likely to besafer than needle injection by avoiding needle stick injury andthe risk of transmission of blood-borne diseases. Noninvasiveimmunization strategies, including EPI and particle-mediatedDNA vaccines, cause no pain and may obviate needle phobiaproblems in children and some adults that are linked to lowvaccination rates. Fifth, epidermal rather than parenteral deliv-ery of adjuvants is likely to result in greater tolerance and theability to use adjuvants that would normally not be consideredfor parenteral or mucosal administration (e.g., CT and LT).Since many adjuvants work by upregulating or activatingAPCs, direct adjuvant delivery to the APC-rich skin may allowfor the use of smaller adjuvant doses and avoid systemic toxic-ity. Finally, dry powder formulations of protein vaccines deliv-ered by EPI demonstrated excellent high-temperature storagestability, possibly eliminating the requirement for an intact coldchain. These advantages may translate into reduced cost ofgoods, increased vaccination rates and improved disease con-trol.
Five-year view & expert opinionChildren in the USA currently receive approximately 11 vac-cines comprising 20 shots by the time they are 18 months ofage. Moreover, several new vaccines are expected to be approvedin the next 5–10 years and introduced into this regime. Nationaland international policy-making bodies are actively seeking nee-dle-free immunization alternatives for widespread implementa-
tion in routine vaccination programs. As reviewed in this paper,a number of such technologies are at preclinical and early clini-cal stages of development and several products are expected toreach late-stage development in coming years. One such tech-nology, EPI, has yielded stronger immune responses to a triva-lent human influenza vaccine in several animal models, includ-ing primates, than traditional needle injection. A human clinicaltrial is currently in progress. The timeline for developing aninfluenza vaccine using EPI may be shortened by employing thecurrently licensed influenza vaccine product but with an alteredformulation and route of delivery. The annual reoccurrence ofwidespread influenza outbreaks and the initial targeting of anadult only (nonpediatric) vaccine may help accelerate clinicaldevelopment and approval.
In addition to prophylactic vaccines, recent progress in theinduction of strong cellular immunity using epidermally-delivered DNA vaccines offers an exciting tool for the devel-opment of candidate therapeutic vaccines for chronic infec-tious diseases, cancer, and allergy. In particular, the use ofgenetic adjuvants in DNA vaccines may allow for the induc-tion of vigorous responses of sufficient quality to exhibit a realtherapeutic effect.
From an industrial perspective, it is less risky to develop nee-dle-free vaccine products using currently approved vaccine anti-gens. However, the application of new delivery technologies tonew antigens and types of vaccines (such as DNA) will ulti-mately yield new tools to combat infectious and neoplastic dis-eases and have a lasting impact on disease control and publichealth.
Information resourcesTABLE 1 shows a list of needle-free skin immunization technolo-gies and institute affiliation.
Table 1. Needle-free immunization technologies and institute affiliation.
Technology Company (website) Lead product candidateEpidermal powder immunization & particle-mediated DNA vaccination
PowderJect Pharmaceuticals, Plc.(www.powderject.com)
Hepatitis B (DNA) and influenza (protein): Phase I
Transcutaneous immunization (topical patch) Iomai Corporation(www.iomai.com)
Tetanus: Phase II
EasyVax(TM) (topical patch) Vaxin, Inc.(www.vaxin.com)
DNA and live-vectored vaccines: preclinical
Macroflux® Skin Interface Technology (micro-needle injection)
Alza Corporation(www.alza.com)
Protein vaccine: preclinical
Micro-fabricated Array (OnVax) BD Technologies(www.BD.com)
DNA and protein vaccine: preclinical
MicroPor™ (heat albation) Altea Genomics, Inc(www.alteatech.com)
DNA vaccines: preclinical
Laser-assisted delivery and micro-needle arrays Norwood Abbey(www.norwoodabbey.com)
Unspecified DNA vaccine: preclinical
Liquid-jet injection Many manufacturers(listed in www.cdc.gov/nip/dev/jetinject.htm)
DNA, protein, live-vectored vaccines
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ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest
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Conclusions & key issues
• Needle-free skin immunization holds great promise for the future of vaccination. It is not simply an alternative vaccination route since needle-free skin immunization may offer important efficacy advantages over traditional needle injection due to the targeted delivery of antigens to antigen-presenting cells (APCs).
• Furthermore, needle-free skin immunization may improve patient compliance in vaccination and reduce the risk of transmitting blood-borne diseases associated with needle-stick injury.
• A number of technologies are in various stages of preclinical and early clinical development.
• Epidermal powder immunzation (EPI) has a distinct advantage over other noninvasive methods in the efficiency of delivering vaccines across the stratum corneum barrier and into the target tissue.
• The impressive preclinical and early clinical results collected thus far with EPI warrants an aggressive product development.
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Affiliations• Dexiang Chen, PowderJect Vaccines Inc., 585
Science Drive, Madison, WI 53711, USA. Tel.: +1 608 231 3150, Fax: +1 608 231 6990, [email protected]
• Yuh-Fun Maa, ALZA Corporation, 1900 Charleston Road, Mountain View, CA 94043, USA. Tel.: +1 650 564 2104, Fax: +1 650 564 2700, [email protected]
• Joel R Haynes, PowderJect Vaccines Inc., 585 Science Drive, Madison, WI 53711, USA. Tel.: +1 608 231 3150, Fax: +1 608 231 3150 [email protected]
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