Towards tailored vaccine delivery: Needs, challenges and perspectives

Journal of Controlled Release 161 (2012) 363–376

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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

Towards tailored vaccine delivery: Needs, challenges and perspectives Jean-Pierre Amorij a, Gideon F.A. Kersten a, Vinay Saluja a, Wouter F. Tonnis b, Wouter L.J. Hinrichs b, Bram Slütter c, Suzanne M. Bal d, Joke A. Bouwstra e, Anke Huckriede f, Wim Jiskoot e,⁎ a

Vaccinology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands Department Pharmaceutical Technology and Biopharmacy, University of Groningen (RuG), Groningen, The Netherlands Department of Microbiology, University of Iowa, Iowa City, USA d Division of Experimental Immunology/Department of Respiratory Medicine, Academic Medical Centre and University of Amsterdam, Amsterdam, The Netherlands e Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Leiden, The Netherlands f Department of Medical Microbiology, Molecular Virology Section, University Medical Center and University of Groningen, Groningen, The Netherlands b c

a r t i c l e

i n f o

Article history: Received 31 October 2011 Accepted 27 December 2011 Available online 5 January 2012 Keywords: Adjuvant Device Formulation Mucosal vaccination Targeting Transcutaneous vaccination

a b s t r a c t The ideal vaccine is a simple and stable formulation which can be conveniently administered and provides life-long immunity against a given pathogen. The development of such a vaccine, which should trigger broad and strong B-cell and T-cell responses against antigens of the pathogen in question, is highly dependent on tailored vaccine delivery approaches. This review addresses vaccine delivery in its broadest scope. We discuss the needs and challenges in the area of vaccine delivery, including restrictions posed by specific target populations, potentials of dedicated stable formulations and devices, and the use of adjuvants. Moreover, we address the current status and perspectives of vaccine delivery via several routes of administration, including non- or minimally invasive routes. Finally we suggest possible directions for future vaccine delivery research and development. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Vaccine delivery: general points to consider . . . . . . . . . . . 2.1. Vaccine design . . . . . . . . . . . . . . . . . . . . . 2.2. Mode of vaccine administration . . . . . . . . . . . . . 2.3. Target population . . . . . . . . . . . . . . . . . . . . 2.4. Delivery concepts and immunological targets . . . . . . . 2.4.1. Targeting to APCs . . . . . . . . . . . . . . . . 2.4.2. Activation of the innate immune system . . . . . 2.4.3. Activation of adaptive immune responses. . . . . 2.4.4. Tailored immune responses . . . . . . . . . . . 2.4.5. Controlled antigen release . . . . . . . . . . . . Current developments and needs . . . . . . . . . . . . . . . . 3.1. Stable vaccine formulations . . . . . . . . . . . . . . . 3.1.1. Perspective . . . . . . . . . . . . . . . . . . . 3.2. Effective, safe and approved adjuvant–antigen combinations 3.2.1. Perspective . . . . . . . . . . . . . . . . . . . 3.3. Vaccine formulations tailored to the route of administration 3.3.1. Injectable vaccines . . . . . . . . . . . . . . . 3.3.2. Transcutaneous vaccine delivery . . . . . . . . . 3.3.3. Oral vaccine delivery . . . . . . . . . . . . . .

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⁎ Corresponding author at: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. E-mail address: [email protected] (W. Jiskoot). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.12.039

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3.3.4. Sublingual and buccal vaccine 3.3.5. Nasal vaccine delivery . . . . 3.3.6. Pulmonary vaccine delivery . 4. Concluding remarks and future directions . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction

2. Vaccine delivery: general points to consider

Among medical interventions vaccination is the one with by far the largest impact. For instance, smallpox with an estimated 50 million cases in the 1950s was eradicated through vaccination in 1977. More recently, by enforcing vaccination of children in priority countries, the WHO Measles Initiative achieved a reduction in global childhood mortality caused by measles from 700,000 in 2000 to 164,000 in 2008 [1]. Currently, most countries have implemented national immunisation programmes which cover a variety of common childhood infections and result in significantly reduced childhood morbidity and mortality [2]. Despite these successes there is still a number of infectious diseases for which no effective vaccine is available (e.g., HIV, malaria), or for which existing vaccines provide insufficient immunity (e.g., tuberculosis, whooping cough) or are unaffordable for those most in need (e.g., pneumomoccal disease). Moreover, we have learned that it is not solely a protective immune response elicited by the vaccine that determines its success. Much depends on willingness of the public to be vaccinated, as well as on the availability of the vaccine and trained personnel to administer it. Not surprisingly, during the last decades an intensified effort has been devoted to the development of safer vaccines (subunit vaccines), adjuvants to enhance the efficacy (dose sparing) and to explore the usage of non-invasive administration routes. A crucial aspect in addressing the challenges in vaccine development is vaccine delivery. Vaccine delivery encompasses (i) administration of the vaccine formulation to specific sites of the body and (ii) delivery of the antigen to and activation of relevant cells of the immune system. Administration of vaccine formulations to specific sites of the body can be achieved by various routes, including intramuscular (i.m.) or subcutaneous (s.c.) injection, transcutaneous delivery, oral delivery, sublingual (s.l.)/buccal delivery, nasal delivery and pulmonary delivery. Each of these immunisation routes requires specially designed formulations (e.g., suspensions, emulsions, powders, tablets) and specially designed delivery devices (like microneedles, nasal sprayers and pulmonary inhalers). Induction of the desired immune response furthermore requires antigen delivery to professional antigen-presenting cells (APCs) and activation of these cells. Live vaccines and vaccines consisting of whole inactivated pathogens have the intrinsic capacity to target and activate these cells. Protein vaccines, however, may need adjuvants which can serve as delivery systems, provide the necessary signals for activation and maturation of APCs or combine both functions [3]. This review addresses vaccine delivery in its broadest scope. Although we focus our discussion primarily on vaccines intended to combat infectious diseases, the vaccine delivery principles discussed generally also apply to other vaccines, such as cancer vaccines and lifestyle vaccines. First, we describe the general points to consider for vaccine delivery, such as vaccine design, mode of vaccine administration, target population and immunological aspects. Next, we discuss the current developments, needs and perspectives for stable vaccine formulations; for effective, safe and approved adjuvant– antigen combinations; and for vaccine formulations tailored to the route of administration. Finally, the main conclusions and overall perspectives will be given.

2.1. Vaccine design

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370 371 372 372 373

For the design of an effective vaccine one has to realise that a vaccine is much more than just an antigen (Fig. 1). Depending on the intrinsic immunogenicity of the antigen and the envisaged route of administration, choices have to be made regarding the (possible) use of an adjuvant serving as delivery system and/or immune potentiator. Table 1 gives an overview of delivery systems and immune potentiators. Examples of typical antigen delivery systems are nanoparticles, microspheres, liposomes, and virus-like particles. Immune potentiators are natural or synthetic compounds that directly activate immune cells through specific receptors and/or pathways [11,56–62]. Delivery system and immune potentiator together determine the magnitude and quality of the innate immune response and the uptake and processing of the antigens by APCs. But, vaccine design encompasses more than choosing the right antigen and adjuvant. The vaccine should have an acceptable shelf-life, which will require formulation excipients like buffers, antimicrobials and stabilisers. Moreover, depending on the formulation and delivery route, a proper delivery device and primary packaging have to be selected, which at the end together with the formulation (formulated antigen, adjuvant and excipients) determine the storage conditions and shelf-life. 2.2. Mode of vaccine administration Because pathogens seldom become extinct, vaccination programmes – especially for childhood vaccination – tend to grow. This will lead to more injections or the development of new combination vaccines, unless non-invasive vaccines are developed. Increasing the number of injections will have the risk of decreased willingness of the public to be vaccinated, whereas new combination vaccines bear the risk of pharmaceutical and immunological interference. Besides factors such as patient compliance and risk of interference, injected vaccines have several limitations and drawbacks, as summarised in Scheme 1. Several of these disadvantages can be circumvented by the use of other vaccination routes, such as mucosal (e.g., oral, s.l., nasal and pulmonary) and transcutaneous administration. Potentially, needle-free vaccines have the advantage that they are easy to use/administer, do not require trained health care workers, are not or minimally invasive, are easy to use on large scale during mass immunisation programmes; and are safe and well perceived by the vaccinee [63–65]. The different administration routes currently explored and their specific requirements with respect to vaccine formulation and delivery will be discussed in detail in Section 3.3. 2.3. Target population Most current vaccines are intended for use in young children. This has several consequences for the strategy how to deliver a vaccine. An overview of different target populations, a selection of their typical characteristics and examples of consequences thereof for vaccine delivery is given in Table 2. The physical, metabolic, immunological and

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extensively in vitro and in animal models. Examples are IgFc — Fc receptor [77,78], mannose — mannose receptor [79,80], carbohydrate — C-type lectin receptors, antibody — DEC205 [81–84], and pathogen associated molecular patterns (PAMPs), which may act as ligands for targeting to pattern recognition receptors (PRRs) [85–87]. Although positive effects of targeting have been demonstrated in experimental animals, there are possibilities for substantial improvement [88]. One reason is that different APC subsets have been and are being identified. A better understanding of the role of these cells may rationalise the choice of targeting ligands. The two main systemic (non-skin) dendritic cell (DC) types, plasmacytoid DCs and myeloid DCs, express different PRR and co-stimulatory molecules [89]. Specific targeting to myeloid DCs or to subsets of this lineage may make the difference between an immune response or the induction of tolerance, or may determine whether the antigen is presented to Th-cells in MHC class II context or whether cross presentation by MHC I molecules is achieved [89].

Fig. 1. Aspects to consider in vaccine design. The key components, antigen and adjuvant (delivery system and/or immune potentiator), must be properly formulated in a container or device, depending in part on the intended delivery route and storage conditions.

psychological processes characteristic for growth from birth into adulthood reveal that children cannot be regarded as small adults. This has implications for the delivery strategy feasible for the intended target group. For example, in the design of an orally delivered vaccine one should address that infants and toddlers are less capable to swallow tablets or capsules. Nasal delivery is well accepted by adults, however in infants and toddlers nasal delivery is related to increased wheezing (as shown for Nasalflu) [66]. Moreover, vaccine producers may be conservative with regard to nasal delivery in this population, because there might be a risk (not proven) that a suckling child will suffocate due to a congested nose. Furthermore, for pulmonary delivery of vaccines in small children the use of spacers (a reservoir for aerosolisation of the vaccine before inhalation) in combination with an inhaler is needed. From the age of 9 years, pulmonary delivery of vaccine powders with dry powder inhalers is technically feasible. Also elderly people should be regarded as a distinct target population. Humans have an immune system that is constantly changing, not only as result of ageing itself but often also by additional underlying diseases. These changes (like differences in TLR expression [67]) have consequences for vaccine delivery. Older people in general develop immunity less efficiently; as a result this population may require stronger, adjuvanted vaccines. 2.4. Delivery concepts and immunological targets With regard to antigen delivery and optimisation of the immune response, several aspects that are considered to influence vaccine immunogenicity should be taken into account (see Table 3). 2.4.1. Targeting to APCs Targeting suggest a process of accumulation of the active pharmaceutical ingredient in certain target organs, cell types or cell compartments. For vaccines this means: lymph nodes, APCs or B-cells, and within cells lysosome or cytoplasm. How specific targeting should be in the context of vaccine delivery is still largely unknown. Excellent preclinical results have been achieved with passive targeting, using cationic particles or antigens, which interact strongly with negative cell surfaces [68–76]. In this context ‘targeting’ means ‘delivery through stickiness’. Other classes of targeted vaccines are licensed: influenza virosomes are thought to work, amongst others, through binding of HA on the virosome surface to sialic acid residues on cell membranes. Other targeting concepts have been evaluated

2.4.2. Activation of the innate immune system To initiate an immune response, the innate immune system needs to be activated. This can be achieved by the presence of PAMPs in the vaccine. PAMPs are ligands for PRRs like Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs), C-type lectin receptors (CLRs) and nucleotide-binding oligomerisation domain-like receptors (NLRs) [90,91]. PRR engagement triggers the production of various chemokines and cytokines and the activation of APCs, which in concert leads to strong activation of the adaptive immune system. Yet, triggering of the innate immune system can also occur in the absence of (known) PAMPs, as demonstrated for example by squalene-in-water formulations which act as strong adjuvants without engagement of any known PRRs. 2.4.3. Activation of adaptive immune responses For successful vaccination both the humoral and the cellular arm of the adaptive immune system need to be activated. This requires (i) recognition of specific epitopes by surface immune globulins (B-cell receptors) on B-cells and (ii) presentation of T-cell epitopes on MHC class I or II molecules of APCs for recognition by T-cell receptors. For vaccine delivery it is important to realise that not all antigen must be taken up by APCs. For a humoral response part of the antigen dose should reach B-cells intact in order to bind to B cell receptors that recognise native epitopes. In other words, there are generally two delivery addresses: APCs and B-cells. Another aspect to consider is the physical form of the vaccine. Multimeric, particulate presentation of adjuvant and antigen lead to high avidity interactions with PRR on APCs or on B-cells which in turn result in their maturation and activation [92]. 2.4.4. Tailored immune responses The spectrum of infectious diseases to be tackled by vaccines is very broad and so are the immune responses which would optimally protect against these diseases. Important input for vaccine design are pathogen characteristics like the composition of the pathogen (components: PAMPs, B-cell epitopes, CD4 and CD8 epitopes), the route of entry (e.g., upper or lower respiratory tract, oropharyngeal, gastrointestinal), the mechanism of invasion (like endosomal escape known for influenza) and the nature of virulence factors involved. Detailed knowledge of the optimal immune responses to a given infection is of crucial importance for guiding vaccine development. Moreover, relevant correlates of protection need to be defined and robust assays for their determination need to be available to allow estimation of vaccine efficacy. 2.4.5. Controlled antigen release The kinetics of antigen availability strongly influence the immune response elicited [92]. Although different concepts have been investigated, currently it is not clear what would be the best way of antigen

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Table 1 Overview of adjuvants and their use in humans. Immune potentiator Bacterial origin Lipopeptides LPS analogues Cell wall components

Flagellin CpG DNA Toxins

Viral origin Double stranded RNA Guanosine analogs Human origin Cytokines Heat shock proteins Vitamin D (calcitriol) Other origin Plant derived

Examples Triacyl lipopeptides (TLR-1/TLR-2 agonists): Pam3Cys Pam3CSK4, Pam-Cys-SK4, PHCSK4 Diacyl lipopeptides (TLR-2/TLR-6 agonists): Pam2Cys, MALP2 or MDHM MPL (licensed), E6020, GLA, AGP, RC-529 (clinical [4]) (TLR-4 agonists) Peptidoglycan (TLR-2/TLR-4 agonist) Muramyl peptides: GMDP (clinical [5]), Murametide, Murapalmitine, Theramide (DTP-DPP), Termurtide (Threonyl-MDP) (clinical [6]), Murabutide (clinical [7]) Muramyl tripeptides: MTP-PE (clinical [8]) Glycolipids: TDM, TDB, MMG, αGalCer CWS (TLR-5 agonist) (TLR-9 agonist) (clinical [9–11]) Cholera toxin, cholera holotoxin, cholera toxin A subunit, cholera toxin B subunit (clinical [12]) Heat labile enterotoxin subunit B, LT (licensed in the past (Nasalflu), ETEC, LTK63 (clinical [13–15]))

(TLR-3 agonist) poly I:C (poly(I:C), poly rA:poly rU (poly-adenylic acid-poly-uridylic acid complex) (TLR-7, 8 agonist) e.g., imidazoquinolines, resiquimod (clinical [16]), imiquimod (clinical [17]), Loxoribine, TMX-201

GM-CSF (clinical [18]), IFN-gamma, IL-1, IL-2, IL-7, IL-12 (clinical [19]), FLT-3 ligand (clinical [20])

Synthetic lipids

Saponins: Quillaja saponin, Quil A, Iscoprep 7.0.3, QS21 (clinical [18,21–26]), many non-Quillaja saponins Inulin, Advax (nanocrystalline inulin) Aluminium phosphate (aluminium hydroxyphosphate) (licensed), aluminium hydroxide (aluminium oxyhydroxide) (licensed), calcium phosphate (licensed in the past) e.g., Avridine, BAY R1005, DDA (clinical [27]), stearyl tyrosine octadecyl tyrosine hydrochloride

Delivery system

Examples

Mineral

Emulsions Oil-in-water emulsions Water-in-oil emulsions

Particulate Lipid based VLP Saponin-cholesterol complexes Polymeric particles, Bacterial ghosts Combination adjuvants

MF59 (licensed), AF03 (licensed), adjuvant system 03 (licensed) IFA (clinical [18], licensed in the past [4,28–32], licensed in Cuba (tumour vaccine)), Montanide ISA 51 (IFA) (clinical [33,34]), Montanide ISA 720 (clinical [35–38]), Adjuvant 65 (clinical [39], licensed in the past [40])

Liposome (clinical [6]), transfersome, NISV, proteosome, OMV (clinical [41,42]), archeasome, cochleate Virosome (licensed [40,43], Ty particles (Ty-VLPs) clinical [44]) ISCOM (clinical [6]), Iscomatrix (clinical [45]), Pluscoms Nanoparticles, microparticles (clinical [6,46,47]) Lactococcus ghosts (clinical) CFA, Gerbu Adjuvant, SAF-1 (clinical [48]), AS01 (clinical [49]), AS02 (clinical [49–54]), AS04 (licensed HPV, HepB [55]), CAF01, IC-31, Ribi adjuvant, Montanide ISA 51 plus GM-CSF (clinical [33])

Abbreviations: AGP: aminoalkyl glucosaminide phosphates; AS01: adjuvant system 01 (liposomes plus MPL plus QS21); AS02: adjuvant system 02 (MPL plus QS21 plus oil in water); AS04: adjuvant system 04 (MPL plus aluminium hydroxide); CAF01: cationic adjuvant formulation (DDA-liposomes plus trehalose dibehenate); CFA: complete Freund's adjuvant; CWS: cell wall skeleton; DDA: dimethyldioctadecylammonium bromide; GM-CSF: granulocyte-macrophage colony-stimulating factor; GMDP: glucosaminylmuramyl dipeptide; IC-31 (cationic peptide KLKL5KLK plus oligodeoxynucleotide ODN1a); IFA: incomplete Freund's adjuvant; MALP2: macrophage-activating lipoprotein; MDHM: mycoplasma-derived high molecular weight material; MDP: muramyl dipeptides (N-acetylmuramylalanyl-d-isoglutamine); MPL: monophosphoryl lipid A; NISV: non-ionic surfactant vesicles (niosomes); OMV: outer membrane vesicle; Poly rA:Poly rU: poly-adenylic acid-poly-uridylic acid complex; Ribi adjuvant: squalene-in-water emulsion plus MPL (plus trehalose dimycolate); SAF-1: Syntex adjuvant formulation (MDP plus Pluronic L-121 plus squalane-in-water); TDB: trehalose-6,6-dibehenate (or α,α′-trehalose 6,6′dibeheneate); TDM: trehalose-6,6-dimycolate (or cord factor); VLP: virus-like particle.

dosing to induce an immune response. Would it be slow continuous release of the antigen (and adjuvant) or increased dosing, like during natural infection [93], or would it be pulsed release, mimicking classical vaccination schemes? And, what is the optimal (continuous or pulsed) release period and how does it depend on the route of administration? It is very difficult to determine this because release profiles are not easily established in vivo. Moreover, when applying sustained antigen release systems, antigen may deteriorate in vivo due to exposure to body temperature and enzymes; antigen loaded microparticles may be phagocytosed by macrophages and fibrous tissue may form an extra barrier. The development of delivery systems through which release can be better controlled in vivo may give new impulses to the development of controlled release vaccines.

3. Current developments and needs 3.1. Stable vaccine formulations The main focus in vaccine development has been put on optimisation of the immunological properties while stability issues are usually minimally addressed. However, inherent to their production process, vaccines are obtained and usually administered in an aqueous environment, in which they are often unstable [94–96]. Vaccines can undergo a wide variety of physical and chemical degradation reactions. For instance, proteinaceous vaccines can undergo conformational changes, oxidation, hydrolysis, deamidation, and aggregation [94,97–104]. Moreover, the particulate nature of practically all

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administration depending on trained health care personnel

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needles as waste / needle stick injuries

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risk of reuse of needles / infections

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liquid based vaccines which depend on a cold chain

-

no mucosal immunity

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limited ease of use for mass vaccination

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combination vaccines (development):

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o

pharmaceutical interference

o

immunological interference

o

need to match immunisation schemes

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needle phobia and related stress

Scheme 1. Limitations and challenges associated with injected vaccines.

vaccines makes them prone to colloidal instability. Therefore, vaccines have to be stored and transported refrigerated, maintaining the so-called cold chain, and even then their shelf-life is limited. Various excipients have been identified, such as sugars and amino acids, that can be used as stabilisers of liquid vaccines [105,106]. Another strategy to improve the stability of vaccines is drying them [66,95,96,107]. In the dry state molecular mobility is strongly reduced, which also reduces degradation reaction rates. However, bringing the vaccine in the dry state, the vaccine is exposed to various harsh conditions, depending on the applied drying method and the formulation (reviewed in [66]), which may deteriorate the vaccine. Amino acids like arginine and proteins like gelatin and serum albumin have been used as stabilising excipients to prevent damage to the vaccine during drying [95]. However, sugars are the most commonly used dessico-protectants. If properly dried, the vaccine is incorporated in a matrix of sugar in the glassy state which provides stabilisation during subsequent storage. Not all sugars are suitable for stabilising vaccines; they should have a high glass transition temperature, low hygroscopicity, low tendency for crystallisation and should not contain reducing groups (if the vaccine is proteinaceous) [108]. Obviously, they should also be non-toxic, readily available and cheap. Often disaccharides like sucrose are used. However, also oligosaccharides and sugar alcohols have been successfully applied.

After reconstitution, the vaccine solution or dispersion should be administered to the subjects as soon as possible, because being again exposed to an aqueous environment, the vaccine is prone to degradation. Alcock et al. coated a filter material with live recombinant viral vectors by atmospheric drying using a mixture of sucrose and trehalose as stabiliser [109]. The authors propose that the filter coated with the vaccine can be combined with a syringe containing an aqueous solution by which reconstitution and injection can be performed in one handling, thereby minimising the time period between reconstitution and administration. Having the vaccine in a dry and stable state is also attractive for the development of various non-invasive dosage forms. Kim et al. successfully coated microneedles with whole inactivated influenza virus vaccine by atmospheric drying using trehalose as a protectant [110]. The microneedle system can be used for dermal administration of the vaccine. Pulmonary influenza vaccine powder formulations made by spray drying and spray freeze drying using inulin (an oligofructose) as stabiliser have been shown to be suitable for pulmonary administration [111]. Burger et al. developed a pulmonary vaccine based on dried live-attenuated measles virus vaccine using super critical fluid technology and myo-inositol (a sugar alcohol) as a stabiliser [112]. Furthermore, it can be envisaged that dry vaccine powders can be used for nasal administration or can be processed into tablets for oral delivery.

Table 2 Target population and consequences for (non-)invasive vaccine delivery. Target population

Age span

Term newborn infants

0–27 days

Infants and toddlers

Children

Adolescents Adults Elderly Persons at risk (COPD, heart patients, …)

Specific characteristics (selection)

Pre-existing immunity from the mother (maternal antibodies) Immature immune system 1–23 months Breast feeding and maternal antibodies Motoric capacities limited 1–12 months nose breathers only 2–11 years State of physical, motoric and cognitive development 12–18 years >18 years b60 years > 59 years

Consequences for vaccine delivery (examples)

Need for strategies that circumvent (systemic) HBsAg for 48 hour old newborns vaccine clearance by maternal antibodies of HepB infected mothers Tailored design of devices, Choice delivery route based on active administration Circumvent nasal and pulmonary route and big tablets (safety) Till 6 years, active administration From 6 years onward, children can comply with instruction Can comply with instruction

General paediatric programmes

Improve immunogenicity by adjuvants

Epidemic influenza (i.m.)

Adjust delivery strategy to underlying disease (safety reasons): COPD: lung disease, avoid pulmonary delivery Heart patients: avoid delivery that requires physical exertion/effort

Epidemic influenza (i.m.)

Independent/(legal) decision making Immunosenescence (related to ageing) Impaired immune system (related to ageing)

Vaccine applied in target population (Netherlands)

General paediatric programmes

HPV Traveller diseases (HAV, HBV, …)

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Table 3 Vaccine delivery: many targets, different requirements. Immune response

To be achieved

How achieved?

Critical delivery aspects

Innate

PAMP–PRR interaction on APCs APC maturation Balanced T-help response Ag processing by APCs MHC II presentation of Th epitopes APC maturation and co-stimulation APC–CD4 T-cell interaction No Ag processing Direct interaction of Ag with B-cell membrane-bound Ig

Immune modulator Multimeric presentation

Type of PAMP Combination of PAMPs Particulate presentation form Targeting to APCs

T-help (Th2 skewed) (see above) Ag processing by APCs MHC I cross presentation of CTL epitopes APC maturation and co-stimulation APC–CD8 T-cell interaction T-help (Th1 skewed) (see above)

Multimeric presentation Delivery of Ag to APCs Endosomal escape

T-help

Antibodies

CTLs

Delivery of Ag to APCs

Unprocessed transfer from SoD to LN Prevention of APC uptake

Native conformation Small (nano-sized) particulate presentation form: particles able to drain to LN Depot/slow release? pH sensitive delivery systems Fusogenic systems

Ag: antigen; APC: antigen-presenting cell; SoD: site of delivery (muscle, subcutaneous, intradermal tissue, mucosa); LN: lymph node.

3.1.1. Perspective Irrespective of the type of vaccine, it should be stable during its intended shelf-life. This may seem trivial but is sometimes overlooked in the search for effective vaccines. Moreover, the availability of heat-stable vaccines would benefit developing countries where cold-chain conditions are not always in place. Therefore, the design of stable vaccine formulations should be integrated early in the development process. 3.2. Effective, safe and approved adjuvant–antigen combinations For many vaccines the antigens as such, especially when highly purified, are not sufficient to induce a protective immune response. Rather, adjuvants are needed to overcome the limited immunogenicity of the antigens. The adjuvants with the longest track record are aluminium salts, in particular aluminium hydroxide and aluminium phosphate. However, aluminium adjuvants are poor inducers of T-cell mediated immune responses and have no potential for noninvasive routes. Moreover, like other adjuvants aluminium salts can bias the type of response that is elicited, a feature that can be employed to improve the protective capacity of the vaccine, but can also have deleterious effects. This became obvious after the dramatic respiratory syncytial virus (RSV) vaccine trials where children vaccinated with aluminium-adjuvanted formalin-inactivated RSV upon subsequent natural infection developed particularly severe symptoms and 2 children died [113,114]. Despite the fact that adjuvants have been in clinical use for almost 90 years and an enormous amount of effort has been put into their further development ever since, the list of clinically approved adjuvants is still very short and most are licensed only in combination with a single antigen for conventional routes of administration (see Table 1). In fact, there are currently no adjuvants for non-invasive vaccine delivery that are approved by regulatory agencies. Several reasons contribute to this slow progress. (1) Prophylactic vaccines are given to a large population of generally healthy individuals. Safety of co-administered adjuvants is therefore of utmost importance. (2) Each adjuvant/antigen combination has to be registered separately and registration is for a single administration route only. (3) Alum has a proven safety track record and works satisfactorily for most conventional vaccines. (4) Animal models are of limited predictive value when it comes to efficacy and safety of adjuvants. The disappointingly low rate of licensure for new adjuvant candidates is in shrill contrast to the urgent need. For difficult-to-develop vaccines like those for HIV, malaria and tuberculosis adjuvants that promote cellular immunity will be essential. Moreover, adjuvants will also be required in vaccines aiming to achieve protective responses against infections in vulnerable individuals with impaired immune

function like the elderly and immunecompromised individuals. The list of adjuvants currently in clinical development comprises many candidates (see Table 1). In how far these substances will fulfil safety and efficacy requirements of regulatory authorities remains to be seen. 3.2.1. Perspective It is encouraging that two of the four adjuvants currently licensed in Europe were marketed recently. It appears that these adjuvants are safe in humans [115]. Improved understanding of how adjuvants work [116] is a key factor for their success and may open the way to the introduction of more licensed adjuvants. This is necessary because there is probably not one universal adjuvant that will work for all vaccines, for all administration routes and for all target populations. There are opportunities for the introduction of adjuvants that are under development, sometimes already for a long time, because it is increasingly possible to unravel their mechanism of action. This is due to the availability of better and more sensitive assays to study adjuvant functions ex vivo and in vivo (in animal models as well as in humans) with multiplex assays and the application of systems biology to understand and predict vaccine efficacy [117]. 3.3. Vaccine formulations tailored to the route of administration 3.3.1. Injectable vaccines 3.3.1.1. Potential. Although one of the most used vaccines in the world is an oral vaccine (OPV, containing attenuated poliovirus), the benchmark for vaccine delivery is injection, mostly i.m., sometimes s.c. The reasons for this are the ease of administration (compared to, e.g., intradermal, intranodal, or intravenous) and the competence of muscles and s.c. tissue, as compared to mucosal surfaces, to drain the antigen to local lymph nodes. 3.3.1.2. Needs and current developments. Although injectable vaccines often work well, there are problems to solve and needs to fulfil. With regard to vaccine administration and antigen delivery these are (i) easy and safe administration, (ii) minimising the number of injections and (iii) dose reduction. 3.3.1.2.1. Ad (i). Easy and safe administration by minimally invasive parenteral administration is an attractive option for substitution of invasive administration using needle and syringe. Multidose fluid jet injectors, used in mass vaccination campaigns between the 1950s and early 1980s, increase flexibility and rule out accidents with needles. However, currently such ways of administration are only acceptable if the perceived pain and local adverse effects are is less than with regular injection. These effects depend

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on the injection depth and velocity, which are determined by jet velocity and nozzle design. The multidose jet injectors were banned after it became clear that cross contamination with, e.g., hepatitis B could occur in subsequent vaccinees. Current jet injectors like Bioject or Pharmajet use monodose containers and are in clinical trials, although multidose jet injectors are being further developed to safer devices [118,119]. There seems to be a trend towards intradermal jet injection in order to achieve dose sparing and reduce pain during injection. Injectable intradermal vaccines using specially designed needles or jet injectors with single dose cartridges have been clinically tested extensively and some of these intradermal vaccines are registered (like influenza vaccine intradermally delivered by short needles — Fluzone®, Intanza ® and IDflu® [120,121]). Needle-free injection of solids is in its infancy. Powder injection is possible, but it is difficult to control powder deposition depth. Another approach is the use of needle-like biodegradable polymeric devices, injected via air pressure [122]. Because of the fast application and low volume this type of injection is less painful compared to needle and syringe use (G. van de Wijdeven, unpublished data). Other advantages of dry injection are the absence of sharp waste and the generally better thermostability of the vaccines. 3.3.1.2.2. Ad (ii). The number of injections can be reduced by combining antigens or developing vaccines with built-in booster capacity. A number of very successful combination vaccines exist, such as trivalent diphtheria–tetanus–pertussis (DTP), hexavalent DTP–inactivated polio–hepatitis B–Haemophilus influenzae, mumps–measles–rubella, and multivalent pneumococcal and meningococcal vaccines. The development of combination vaccines is not trivial and bears several challenges and risks. Immunological interference [123] may require dose optimisation of the individual antigens and the adjuvant. Differences in stability profiles sometimes ask for a compromise on formulation composition or pH (pharmaceutical interference). Although these issues often can be solved to satisfaction, some restrictions remain because they are inherent to the combination, e.g., the immunisation schemes of the different components should match or have to be adjusted. The use of single-shot vaccines with built-in booster capacity is a long sought delivery method. The concept is based on injectable polymeric microspheres with antigen incorporated in or adsorbed to the particles. Although several groups have addressed singleshot vaccines with built-in booster capacity [124–127], there are no marketed products yet and we are not aware of any clinical studies. It may be that the concepts that have been investigated are too simple and not sufficiently developed. Although supposed critical variables like particle size, release pattern and type of polymer have been addressed, it is not entirely clear yet what the specifications of a single-shot vaccine should be: burst release or continuous release, particle uptake by APCs or extracellular antigen release, etc. It appears to be important that at least part of the microspheres should drain directly to the lymph nodes as opposed to local uptake by APCs [92] and an increasing antigen load over time favours T-cell responses [93]. Better insights into this delivery aspect would give a better basis to further development of single-shot vaccine delivery systems. Other potential problems that hardly have been addressed are antigen stability and in vivo release profiles. For human application a slow release vaccine should probably be stable and available in vivo for weeks to months. Body temperature and other conditions like the presence of proteolytic enzymes may destabilise or degrade antigen during the release period. Local conditions like particle encapsulation by fibrous tissue may result in very different bioavailability as predicted from in vitro release profiles. Pharmacokinetic– pharmacodynamic (PKPD) studies are therefore important to perform with this type of delivery systems. 3.3.1.2.3. Ad (iii). Although vaccine doses are already low (tens of micrograms) as compared to other biologicals (typically milligrams), there is a need for some vaccines to decrease the dose further or to

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improve potency. This is important because some vaccines are expensive to produce or in short supply (e.g., pandemic influenza vaccines, inactivated polio vaccine). Also, new vaccines are often better defined subunit vaccines with a relatively low intrinsic immunogenicity. Sometimes the immunogenicity is improved via multimeric presentation. Examples are vesicular formulations or viruslike particles (VLPs; influenza virosomes, human papillomavirus vaccine). Other ways to increase immunogenicity are the use of adjuvants. Unfortunately, the number of adjuvants in licensed vaccines is still very limited as discussed above (Section 3.2). 3.3.1.3. Perspectives. For inactivated vaccines i.m. and s.c. administration currently are the predominantly used routes. These routes will remain important for many years to come. Minimally invasive delivery techniques are currently entering the clinical phase both in developed and developing countries, which may contribute to better compliance by vaccinees. 3.3.2. Transcutaneous vaccine delivery 3.3.2.1. Potential. Transcutaneous immunisation is an appealing alternative to the classical injected vaccines. 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 APCs than the muscle or s.c. tissue and thereby offers the possibility to induce a more effective immune response. The epidermis contains the Langerhans cells and the dermis is populated with at least two different subsets of dermal DCs [128]. These APCs complement each other and make it possible to induce a potent immune response after transcutaneous vaccination. 3.3.2.2. Needs and current developments. The skin's function is to protect the body from the surrounding environment and the skin is therefore equipped with a formidable barrier, the stratum corneum. It is almost impossible to induce an immune response through transcutaneous vaccination without disrupting this barrier. Therefore, effective, safe, and convenient methods to achieve disruption of the stratum corneum are needed. There are many different methods to disrupt the skin barrier as was recently reviewed [129]. Intercell developed a transcutaneous vaccination method based on skin abrasion (removal of the stratum corneum) that showed promising results in a phase II trial against traveller's diarrhoea [130], but its development was discontinued after failing to meet efficacy endpoints in the following phase III trial. This illustrates the challenges in developing a transcutaneous vaccine. An attractive way to deliver vaccines into the skin more efficiently is by using microneedles: small needles that are generally shorter than 1 mm, which can penetrate the stratum corneum and reach the epidermis or the dermis. Most microneedle technologies, as discussed elsewhere in this issue [131], are still in the preclinical phase and the optimal microneedle strategy (material, shape) to deliver a vaccine into the skin has not yet been established. One elegant strategy is to use dissolvable microneedles encapsulating the vaccine. Recently, Sullivan et al. showed that immunisation with dissolvable microneedles containing inactivated influenza virus in mice resulted in a protective immune response, superior to that obtained after i.m. injection of the same dose [132]. Another strategy is to coat the antigen onto microneedle arrays. Both longer sparsely packed and shorter densely packed microneedle arrays have been proposed [133,134], the latter showing a need for increasing the application speed of the microneedles to optimise the delivered dose of the vaccine. These two utilisations of microneedles, but also the more straightforward manner of pre-treating the skin with solid microneedles [75] or using hollow microneedles to inject the vaccine into the (epi)dermis [120], are currently being exploited by a number of research groups and companies. Until today only limited information

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is present on issues related to the induction of local inflammation by the different transcutaneous delivery approaches. The only paper reporting on skin irritation (side effect) is from our group. This paper shows that microneedle arrays have very limited irritation in human skin [135]. According to a survey on the public opinion (health care professionals and general public) about microneedles, the side effects of microneedles were generally assumed to be less compared to those of hypodermic needles [136]. The coming years will show if effective and safe microneedle-based vaccines can be introduced to the market. Next to skin disruption, proper vaccine formulation is essential to ensure the stability and immunogenicity of a transcutaneous vaccine. However, in the transcutaneous vaccination field vaccine formulation has received modest attention so far. Vesicles and nanoparticles have been developed to encapsulate the antigen and have been delivered with the aid of microneedles, but this approach proved unsuccessful in animal models, most probably because the size (150–300 nm) of the particulate formulations is a limiting factor [76,137]. Recently, smaller (80–100 nm sized) VLPs in combination with microneedles proved suitable to induce a protective immune response in mice challenged with influenza virus [138]. Formulation of the antigen allows for inclusion of an adjuvant to steer the type of immune response that is elicited. Adjuvants can improve the potency of the vaccine, but, especially for the transcutaneous route, a small adjuvant–antigen entity is preferred. This is supported by a recent study in mice showing that small antigen–adjuvant conjugates of 28 nm applied on microneedle pre-treated skin induced higher antibody titres compared to 300-nm sized nanoparticles of the same components [75]. This opens new opportunities to design potent antigen–adjuvant conjugate formulations for transcutaneous delivery. 3.3.2.3. Perspectives. Transcutaneous vaccination research still has several hurdles to overcome before an ideal vaccine will be available. However, by combining a proper way to achieve skin disruption with a vaccine formulation that can induce the necessary type of immune response transcutaneous vaccination has great potential. 3.3.3. Oral vaccine delivery 3.3.3.1. Potential. Oral vaccination has the advantage of being needlefree and it can induce humoral and cellular immune responses at the site of delivery as well as at some (limited) distant mucosal compartments. Orally delivered antigens are processed by two different pathways, i.e. uptake by (i) microfold cells (M cells) present primarily in the follicle-associated epithelium covering the Peyer's patches, and (ii) interstitially located DCs in the intestine. M cells in Peyer's patches are known to take up antigens and transcytose them to underlying APCs [139,140]. 3.3.3.2. Needs and current development. OPV is the most successful oral vaccine up until now. However, the success of OPV could not be translated to other vaccines. Like OPV, almost all of the other approved oral vaccines (like those against typhoid fever and rotavirus) are live attenuated bacteria or viruses. These vaccines are stable under acidic conditions (OPV) or formulated in bicarbonate buffers in order to protect the antigens from harsh gastric conditions. A lyophilised live attenuated Shigella vaccine, reconstituted in bicarbonate buffer, is currently in Phase II clinical trials [141,142]. However, the use of live attenuated vaccines is associated with the risk of reverting to a pathogenic strain. In fact, the major drawback of OPV is its ability to cause vaccineassociated poliomyelitis. This occurs in about one case per 1.4 million after the first dose and one case per 2.4 million after the second dose [143] and therefore it will be impossible to eradicate polio with OPV. Moreover, it has been reported that oral live attenuated vaccines are less immunogenic in developing countries possibly due to differences in the intestinal barrier caused by small bowel bacterial overgrowth and heavy intestinal helminth infestation [142,144]. Therefore,

controlled clinical trials should be performed to study the influence of dietary and environmental factors on the intestinal barrier. The only approved oral vaccines that are not based on live attenuated pathogens are cholera vaccines consisting of whole inactivated bacteria. A cholera vaccine registered in Europe contains 4 types of Vibrio cholerae bacteria (heat inactivated O1 Inaba, formalin inactivated El Tor Inaba, heat inactivated Ogawa and formalin inactivated Ogawa) and 1 mg recombinant cholera toxin subunit B (CTB) and is given in an alkaline buffer [142]. An oral vaccine approved in India is based on two types of inactivated cholera bacteria (V. cholerae O139 and O1), but does not contain CTB. Rather, the vaccine contains antigens that have a relatively high intrinsic immunogenicity and by themselves act as strong mucosal adjuvants. These vaccines are predominantly given to healthy adults. Other approaches used for oral vaccination but still awaiting approval utilise live vector vaccines, VLP vaccines, adenovirus-based vaccines, plant-based vaccines and M cell targeting concepts (as reviewed in [142] and [145]). Attention has been given to achieving sufficient antigen stability in harsh gastric environment and overcoming oral tolerance due to repeated administration of high doses of antigen. Live attenuated viral vectors are tested for oral delivery of expressed antigens, as these vectors can protect the antigens from acidic conditions and also act as immune potentiators. However, pre-existing immunity to these vectors should be studied, since this may prevent optimal delivery of the antigens. Particulate delivery approaches like vesicles (e.g., liposomes, bilosomes and archeaosomes), polymeric (e.g. PLGA, chitosans) microparticles, VLPs and ISCOMs have been tested in preclinical and clinical studies. These adjuvants can prevent the degradation of antigen in the gastrointestinal tract and act as immune potentiators. Some of these formulations are thought to deliver the antigen to the M cells and/or show slow release of the antigen. However, to which extent these formulations deliver the antigen to M cells and which amount of antigen is actually taken up is in general not known. In addition, most preclinical proofs of concept, unfortunately mostly in murine models, have been obtained with high doses of antigen and adjuvants. Although many strategies have been tested, no strategy has been able to tackle the obstacles for oral delivery with great success. To our knowledge no approach has passed phase I clinical testing until today. Recently, it was shown that delivery of antigen to different parts of the gastrointestinal tract of mice induced different phenotypes of immune response, which was also dependent on the type of adjuvant used [146–148]. Therefore, a better understanding of the intestinal mucosa is required in order to design promising approaches for targeted vaccine delivery, like using enteric coatings that dissolve at a specific site of the GI-tract, for induction of optimal immune responses. 3.3.3.3. Perspective. From a user perspective oral administration is the most accepted and easiest form of delivery. This makes oral vaccines attractive for developed and developing parts of the world. Some live attenuated oral vaccines have reached the market. However, despite numerous efforts there is no single oral subunit vaccine on the market yet. This reflects the enormous hurdles that have to be overcome to develop such a vaccine. In the search for effective oral subunit vaccines lessons should be learned from the successful live attenuated vaccines. This should lead to insights into which gastrointestinal regions and which cells should be targeted, what adjuvants should be included, what kind of delivery mechanisms could be employed (e.g., cell-penetrating ligands, specific receptor ligands), and which vaccination schedule is needed. 3.3.4. Sublingual and buccal vaccine delivery 3.3.4.1. Potential. The s.l. and buccal routes have been used for many years to deliver low molecular-weight drugs to the bloodstream.

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Moreover, for antigen delivery these routes are thought to be effective for regulating systemic antibody responses, including prevention/suppression of IgE responses, and are being exploited for the treatment of type I allergies. It is shown that the delivery of allergens via the s.l. route can activate regulatory T cells that can suppress undesired immune reactions [149]. Currently, this has resulted in several approved s.l. products for immunotherapy of allergy: SLITone®, Sublivac®, Grazax®, Oralair®, AllerSlit®forte. An advantage over the oral transmucosal route is that the low gastric pH is avoided and that the enzymatic activity in the mouth is relatively low. Moreover, s.l. administration of live or inactivated influenza virus with cholera toxin from V. cholerae (CT) as an adjuvant induced immune responses in mucosal and in extra-mucosal tissues and conferred protection upon influenza virus challenge in mice [150,151]. Inactivated vaccines (VLPs, inactivated viruses) tested preclinically for s.l. immunisations are able to induce secretory IgA (sIgA) antibodies as well as CTL responses in different mucosal compartments [151]. Desvignes et al. showed that DCs of the buccal mucosa and Langerhans cells of the skin of mice share a similar phenotype and proposed that immunisation through the buccal mucosa, which allows antigen presentation for efficient priming of systemic class I-restricted CTLs, may be a valuable approach for single-dose mucosal vaccination [152]. This was supported by Etchart et al. who found that buccal delivery of a viral protein, measles virus nucleoprotein, to DCs of the buccal mucosa of mice induces in vivo priming of protective anti-viral CTLs [153]. In preclinical studies it was shown that s.l. delivered antigens are taken up via the stratified s.l. mucosa by DCs that migrate to draining lymph nodes and present the antigenic peptides to immune cells [152]. In addition, Song et al. showed that inactivated or live influenza virus did not migrate to or replicate in the central nervous system after s.l. administration in mice [151]. These results suggest that vaccines could be safely delivered via the s.l. route. Moreover, because of ease of administration s.l. and buccal vaccine delivery could be a preferred route of immunisation for children and especially infants because of ease of administration. 3.3.4.2. Needs and current developments. Several delivery systems, such as sprays, solutions, adhesive films and lollipops have been developed for s.l. or buccal delivery of small molecules [154–156]. However, the development of s.l. and buccal vaccines is still in a juvenile phase. Only liquid formulations containing vaccine and adjuvant have been used in animals until today. The efficiency of antigen uptake and transport, including the role of residence time has not been studied in detail until today. For s.l. vaccination in a clinical setting, formulations are required that guarantee prolonged residence time and enhanced permeability of the mucosal surfaces for the antigens. In most in vivo studies for s.l. immunisation CT-based adjuvants have been used [151,157,158]. Recently, it was shown that mice immunised s.l. with Salmonella antigens mixed with CpG as an adjuvant were 100% protected against a lethal challenge of live Salmonella enteritidis [159]. This vaccine should be evaluated clinically for safety and efficacy in human. For other future developments it might be needed to evaluate also new classes of adjuvants like other TLR agonists for vaccination with purified antigens via the s.l. and buccal routes. 3.3.4.3. Perspective. The potential of s.l. and buccal vaccination of humans has still to be proven. As with other mucosal routes safe and potent (new) adjuvants for s.l. and buccal immunisation are required. In addition to factors relating to safety and costs, the ideal delivery systems for vaccination should provide prolonged exposure of the antigen to the mucosal tissue and increased permeability of this mucosal tissue while at the same time ensuring the preservation of the antigen's immunogenicity and user acceptability.

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3.3.5. Nasal vaccine delivery 3.3.5.1. Potential. Nasal vaccination has the potential to address all the prerequisites for a successful needle-free vaccine as described in Section 1 and may open the way to better vaccine efficacy. As the respiratory tract is a common site of entrance for pathogens, immune surveillance in the nasal cavity is high. Therefore all the machinery to induce a potent immune response is present on or just beneath the nasal epithelial linings [64]. Additionally, mucosal administration can lead to local production of antigen specific sIgA, which can prevent pathogens from colonising mucosal surfaces. 3.3.5.2. Needs and current developments. Although the advantages of nasal vaccination are abundant, development of nasal vaccines is not trivial. During the past few decades only 2 intranasal (influenza) vaccines have been introduced to the market, one of which (Flumist) has established itself as a major competitor to its injectable counterpart. Flumist is a live trivalent vaccine containing the recommended seasonal influenza subtypes (both A and B) and has been attenuated (cold adapted) to only colonise the nasal epithelium and not spread to the lower region of the respiratory tract. Flumist has shown to elicit protective antibody titres in humans, with little adverse events [65]. Interestingly, an increasing number of studies reports a superior influenza specific CTL response, indicating that this vaccine activates both the humoral and the cellular arm of the immune system [160]. The live-attenuated character of the vaccine circumvents the major obstacles for efficacious nasal vaccines. Whereas substances in the nasal cavity are generally cleared within minutes due to mucociliary clearance, the persistence of Flumist's attenuated virus circumvents this problem and allows a continuous presence of antigen for 2–3 days. Moreover, influenza viruses naturally infect epithelial cells using their HA, making the uptake into the epithelium highly efficient. Finally, influenza virions contain a variety of natural adjuvants, like Toll like receptor (TLR) ligand TLR7, RIG-1 and can activate the inflammasome [161]. This allows effective maturation of mucosal APCs in an environment that is usually more prone to tolerance than immunity. The downside of using live attenuated influenza vaccines (LAIV) is, however, that it precludes application to elderly (> 49, LAIV less immunogenic), young children (b2 years, risk of wheezing) and immuno-compromised individuals (risk of infection), exactly those groups that are recommended to receive influenza vaccination. A nasal subunit vaccine for all age groups would therefore be highly desirable. Not surprisingly, the design of nasal subunit formulations has been focused on increasing the nasal residence time, increasing the uptake by the epithelium/APCs and application of adjuvants. The application of mucoadhesive substances, either in soluble form or elaborately formulated into microparticles [162], nanoparticles [163] or nanogels [164], has been shown to increase the nasal residence time of the antigen (ranging from hours to days) and improve the immunogenicity of nasally applied antigens. To enhance the uptake of the antigen, absorption enhancers like chitosan derivatives have been explored, although the immuneenhancing effect of these compounds could be attributed to their mucoadhesiveness as well. A similarly effective approach is the use of particulate formulations. Encapsulation of antigens in liposomes [165], virosomes [166], nanoparticles [167] or microparticles [168] generally increases the resulting immune response after nasal administration. It is argued that this is due to specialised epithelial cells referred to as M cells, which preferentially transcytose particulate matter from the luminal to the subepithelial regions of the epithelium [169]. Although enhanced M-cell transport may contribute, it is also clear that particles enhance the uptake of antigen by DCs and B-cells [170], which may contribute to the enhanced immune response. Finally, application of immune-potentiating

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adjuvants is necessary, but also may bear some risk as adjuvants generally walk a fine line between immune potentiation and toxicity. For instance, an inactivated nasal influenza vaccine supplemented with a heat labile enterotoxin (LT) from an aseptic E. coli strain had to be withdrawn from the market after reports of Bell's palsy [171], which was attributed to uptake of LT via the olfactory nerves. A more recent clinical trial using a detoxified LT analogue (LTK63) had to be suspended due to similar side effects [172]. Although our knowledge about the innate immune system is steadily increasing and there is a growing arsenal of approved adjuvants for systemic administration, there is an urgent need for safe and effective mucosal adjuvants (cf. Section 3.2). 3.3.5.3. Perspective. The example of Flumist demonstrates the great potential of the nasal route for immunization. However, development of a nasal subunit vaccine appeared to be tough and has yet to result in licensed products for human use although various approaches have individually shown promise. Therefore the major challenge ahead is how to formulate an antigen with various components such to achieve the ideal (i.e., efficacious, safe, stable and easy to administer) nasal vaccine. 3.3.6. Pulmonary vaccine delivery 3.3.6.1. Potential. The respiratory tract is a common site of entrance for pathogens, in which immune surveillance is high. As a result, physiological prerequisites for successful pulmonary vaccination are present. The human lungs have a very large vascularised surface area and contain highly heterogeneous APCs, like respiratory tract DCs and alveolar macrophages [173]. In addition, bronchus-associated lymphoid tissue in the lungs is part of the common mucosal immune system and has the potential, to elicit systemic immunity and local immunity (including sIgA) in the respiratory tract as well as local immunity on more distant mucosae [174,175]. 3.3.6.2. Needs and current developments. The immunological potential of pulmonary vaccination is demonstrated by the fact that (1) several veterinary vaccines are delivered by the pulmonary route already for several decades, such as live Newcastle disease vaccine for poultry, and (2) several aerosol based vaccinations in humans were recognised as promising by the Soviet Union and the USA up to 40 years ago for diseases like plague, tularemia, brucellosis, anthrax, tuberculosis, rubella [176]. However, no pulmonary human vaccine has reached the market until today. Some issues have to be resolved for successful application of pulmonary vaccination. First, for targeting the appropriate parts of the lung, aerosols with a proper particle size and density should be applied. Usually, particles or droplets with an aerodynamic diameter in the range of 1 to 5 μm give the most efficient deposition into the deep lungs [177]. The particle size of the delivered aerosol is dependent on the formulation and inhaler device used [174]. Inhalers can be distinguished in three categories: nebulisers, pressurised metered-dose inhalers (pMDIs) and dry-powder inhalers (DPIs). A device for pulmonary vaccination should disperse the vaccine formulation in the right particle size and should be easy to use. Secondly, more immunological understanding of the respiratory tract is needed in order to develop optimal targeting strategies to different anatomical regions of the lungs (main trachea, bronchus, bronchiole and/or alveoli) for pulmonary vaccines. Targeting DCs in different compartments of the respiratory tract, such as conducting airways and the lung parenchyma, is appealing owing to different characteristics and functionalities of DC populations in these compartments, however the optimal region is unknown to date. Finally, little is known about the safety profile of pulmonarily applied vaccines, above all in young children and persons with underlying conditions such as chronic obstructive lung disease. Clinical studies

need to be performed for testing safety profiles of vaccine administration to the lungs first, but also need to address the site of deposition. In recent years, mainly pulmonary vaccination against measles, tuberculosis and influenza have been investigated. Hickey and coworkers showed that spray dried BCG could protect guinea pigs against challenge with virulent Mycobacterium tuberculosis to a higher extent than s.c. injected BCG vaccine [178]. Pulmonary influenza vaccination with spray (freeze) dried powders has shown to induce systemic antibodies as well as sIgA in nose and lungs of mice [111,179,180]. Moreover, pulmonary delivered whole inactivated influenza vaccine showed protection (reduced lung virus load) in mice upon challenge [180]. Pulmonary administration of dried measles vaccine formulations has been successfully tested in cotton rats [181] and in rhesus macaques [182]. In the latter study, complete protection was shown against challenge. Results of clinical evaluation of these powders are expected in the upcoming years. The only successful clinical evaluation of pulmonary vaccination on large scale thus far is the measles vaccination of school children. In this study, a liquid live attenuated measles vaccine formulation was delivered into the lungs [183]. Clinical follow up studies on measles as part of the Measles Aerosol Initiative (started in 2002 by WHO [184]), however, resulted in confusing and disappointing results [183]. These disappointing results might be related to the time-consuming delivery of the reconstituted vaccine and the limited stability of the vaccine after reconstitution of the lyophilised vaccine-cake. Furthermore, the devices used may have performed suboptimally in terms of reproducible lung deposition and/or may have resulted in degradation of vaccine caused by shear stresses. In addition, recently a vibrating mesh liquid inhaler system, designed for use in mass vaccination campaigns, has been used to deliver aerosolised measles vaccine to a range of subjects from adults to infants between 9 and 12 months of age in trials in India. The vaccine is administered within 6 h after reconstitution, and dosing time is 30 s. Experience to date suggests that this vaccine elicits serum titres comparable to or greater than those achieved with injected measles vaccine. Inhalation was safe and well tolerated, with a favourable cost per dose to injections. The goal is to license this system for administration of inhaled measles vaccine in the developing world (personal communications, Dr. James B. Fink). Dry powder inhalation of measles vaccine has gained a lot of attention with respect to circumventing stability problems associated with pulmonary delivery of reconstituted vaccines. For this purpose powders have been produced by drying methods like supercritical fluid drying as well as spray drying [181,182]. 3.3.6.3. Perspective. The pulmonary route for vaccination has potential and may become reality if the measles aerosol project reaches its ultimate goal. This success will depend on formulation design as well as inhaler design. For general pulmonary vaccination worldwide, however, the major challenge is to achieve general acceptance based on improved insights into efficacy and safety as well as proof of easy and robust administration of pulmonary vaccines. 4. Concluding remarks and future directions With the significant progress made during the past decade regarding our insights into immune mechanisms and modes of action of adjuvants, vaccine delivery has become a mature science with a potentially huge medical and economic impact. To further advance the field, much can be gained by better identifying the immune mechanisms most relevant for protection against a given infection or disease, defining clear correlates of protection and developing robust assays for their determination. This knowledge combined with optimisation of vaccine delivery, in terms of administration route, delivery system and immune modulator, and stable formulation, will allow the generation of better vaccines.

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Although the number of clinically tested adjuvants is substantial, clinical research on adjuvants for non-invasive delivery, especially in target populations such as infants and children, is still very limited and requires further efforts. Highly challenging is the development of minimally and noninvasive transcutaneous and mucosal delivery systems for vaccines. One reason is that there are still several gaps in fundamental understanding of vaccine delivery. Especially, immunological knowledge of the different routes of administration in humans is limited. For instance, levels of TLR expression in the different anatomical regions (e.g., mucosae, skin) in human are not yet fully revealed. The presence of specific DC subtypes, macrophages or other types of APCs and other immune cells is route-specific. In this respect, ex vivo use of human material is still very limited and deserves further exploration. The same holds true for exploitation of humanized animal models. Another gap in our current understanding is the limited knowledge about PKPD of vaccine delivery, a barely explored area of research. Importantly, PKPD relationships may vary depending on the target population. In vivo imaging tools and quantitative biodistribution studies may help to unravel PKPD of vaccines. A new direction in preclinical research, partially taken up by different research groups, is the immunological understanding of age-related immunity. Preclinical research using “infant” and “elderly” animal models is on its way [185–191]. Selecting the proper vaccine delivery strategy should be based on the delivery site and the pathogen in question. The optimal formulation, presentation form (solution, particles or conjugates) and its physicochemical characteristics (e.g., particle size, surface charge and hydrophobicity, mucoadhesiveness, diffusion characteristics) will depend on the route of delivery and the selection of the adjuvant also depends on the type of response necessary to combat the pathogen or disease. Depending on the delivery site, custom made devices (like microneedles or inhalers) are needed. In the choice of the route of delivery and the type of device, attention should be paid to the limits (passive administration, active administration, physiology, etc.) set by the intended target population. Upfront “market research” and clinical placebo trials in the target groups may add to vaccine delivery success at the end. Last but not least, invasive as well as non-invasive vaccine delivery concepts require not only detailed knowledge of immunological principles but also advanced pharmaceutical technologies (such as formulation processes, stabilisation strategies and design of delivery devices) and regulatory insights in order to avoid a high risk of failure in the development phase. Therefore, a concerted multidisciplinary approach of immunologists, pharmaceutical scientists, clinical researchers and regulatory scientists is needed to make the leap from promising vaccine delivery concepts to potent and safe registered vaccines that will contribute to improved public health in the near future.

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