Needle-free influenza vaccination

www.thelancet.com/infection Vol 10 October 2010 699

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Lancet Infect Dis 2010; 10: 699–711

This online publication has been corrected. The corrected version fi rst appeared at TheLancet.com/infection on October 25, 2010

Department of Pharmaceutical Technology and Biopharmacy (J-P Amorij PhD, W L J Hinrichs PhD, Prof H W Frijlink PhD) and Department of Medical Microbiology, Molecular Virology Section, University Medical Centre GrÖningen (Prof J C Wilschut PhD, A Huckriede PhD), University of GrÖningen, GrÖningen, Netherlands; and Research and Development Unit, Netherlands Vaccine Institute, Bilthoven, Netherlands (J-P Amorij)

Correspondence to:Dr Anke Huckriede, Department of Medical Microbiology, Molecular Virology Section, PO Box 30001, EB88 9700, RB, GrÖningen, [email protected]

Needle-free infl uenza vaccinationJean-Pierre Amorij, Wouter L J Hinrichs, Henderik W Frijlink, Jan C Wilschut, Anke Huckriede

Vaccination is the cornerstone of infl uenza control in epidemic and pandemic situations. Infl uenza vaccines are typically given by intramuscular injection. However, needle-free vaccinations could off er several distinct advantages over intramuscular injections: they are pain-free, easier to distribute, and easier to give to patients, and their use could reduce vaccination costs. Moreover, vaccine delivery via the respiratory tract, alimentary tract, or skin might elicit mucosal immune responses at the site of virus entry and better cellular immunity, thus improving eff ectiveness. Although various needle-free vaccination methods for infl uenza have shown preclinical promise, few have progressed to clinical trials—only live attenuated intranasal vaccines have received approval, and only in some countries. Further clinical investigation is needed to help realise the potential of needle-free vaccination for infl uenza.

Introduction Infl uenza is a major cause of morbidity, mortality, and economic loss that aff ects about 5–10% of the world’s population each year.1 Epidemic infl uenza strains can cause life-threatening disease in immunocompromised individuals. Pandemic infl uenza viruses have caused substantial morbidity and mortality worldwide, and are a constant public health threat. Vaccination is the main method of infl uenza prevention and is the most cost-eff ective measure for control of the disease. Vaccination against epidemic infl uenza strains, usually with split or subunit vaccine formulations, is recommended in the European Union for people with chronic diseases, people older than 65 years of age, immunocompromised patients, residents of nursing homes, and health-care workers.2 Vaccination is also the main method of prophylaxis to protect against pandemic infl uenza.3 Prepandemic mock-up vaccines based on infl uenza A H5N1 virus strains, formulated as adjuvanted split or whole inactivated virus (WIV) vaccines, received approval in Europe at the beginning of 2009.4 During the 2009 H1N1 infl uenza pandemic, these newly approved vaccine formulations and conventional H1N1 vaccines were used in accordance with national immunisation plans worldwide.

Conventional needle-based intramuscular infl uenza vaccines stimulate the production of serum antibodies, which by transudation to the lungs protect the lower respiratory tract against infl uenza infection.5 However, protection against initial infection of the upper respiratory tract is poor because of an absence of antibody induction in the nasal mucosa. Other disadvantages of needle-based intramuscular vaccines are safety problems (ie, the risk of needle stick injuries and the risk of infection by reuse of needles), low acceptance among patients with needle phobia, and logistic challenges in mass vaccination programmes. Several of these problems can be avoided with the use of needle-free vaccination systems.6–12 Systems in development can be classifi ed according to the route by which they are given, either via mucosal tissues (nasal, pulmonary, oral cavity, or gastrointestinal) or via the skin (intradermal or transcutaneous). Each of these routes has its own advantages and disadvantages related to ease of vaccination and the type of immune response elicited.

In this Review, we discuss the development of needle-free infl uenza vaccines and give an overview of approaches that have entered clinical assessment, such as live-attenuated infl uenza virus (LAIV), WIV, and split-virus and subunit vaccine formulations.

Development of non-parenteral infl uenza vaccines Need for improved vaccines WHO urges countries to introduce and broaden infl uenza vaccination programmes to achieve high vaccination coverage in at-risk populations.13 Major issues such as vaccine acceptance and cost have to be addressed before this can be achieved. Needle-free vaccination might reduce costs because, unlike needle-based vaccination, it does not require trained health-care personnel. Furthermore, people with a phobia of needles will be more likely to accept needle-free vaccination than they would injection-based vaccination. In a study by Sendi and colleagues,14 1552 (97%) of 1600 participants chose an intranasal vaccine spray when given the choice between the spray and an intramuscular injection. Vaccine acceptance would also benefi t from improved eff ectiveness. Intramuscular vaccines, which induce only systemic IgG responses, protect from severe complications of infl uenza but not necessarily from early disease symptoms. In addition to serum IgG, vaccines given via mucosal tissues can also induce local IgA responses in the upper respiratory tract, which can neutralise the virus at the point of entry. IgA is more cross-reactive than IgG and can provide some protection against drift and even shift virus variants.15–19

Needle-free vaccination, especially if there is no requirement for trained health-care personnel, would be ideal for mass vaccination campaigns. Logistic problems associated with supply and disposal of syringes and needles and safety risks associated with injections would be reduced.20 Compared with injection, most methods of non-parenteral vaccination could improve the speed of distribution.21 Vaccine logistics can be further simplifi ed by the use of dry vaccine formulations, which, when packaged, can be smaller and lighter than liquid formulations, and might not require a cold chain for storage and distribution.22

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Adjuvant system Vaccine type

Animal model

Immune response detected Protection (challenge) Clinically assessed

Nasal

Joseph47 Ceramide carbamoyl-spermine / cholesterol lipids Subunit Mice HI, IgG(2a), sIgA, interferon γ/proliferation

Homologous No

Ko48 α-galactosylceramide Subunit Mice IgG(2a), sIgA, interferon γ/proliferation

Homologous No

Youn49 α-galactosylceramide WIV Mice IgG(2a), sIgA, interferon γ, proliferation

Homologous No

Shim50 Poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP)

WIV Mice IgG, sIgA, interferon γ Homologous No

Guy51 Cationic cholesterol (DC-Chol) Split Mice HI, IgG(2a), sIgA ·· No

Nishino52 Surfacten (modifi ed bovine pulmonary surfactant) Split Minipigs HI, sIgA, cross-neutralising IgA ·· No

Bracci53,54 Type I interferon (interferon-α) Subunit Mice IgG(2a), sIgA Homologous Yes

Arulanandam55 Interleukin 12 Subunit Mice IgG(2a), sIgA, interferon γ Homologous No

Joseph56 Oligodeoxynucleotides-vaccine encapsulated in large multilamellar liposomes

Subunit Mice HI, IgG(2a), sIgA, proliferation Homologous NCT00197301

Ichinohe19,57 Poly I:Poly C12U, dsRNA WIV Mice IgG, sIgA, interferon γ, proliferation

Heterologous No

Song58 Mistletoe lectin C WIV Mice IgG(2a), sIgA, interferon γ, proliferation

HomologousHeterologous (cholera toxin-like)

No

Hasegawa18 Surf clam (microparticles) Split Mice IgG sIgA Cross-protection against variant infl uenza viruses.

No

Zanvit59 Delipidated bacterium Bacillus fi rmus WIV Mice IgG sIgA Homologous and heterologous No

Plante60 Proteosomes from Neisseria meningitidis Subunit Mice HI, IgG, sIgA, interferon γ Homologous No

Lovgren,61 Sjolander,62 Coulter,63 Helgeby,64 Hu65

ISCOMs saponin Quil-A+cholesterol Split Mice HI, IgG(2a), sIgA, CTL, interferon γ

Homologous No

Coulter,63 Scheerlinck66 ISCOMATRIX (empty ISCOM admixed with vaccine) Split MiceSheep

HI, sIgA ·· No

Read,67 Bacon,68 Amidi,69 Illum70

Chitosan or chitosan derivatives Subunit/split/WIV

Mice HI, IgG, sIgA, interferon γ Homologous Yes

Huang,71 Garmise72 Intranasal powder formulation co-formulated with chitosan

WIV Rats HI, IgG(2a), sIgA ·· No

Pulmonary

Smith73 1,2-dipalmitoylphosphatidyl-choline, distearoylphosphatidyl-choline and tyloxapol (spray-dried powder)

WIV or split

Rats HI, IgG, no sIgA ·· No

Sublingual

Song74 mCTA-cholera toxin B WIV Mice IgG(2a), sIgA, interferon γ, CD4 and CD8

Heterologous No

Oral

McCluskie75 Cpg, non-CpG oligodeoxynucleotides Subunit Mice IgG(2a)/sIgA ·· No

Katz76 LT Split Mice HI, IgG, sIgA, interferon γ, CTL Homologous No

Amorij34 LT, gastric vs colonically Subunit Mice HI, IgG(2a), sIgA, interferon γ Intracolonic: Th1-skewing Intragastric: increased T-helper responses; no Th1-skewing

·· No

Barackman,77 Lu78 LT derivatives Split Mice HI, IgG, sIgA, interferon γ ·· No

Conacher,79 Kunzi80 Deoxcycholate (in bilosomes) Subunit Mice/ferret HI, IgG2a, sIgA, interferon γ Homologous (ferret) No

Intradermal

Skountzou81 Cholera toxin transcutaneous immunisation WIV Mice IgG, sIgA, interferon γ Homologous No

Chen82,83 Cholera toxin B LT-mutants QS-21 adjuvant epidermal powder immunisation

Split Mice HI, IgG, sIgA, antibody affi nity Homologous No

Chen83 QS-21 adjuvant epidermal powder immunisation Split Macaques HI HomologousVariable virus titres No conclusion

No

WIV=whole inactivated virus. HI=haemagglutination inhibition. LT=heat-labile enterotoxin. CTL=cytotoxic T lymphocytes. ISCOM=immunostimulating complex.

Table 1: Preclinical assessments of adjuvants for needle-free infl uenza vaccination


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Advances in immunological knowledge The identifi cation of pathogen-associated molecular patterns that stimulate and shape the adaptive immune response via interaction with host pattern recognition receptors, has provided a rational basis for the development of new adjuvants.23,24 Moreover, the mechanisms by which traditional adjuvants work are being elucidated.25,26 Understanding of the initiation of adaptive immune responses at mucosal sites and in the skin, antigen processing and presentation, and B-cell activation has also improved.27–29 Topological issues in immunity have received attention and have enabled research into the role of the site of antigen delivery in eliciting an immune response.30–34 These general insights can now be implemented and can guide the development of new vaccines.

Technical progress Progress has been made in the development of nebulisers and inhalers for vaccination via the respiratory tract, and microneedle arrays, patches, and jet injectors for dermal vaccine delivery.35 Dry powder vaccine formulations have high stability and enable new or improved vaccine targeting strategies. The powders are usually produced by freeze-drying in the presence of stabilising sugars.22 Advanced particle engineering techniques allow the generation of powders with predefi ned physicochemical characteristics (eg, particle size, density, aerodynamic behaviour), which can be tailored to specifi c methods of vaccination. Moreover, special formulations, such as mucoadhesive compounds and gel-forming powders, can improve the local availability of the delivered vaccine and thereby vaccine effi cacy. Formulation of vaccine powder as coated tablets enables delivery of oral vaccination to desired sites in the gastrointestinal tract.36

Limited eff ectiveness A major drawback of needle-free vaccines is that most have low eff ectiveness. Dermal vaccination with doses similar to or even lower than those used in parenteral vaccination might be feasible with use of proper delivery methods or suitable adjuvants. However, mucosal vaccination, unless done with a live attenuated vaccine, requires large amounts of antigen and usually several doses to achieve the required haemagglutination inhibition titres in serum. For immunisation programmes against epidemic strains, and especially against pandemic strains, mucosal vaccination with inactivated vaccines is therefore impractical because of fi nancial and logistic reasons.

The absence of approved adjuvantsDevelopment of adjuvants for clinical use is a very slow process because of the low predictive value of data from studies in animals. In Europe, the only adjuvants licensed for use in clinical vaccines are aluminium hydroxide, AS04 (aluminium hydroxide with monophosphoryl-lipid A), the oil-in-water emulsions MF59 and AS03, and

infl uenza virosomes. In the USA, only aluminium hydroxide and AS04 have obtained approval.37 For intranasal vaccination these adjuvants seem to be ineff ective (alum, MF59, infl uenza virosomes)38–41 or have not been clinically tested (AS03, AS04). An Escherichia coli heat-labile enterotoxin formulated in a nasal infl uenza vaccine, is one of the few adjuvants that reached the market, but it had to be withdrawn because of risk of Bell’s palsy as an adverse outcome of vaccination.41–44 The clinical assessment of more suitable, detoxifi ed heat-labile enterotoxin variants is underway.45,46 Compared with intranasal vaccination, there has been little assessment of possible adjuvants for transcutaneous vaccination. Many adjuvants for needle-free infl uenza vaccination are in preclinical assessment (table 1).

The process of approving new adjuvants is slow, mainly because of regulatory concerns about safety and universality (ie, whether adjuvant activity and side-eff ects vary with the type of antigen used and the site of vaccine delivery).84 Consequently, the fi nal combination of adjuvant, antigen, and route of vaccination has to be licensed by a time-consuming, case-by-case process. This cautious approach is warranted because vaccines are given to a large and predominantly healthy population, and toxic adjuvants might put patients at risk.44,85,86 More information about the biological properties of formulations of antigens and adjuvants is urgently needed for successful development and faster licensing of new adjuvants, as are quality control tests, and in-vitro or in-vivo models that predict safety and effi cacy.

Restricted predictive value of studies in animals Development of eff ective and safe needle-free infl uenza vaccines, whether adjuvanted or not, requires the assessment of vaccine eff ects and protection in animals. The main species used for the development of infl uenza vaccines are mice, ferrets, and, to a lesser extent, macaques. Each of these animal species has advantages and disadvantages related to ease of handling, costs, and the availability of reagents for characterisation of their immune response.87,88 However, infl uenza infection, pathogenicity, and symptomatology in any of these animals only partly overlaps with that in human beings, which limits the predictive value of vaccination results from studies in animals. Nevertheless, results from studies in animals determine whether or not an approach will be tested in clinical trials.88 Ultimately, estimation of vaccine eff ectiveness in human beings is based on a weight-of-evidence approach87 that takes into account the evidence of protection in one or two animal species and compliance with relevant correlates of protection in the clinical situation.

Poorly defi ned correlates of protection Haemagglutination inhibition serum titres are currently the only validated correlate of protection for infl uenza vaccines. On the basis of a titre of 40, which is regarded


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as protective, the European Medicines Agency formulated criteria that infl uenza vaccines have to fulfi l for registration (table 2).89 Consequently, new infl uenza vaccines have to induce adequate haemagglutination inhibition serum titres even though other immune responses might be just as or even more relevant for protection from infection.

Secretory IgA, rather than nasal or serum IgG or IgM, is a major mediator of protection against nasal challenge with infl uenza virus.90–92 Accordingly, suffi cient protection might be achieved by mucosal IgA induced by oral, pulmonary, or nasal vaccination even in the absence of protective haemagglutination inhibition titres in serum. This issue needs to be urgently addressed in clinical trials.

Cellular immune responses can also contribute to protection although they cannot provide sterilising immunity.93 In elderly people, T-cell responses seem to better predict protection from disease than do antibody titres.94 Intranasal vaccination with LAIV and dermal vaccination with WIV induce T cells but are less eff ective than intramuscularly injected vaccines in eliciting haemagglutination inhibition titres.95,96 Before criteria for these new correlates of protection can be defi ned, standardised methods have to be developed to reliably sample mucosal tissue and measure T-cell and local antibody responses.97

Mucosal immunisation Because of their large size and immunological competence, mucosal tissues are attractive target sites for vaccination. Moreover, mucosal vaccination can elicit local immune responses, which can protect against infection at the point of virus entry. Because mucosal surfaces are generally exposed to many environmental antigens, tolerance mechanisms prevent an over-reaction of the immune system. For successful vaccination these tolerance mechanisms have to be overcome, which requires the use of strong mucosal adjuvants unless vaccination is done with live attenuated viruses.98

Intranasal antigen delivery So far, vaccination via the intranasal route, which targets the nasal-associated lymphoid tissue, is the only approach for mucosal vaccination that has been successfully used for commercial infl uenza vaccination. At present, all intranasal infl uenza vaccines on the market are LAIV vaccines. The attenuated viruses have six RNA segments from a cold-adapted virus strain that encode the viral polymerases, nucleoprotein, the matrix proteins, and

non-structural proteins, and two segments from circulating strains that encode haemagglutinin and neuraminidase.99,100 In Russia, LAIVs have been routinely used for many years. In 2003, an LAIV vaccine (FluMist) was approved in the USA for annual infl uenza vaccination of people aged 2–49 years. The vaccine is given as large droplet aerosol that deposits in the nasopharynx.99,100

By contrast with parenteral vaccines, which induce only systemic immune responses, intranasally delivered LAIV vaccines induce both systemic and broad mucosal immune responses.100 Although inactivated intramuscular vaccines elicit higher haemagglutination inhibition titres than do LAIV vaccines,101 they are both similarly eff ective in preventing infl uenza illness from homologous virus infections.90,102 LAIV vaccines can also provide immunity against heterologous virus strains, possibly mediated by mucosal IgA or cytotoxic T lymphocytes.100,103 LAIVs are very eff ective in priming immune responses in young children and adults.101,104 However, in elderly people, one intranasal LAIV vaccination is not more eff ective than an intramuscular vaccination.101

The use of inactivated vaccines for intranasal vaccination circumvents any safety concerns associated with LAIVs and has been explored in several clinical studies. Greenbaum and colleagues105 noted that nasal vaccination with 20 μg or 40 μg WIV resulted in a four-fold increase in haemagglutination inhibition titres in 30–40% of the individuals in the study. However, such large increases of haemagglutination inhibition titres were mostly recorded in individuals with low titres before vaccination. In 31–44% of people who received vaccines a local antibody response was recorded.105,106 Samdal and co-workers107 noted protective haemagglutination inhibition titres in 80% of volunteers after four intranasal doses of WIV (total dose 84 μg of haemagglutinin). In elderly people, two nasal vaccinations with WIV resulted in an increase in mucosal antibody titres by up to three times, but only small serum response rates.108 When compared with WIV vaccines, subunit and split infl uenza vaccines seem to be poorly immunogenic in clinical trials and will require the use of special delivery systems or mucosal adjuvants to be eff ective.40,109 Accordingly, recent studies have focused on adjuvanted or specially formulated vaccines.

The most potent mucosal adjuvants are bacterial enterotoxins such as E coli heat-labile enterotoxin and cholera toxin. Use of these adjuvants in human beings is, however, hampered by their toxic properties. Detoxifi ed variants of cholera toxin and heat-labile enterotoxin retain their adjuvant properties and improve the immunogenicity of infl uenza vaccine when given intranasally to animals.45,110–112 Stephenson and colleagues46 showed induction of mucosal immune responses but not serum IgG titres in human beings by use of a new nasal infl uenza vaccine formulation that consisted of a subunit vaccine, a detoxifi ed heat-labile enterotoxin, and a nano-sized biovector (a positively charged polysaccharide core enclosed by a phospholipid-cholesterol double layer). In view of the

Age 18–60 years old Age >60 years old

Seroconversion (HI titre <40 to ≥40 or four-fold rise) >40% >30%

Mean fold increase in geometric mean HI titre >2·5 >2

Seroprotection (HI titre ≥40) >70% >60%

HI=haemagglutination inhibition.

Table 2: European Medicines Agency criteria for infl uenza vaccines


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adverse outcomes seen with vaccines adjuvanted with the wild-type E coli heat-labile enterotoxin, vaccines containing endotoxin adjuvants might not receive approval easily.

Several experimental adjuvants for intranasal infl uenza vaccination have been assessed in clinical trials. MF59 is inactive as an intranasal adjuvant in human beings, despite its strong adjuvant activity when injected intramuscularly and its mucosal adjuvant function in mice.40,113 Two doses of lipid or polysaccharide carrier molecules with split infl uenza vaccine stimulated only slight serum antibody responses but substantial increases in nasal antibody responses in healthy adults.114 Two nasal vaccinations that combined chitosan with inactivated infl uenza vaccine induced suffi cient seroprotection and seroconversion rates, but low haemagglutination inhibition titres.67,70 Chitosan-like molecules have mucoadhesive properties, which increase the time that the vaccine antigen remains in the patient’s nose and improves mucosal barrier penetration.115 Johnson and colleagues116 have investigated intranasal vaccination with type 1 interferon in combination with WIV in a clinical trial, but antibody titres in sera and nose swabs were lower than or similar to those achieved by intranasal vaccination with non-adjuvanted WIV. Various other approaches for nasal vaccination against infl uenza have been assessed in preclinical experiments (table 1).

In addition to liquid formulations, powders have also been developed for nasal delivery of infl uenza vaccines and have shown promising results in animals.71,117–120 Some powder formulations remain in the nasal cavity for longer periods than do liquid formulations, which might translate into higher bioavailability and immune responses.121,122 However, clinical assessment of intranasal infl uenza vaccine powders has not started.

Because there is a non-ciliated area in the anterior part of the nasal cavity and a ciliated area in the posterior parts, the site of vaccine deposition is important when considering mucociliary clearance of vaccine from the nose.31 The site is determined by factors associated with the delivery device and formulation, such as the velocity at which an aerosol is delivered and the size of the droplets or particles in the formulation (fi gure 1).32 For aerosols or particles larger than 50 μm, intranasal delivery is highly reproducible and independent of the vaccine recipient’s control of breathing, because the site of deposition is governed by inertial impaction (heavy aerosol particles collide with the nasal mucosa rather than follow the streamline direction of the inhaled air).123 A range of devices has been developed for intranasal delivery.71,117,124

Pulmonary antigen delivery Lungs are highly vascularised, have a large absorptive surface area because of the alveoli structure,116,125 and contain bronchoalveolar lymphoid tissue.126 Furthermore, local antigen-presenting cells, such as diff erent types of macrophages and dendritic cells, are ideally located for antigen sampling and subsequent presentation to

T cells.127–129 Additionally, pulmonary vaccination has the advantage of inducing both systemic and local immunity (IgA and IgG) in the respiratory tract.130

Pulmonary delivery of infl uenza vaccine suspensions was investigated in the 1960s and 70s in several clinical studies. Kasel and colleagues131–133 assessed aerosol immunisation with a classic inactivated infl uenza vaccine. Vaccine droplets ranging in size from 1 μm to 100 μm were sprayed into patients’ posterior oropharynx during rapid, deep inhalation and then sprayed into their noses. After two immunisations, substantial levels of cross-reactive mucosal antibodies and satisfactory protection were achieved.131,133–136 In several studies136–138 the protective effi cacy of inhaled infl uenza vaccines was similar to that of an intradermal vaccine. Moreover, even in a season when the vaccine strain did not match with the circulating infl uenza strain, pulmonary immunisation resulted in a protection rate of 47% (compared with 60% protection against infection with the homologous strain observed in a previous season).137 Despite these successful clinical trials, no pulmonary infl uenza vaccines are commercially available, possibly because of the absence of eff ective inhaler systems and insuffi cient data on the safety of pulmonary antigen delivery.

More recently, the pulmonary route for vaccination against infl uenza has been explored again in various preclinical studies. These studies show that pulmonary immunisation is more eff ective than intranasal

Figure 1: Intranasal antigen deliveryDevice for intranasal delivery of powder (A). The vaccine is held in a capsule sealed with rupturable fi lm. Particle size distribution of freeze-dried whole inactivated virus for intranasal vaccination (B). Adapted from reference 71 with permission from Elsevier.

HousingSyringe Rupture film

Rupture film Capsule

A

B

1 10 100 10000

1

2

3

4

5

6

7

8

9

10

Freq

uenc

y (%

)

Particle size (μm)


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immunisation and can be done with a small amount of antigen in combination with a strong adjuvant, especially when targeted to the deep lungs.30,73,139

Stable dry-powder vaccine formulations for pulmonary delivery of infl uenza vaccine might have several advantages. Dry-powder inhalation is more reproducible and effi cient than is vaccination with liquid aerosols.35 Furthermore, in a dry-powder vaccine, particle properties can be controlled to ensure optimum inhalation and accurate targeting to the desired lung areas. Recent studies in animals, involving, among other vaccines, an inulin-stabilised, freeze-dried infl uenza subunit vaccine, showed promising results.73,140 No data are available on the effi cacy of infl uenza vaccine powder inhalation in human beings.

Inhalers that can guarantee substantial and reproducible deep lung deposition of vaccine will defi nitely contribute to the development of pulmonary vaccination. Available inhalation devices for liquid vaccines include nebulisers, medical metered-dose inhalers, and soft-mist inhalers. Of these, soft-mist inhalers might be the most suitable because they produce low-velocity aerosols thus preventing the high shear forces characteristic of nebulisers, and do not require the propellants needed for metered-dose inhalers that might damage vaccines.35,141 However, the denaturing eff ects of aerosol generation on the vaccine compound, in particular the eff ect of large air-liquid interfaces, should be assessed before clinical trials are done. Dry-powder inhalers combine the advantages of stable vaccine formulations with simple and fast delivery and, potentially, high lung deposition. A prototype dry-powder inhaler is the single-use disposable inhaler Twincer (University of Groningen, Netherlands; fi gure 2), an inexpensive device with good

dispersion of powder doses and good moisture protection of the powder formulation.142

The main safety concern for pulmonary infl uenza vaccination is exacerbation of respiratory diseases, such as chronic obstructive pulmonary disease, allergic asthma, or pneumonia.143 Meyer and colleagues144 showed that pulmonary vaccination with pneumococcal polysaccharide is safe in patients with chronic obstructive pulmonary disease and that it induces rapid serum antibody responses. Minne and co-workers145 showed that in asthmatic (ovalbumin-sensitised) mice, pulmonary vaccination with seasonal infl uenza vaccine does not lead to asthma exacerbation. However, more progress has to be made in the safety assessment of pulmonary vaccination.

Antigen delivery to the oral cavity Delivery to the oral cavity via the sublingual, buccal, or gingival route provides excellent accessibility and avoids the potential degradation of vaccine compounds that occurs with delivery via the gastrointestinal tract.146 The oral cavity contains intraepithelial and submucosal immune cells and lymphoid tissues (palatine, lingual tonsils, and adenoids), which, like the nasopharyngeal tonsils, are part of Waldeyer’s ring.147 However, the immunological processes within the oral cavity and, in particular, the importance of the tonsils in antibody induction are poorly understood.148–150

Only one clinical study has investigated oral cavity immunisation against infl uenza. In this phase 1–2 trial, healthy adult volunteers were immunised four times at 1-week intervals with WIV (360 μg hemagglutinin in total), by spraying 100 μL vaccine suspension into their mouths.151 No substantial increase in salivary or nasal IgA antibodies was recorded, but serum haemagglutination inhibition antibodies were induced in 56 of 75 volunteers after only two doses. Whether this response was because of antigen deposited in the oral cavity or by inhaled antigen needs to be investigated.

Sublingual antigen delivery has received much attention for use in allergen-specifi c immunotherapy.152 Clinical trials with sublingual infl uenza vaccines have not been done. In mice, sublingual vaccination with live infl uenza virus and adjuvanted WIV was shown to be safe and to induce substantial immune responses.74,153

Gastrointestinal antigen delivery Oral vaccinations are ideal for immunisation programmes because they are easy to give and well accepted by most of the population. Orally delivered vaccines target the gut-associated lymphoid tissue in the gastrointestinal tract, which consists of Peyer’s patches, the appendix, solitary lymphoid nodules, and isolated lymphoid follicles. Few vaccines, all of which are live attenuated vaccines, are given orally and oral vaccination with inactivated vaccines seems to be very diffi cult because of vaccine instability in the acidic and proteinase-rich environment of the gastrointestinal tract.7

Figure 2: The Twincer (University of GrÖningen, Netherlands),A disposable dry powder inhaler that consists of three specifi cally formed plastic plates. A blister containing the vaccine powder is located between the plates and can be opened by removal of a pull-off strip.


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In a study by Avtushenko and co-workers,154 volunteers who received either one or two oral vaccinations with split infl uenza vaccine (140 μg haemagglutinin), which was formulated as an emulsion, only sporadically developed serum antibodies. After one vaccination, levels of secretory IgA in nasal secretions and saliva were increased. However, after two vaccinations this response was no longer seen, possibly because of oral tolerance caused by repeated ingestion of substantial doses of antigen. The researchers concluded that the dose of antigen needs to be optimised to ensure a suffi cient immune response. Other clinical studies used high doses of antigen to ensure suffi cient absorption to M cells.154–156 However, none of these vaccinations resulted in detectable IgG responses, whereas IgA titres in both saliva and nasal lavage fl uids were substantially increased. Whether these IgA antibodies alone could provide adequate protection against infl uenza infection is not known. Current preclinical research on liquid oral infl uenza vaccination mainly focuses on the use of adjuvants and complex vaccine formulations to achieve satisfactory serum antibody titres.

Moldoveanu and colleagues155 vaccinated healthy adults orally with split infl uenza vaccine air-dried with D-xylose and formulated as enteric-coated gelatin capsules (ten doses, 150 μg haemagglutinin per strain). Serum antibodies of all isotypes were only slightly increased at 21 days after ingestion of the last vaccine dose. However, in saliva and nasal lavage, antigen-specifi c IgA and IgG responses were detected. Similarly, oral vaccination of volunteers with enteric-coated alum-absorbed WIV (six doses, 24 μg haemagglutinin in total) did not induce serum antibody titres, but resulted in a slow but substantial rise of IgA-specifi c antibodies in tears, saliva, and nasal secretions.157

Data from studies in animals indicate that the site of vaccine release within the alimentary tract might be of major importance and imply that the colon could be preferable to the upper gastrointestinal tract for vaccination.33,34 However, no clinical studies have been done to investigate this issue. To reliably target specifi c sites in the gastrointestinal tract, tablets or capsules need to be developed that contain dried vaccine and have special coatings that allow antigen release at the desired location of the gastrointestinal tract.36,158

Despite many years of research into oral infl uenza vaccination there has not been convincing data. More information is needed about the mechanisms of oral tolerance and how they can be overcome, and also on the immunological properties of diff erent parts of the alimentary tract. With this information, new adjuvants and vaccine formulations with targeted antigen release can be developed that might eventually enable infl uenza vaccination via the oral route. Alternatively, live vaccine vectors, either bacterial or viral, that encode relevant infl uenza proteins might contribute to a successful oral vaccination strategy.

Immunisation via the skin The skin is an easily accessible and highly immuno-competent organ and therefore very attractive for immunisations.7 The skin is divided into the stratum corneum, which consists of cornifi ed keratinocytes, the epidermis, which has live keratinocytes and a large number of antigen-presenting Langerhans cells, and the dermis, which also contains antigen-presenting cells and supplies the epidermis through, among other routes, blood vessels and the lymphatics.159,160 The intact stratum corneum is an eff ective barrier that prevents penetration of foreign molecules larger than 500 Da. However, once the stratum corneum has been penetrated, antigens as large as 1×10⁶ Da are capable of reaching antigen presenting cells in the epidermis and dermis.161 Accordingly, research eff orts have focused on the development of devices suitable for delivery of vaccines through the stratum corneum.

Delivery of liquid vaccine formulations Traditional needles have been used for intradermal delivery of various vaccines in several clinical trials.21,80,162–172 In general, these trials show that a low-dose intradermal vaccine (two-thirds to one-fi fth of the normal dose) is as eff ective as the conventional full dose intramuscular vaccine in terms of seroconversion, fold increase in geometric mean haemagglutination inhibition titre and seroprotection. The few studies that compare intradermal with intramuscular injection at the same dose do not suggest that intradermal is any more effi cient (in terms of dose-sparing) than intramuscular.

Irrespective of dose-sparing potential, the technique for intradermal delivery with traditional needles is technically demanding and is therefore not ideal for routine vaccinations.173 Intradermal vaccination can be done with specially designed needles that allow a controlled depth of skin penetration174 and that are suitable for intradermal vaccination with infl uenza vaccines.175 One such device, the BD Soluvia (BD Medical Pharmaceutical Systems, Franklin Lakes, NJ, USA), proved especially promising in phase 2 and phase 3 clinical trials.165,176,177 In Europe, an intradermal infl uenza vaccine with this device has been approved by the European Medicines Agency.178

Another approach for intradermal vaccine delivery makes use of arrays of pointed microneedles (25–50 μm) that can penetrate the stratum corneum. These arrays are designed to target Langerhans cells in the epidermis, but do not reach the nerves in the underlying tissue and therefore elicit little sensation and no pain.179,180 Because of their ease of use, microneedle arrays can be given by people with little medical training.8 In a recent clinical trial, individuals who received doses of 3 μg or 6 μg haemagglutinin per infl uenza strain via a four-needle array, the MicronJet device (NanoPass Technologies Ltd, Rehovot, Israel), developed similar haemagglutinin inhibition titres as did those who received 15 μg by conventional intramuscular injection.181


Review

Jet injectors are an alternative option to deliver vaccine to epidermal tissue (and also to deeper subcutaneous or intramuscular tissue). This method is especially suitable for mass vaccination.182,183 Older, multiuse nozzle jet injectors were associated with rare inadvertent transmission of blood-borne infections. However, new generation jet injectors, like Biojector 2000 (Bioject Medical Technologies, Tualatin, OR, USA), Pharmajet (Pharmajet, Golden, CO, USA), and Injex (Equidyne Systems, Fullerton, CA, USA) use disposable cartridges and thus avoid the risk of cross-contamination. Immunisation with these devices has the same effi cacy as the traditional intramuscularly injected infl uenza vaccine.184

Topical immunisation via the skin can also be done by use of patches.11,185,186 The stratum corneum can be penetrated by means of hydration, mechanical disruption, electroporation, or the use of carrier systems; each of these techniques can be used alone or in combination.8 Hydration results in swelling of keratinocytes and pooling of fl uid in the intracellular spaces, which allows antigens to pass the stratum corneum more easily.187 In a small phase 1 clinical trial, skin surface stripping before

patch-assisted immunisation with a standard dose of infl uenza subunit vaccine induced not only CD4 but also CD8 cellular responses. However, neutralising antibody titres were recorded in only two of seven vaccine recipients.188 Further study is being done in animals to develop more eff ective immunisation patches.189

Delivery of dry vaccine formulations A jet injector for powders, the PowderJect (PowderJect Pharmaceuticals, Oxford, UK; fi gure 3), accelerates powder particles to such a speed that they can perforate the stratum corneum and reach the epidermis.190,191 The physical properties of the powder, especially the size and density of the particles, are of high importance to ensure delivery to the epidermis. Production of vaccine powders with the right physical properties is challenging.22 In a phase 1 trial, a powdered trivalent subunit infl uenza vaccine (15 μg per strain) was safe and elicited similar rates of seroconversion (75–100%) and geometric mean titres as standard intramuscular vaccines.192 However, clinical trials on a larger scale are needed to prove the eff ectiveness and safety of dry vaccine formulations. In animals, epidermal powder immunisations elicit mucosal immune responses;83,193 their potential to do so in human beings should be assessed. Studies in animals show that the inclusion of adjuvants might improve the effi cacy of vaccination with PowderJect.82,83

Microprojection arrays have also been developed for delivery of solid antigens, including infl uenza WIV.194–196 A simple controllable method can be used to coat microneedles197 with infl uenza vaccines for delivery into the skin (fi gure 4). Kim and co-workers196 showed that microneedles coated with trehalose-stabilised WIV induced longer immunological memory in mice than did conventional vaccination.196 Also, dissolvable microneedles that consist of a dried hydrogel are a safe and painless alternative to hypodermic needle injection for proteins and vaccines.198

Conclusion Infl uenza vaccines that can be given via the respiratory or the alimentary tract, or that can be delivered through the skin without use of needles, are alternatives to traditional, intramuscularly injected vaccines. Mucosal vaccine delivery can induce local immune responses at the point of virus entry, but, if not done with live attenuated virus, requires large amounts of antigen and several vaccinations. Immunisation via the skin might have the potential to induce robust systemic immune responses with just one low dose of infl uenza vaccine. For both mucosal and transcutaneous delivery, the use of dry vaccine powders seems especially promising.

So far, the only commercially available needle-free infl uenza vaccines are intranasal vaccines with live attenuated virus. For other needle-free vaccination strategies, additional clinical research is needed before any decision can be made on their usefulness in human

Figure 3: PowderJect ND5.2, a prototype single-use delivery device for epidermal powder immunisationVaccine particles entrapped between polycarbonate membranes are propelled via the nozzle by release of helium from a microcylinder. Adapted from reference 192 with permission from Elsevier.

Helium microcylinder

Actuation button

Vaccine cassetteSilencer

Nozzle

Figure 4: Microneedle array uniformly coated with a model drug (ribofl avin)Imaging by brightfi eld microscopy shows coating of microneedle shafts of an array of 50 microneedles (A) and a single microneedle (B). Adapted from reference 197 with permission from Springer.

A

500 μm

B

100 μm


Review

beings. Re-evaluation of old clinical studies, the use of smart delivery technologies, site-specifi c vaccine delivery, introduction of new criteria for vaccine immunogenicity, and the design of more eff ective vaccine formulations together with new, safe adjuvants will further aid the development of needle-free infl uenza vaccines.

Contributors JP-A had the idea for the review and prepared the tables. J-PA and AH

did the data search, determined the set-up, and wrote the paper. WLJH,

HWF, JCW revised the paper. All authors participated in data analysis

and interpretation.

Confl icts of interest JCW is a member of the Dutch Health council (Nederlandse

Gezondheidsraad). All other authors declare that they have no confl icts

of interest.

References1 WHO. WHO Media Infl uenza Factsheet N°211 (2003). http://www.

who.int/mediacentre/factsheets/2003/fs211/en/(accessed July 1, 2010).

2 The European agency for the evaluation of medicinal products (EMEA): committee for proprietary medicinal products (CPMP). Guideline on dossier structure and content for pandemic infl uenza vaccine marketing authorisation application (2004). http://archives.who.int/prioritymeds/report/append/62EMEAguidelines.pdf (accessed July 1, 2010).

3 The European Agency for the Evaluation of Medicinal Products (EMEA). http://www.ema.europa.eu/docs/en_GB/document_library/Scientifi c_guideline/2009/09/WC500003869.pdf (accessed July 1, 2010).

4 The European Agency for the Evaluation of Medicinal Products (EMEA). http://www.ema.europa.eu/ema/index.jsp?curl=pages/special_topics/general/general_content_000267.jsp&murl=menus/special_topics/special_topics.jsp&mid=WC0b01ac058004b634 (accessed July 1, 2010).

5 Wilschut J, McElhaney JE, Palache AM. Rapid Reference Infl uenza: 2nd edn. London: Mosby/Elsevier Science, 2006.

6 Kersten G, Hirschberg H. Needle-free vaccine delivery. Expert Opin Drug Deliv 2007; 4: 459–74.

7 Mitragotri S. Immunization without needles. Nat Rev Immunol 2005; 5: 905–16.

8 Giudice EL, Campbell JD. Needle-free vaccine delivery. Adv Drug Deliv Rev 2006; 58: 68–89.

9 Azad N, Rojanasakul Y. Vaccine delivery—current trends and future. Curr Drug Deliv 2006; 3: 137–46.

10 O’Hagan DT, Rappuoli R. Novel approaches to vaccine delivery. Pharm Res 2004; 21: 1519–30.

11 Kersten G, Hirschberg H. Antigen delivery systems. Expert Rev Vaccines 2004; 3: 453–62.

12 Nugent J, Po AL, Scott EM. Design and delivery of non-parenteral vaccines. J Clin Pharm Ther 1998; 23: 257–85.

13 WHO. WHO position paper (macroepidemiology). Wkly Epidemiol Rec 2005; 80: 279–87.

Search strategy and selection criteria

We searched Medline, Current Contents, and Scopus with the search terms “vaccine”, “infl uenza”, “delivery or administration”, “needle-free”, “non-parenteral”, “nasal”, “oral”, “rectal”, “pulmonary”, “sublingual”, “buccal”, “mucosal”, “dermal”, “intradermal”, “cutaneous”, and “transcutaneous”. From the retrieved papers we selected those describing alternative routes for infl uenza vaccine delivery. Preference was given to clinical studies. Studies on alternative routes of infl uenza vaccination based on recombinant proteins, recombinant viruses, or plasmid DNA were excluded.

14 Sendi P, Locher R, Bucheli B, Battegay M. Intranasal infl uenza vaccine in a working population. Clin Infect Dis 2004; 38: 974–80.

15 Liew FY, Russell SM, Appleyard G, Brand CM, Beale J. Cross-protection in mice infected with infl uenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur J Immunol 1984; 14: 350–56.

16 Tumpey TM, Renshaw M, Clements JD, Katz JM. Mucosal delivery of inactivated infl uenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal infl uenza A H5N1 virus infection. J Virol 2001; 75: 5141–50.

17 Asahi-Ozaki Y, Yoshikawa T, Iwakura Y, et al. Secretory IgA antibodies provide cross-protection against infection with diff erent strains of infl uenza B virus. J Med Virol 2004; 74: 328–35.

18 Hasegawa H, Ichinohe T, Strong P, et al. Protection against infl uenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant. J Med Virol 2005; 75: 130–36.

19 Ichinohe T, Tamura S, Kawaguchi A, et al. Cross-protection against H5N1 infl uenza virus infection is aff orded by intranasal inoculation with seasonal trivalent inactivated infl uenza vaccine. J Infect Dis 2007; 196: 1313–20.

20 Ekwueme DU, Weniger BG, Chen RT. Model-based estimates of risks of disease transmission and economic costs of seven injection devices in sub-Saharan Africa. Bull World Health Organ 2002; 80: 859–70.

21 Sugimura T, Ito Y, Tananari Y, et al. Improved antibody responses in infants less than 1 year old using intradermal infl uenza vaccination. Vaccine 2008; 26: 2700–05.

22 Amorij JP, Huckriede A, Wilschut J, Frijlink HW, Hinrichs WL. Development of stable infl uenza vaccine powder formulations: challenges and possibilities. Pharm Res 2008; 25: 1256–73.

23 van Vliet SJ, den Dunnen J, Gringhuis SI, Geijtenbeek TB, van Kooyk Y. Innate signaling and regulation of dendritic cell immunity. Curr Opin Immunol 2007; 19: 435–40.

24 Geeraedts F, Goutagny N, Hornung V, et al. Superior immunogenicity of inactivated whole virus H5N1 infl uenza vaccine is primarily controlled by Toll-like receptor signalling. PLoS Pathog 2008; 4: e1000138.

25 Mosca F, Tritto E, Muzzi A, et al. Molecular and cellular signatures of human vaccine adjuvants. Proc Natl Acad Sci USA 2008; 105: 10501–06.

26 Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 2009; 9: 287–93.

27 Jensen PE. Recent advances in antigen processing and presentation. Nat Immunol 2007; 8: 1041–48.

28 Harwood NE, Batista FD. New insights into the early molecular events underlying B cell activation. Immunity 2008; 28: 609–19.

29 Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 2006; 6: 148–58.

30 Minne A, Louahed J, Mehauden S, Baras B, Renauld JC, Vanbever R. The delivery site of a monovalent infl uenza vaccine within the respiratory tract impacts on the immune response. Immunology 2007; 122: 316–25.

31 Tafaghodi M, Abolghasem Sajadi Tabassi S, Jaafari MR, Zakavi SR, Momen-Nejad M. Evaluation of the clearance characteristics of various microspheres in the human nose by gamma-scintigraphy. Int J Pharm 2004; 280: 125–35.

32 Vidgren MT, Kublik H. Nasal delivery systems and their eff ect on deposition and absorption. Adv Drug Deliv Rev 1998; 29: 157–77.

33 Meitin CA, Bender BS, Small PA Jr. Enteric immunization of mice against infl uenza with recombinant vaccinia. Proc Natl Acad Sci USA 1994; 91: 11187–91.

34 Amorij JP, Westra TA, Hinrichs WL, Huckriede A, Frijlink HW. Towards an oral infl uenza vaccine: Comparison between intragastric and intracolonic delivery of infl uenza subunit vaccine in a murine model. Vaccine 2007; 26: 67–76.

35 Frijlink HW, De Boer AH. Trends in the technology-driven development of new inhalation devices. Drug Discovery Today: Technologies 2005; 2: 47–57.

36 Schellekens RC, Stellaard F, Mitrovic D, Stuurman FE, Kosterink JG, Frijlink HW. Pulsatile drug delivery to ileo-colonic segments by structured incorporation of disintegrants in pH-responsive polymer coatings. J Control Release 2008; 132: 91–98.


Review

37 Glenn GM, O’Hagan DT. Adjuvants: progress, regress and pandemic preparedness. Expert Rev Vaccines 2007; 6: 651–52.

38 Smith DJ, King WF, Barnes LA, Trantolo D, Wise DL, Taubman MA. Facilitated intranasal induction of mucosal and systemic immunity to mutans streptococcal glucosyltransferase peptide vaccines. Infect Immun 2001; 69: 4767–73.

39 Oien NL, Brideau RJ, Walsh EE, Wathen MW. Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit. Vaccine 1994; 12: 731–35.

40 Boyce TG, Hsu HH, Sannella EC, et al. Safety and immunogenicity of adjuvanted and unadjuvanted subunit infl uenza vaccines administered intranasally to healthy adults. Vaccine 2000; 19: 217–26.

41 Gluck U, Gebbers JO, Gluck R. Phase 1 evaluation of intranasal virosomal infl uenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers. J Virol 1999; 73: 7780–86.

42 Glueck R. Pre-clinical and clinical investigation of the safety of a novel adjuvant for intranasal immunization. Vaccine 2001; 20 (suppl 1): S42–44.

43 Huckriede A, Bungener L, Stegmann T, et al. The virosome concept for infl uenza vaccines. Vaccine 2005; 23 (suppl 1): S26–38.

44 Mutsch M, Zhou W, Rhodes P, et al. Use of the inactivated intranasal infl uenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 2004; 350: 896–903.

45 Pine S, Barackman J, Ott G, O’Hagan D. Intranasal immunization with infl uenza vaccine and a detoxifi ed mutant of heat labile enterotoxin from Escherichia coli (LTK63). J Control Release 2002; 85: 263–70.

46 Stephenson I, Zambon MC, Rudin A, et al. Phase I evaluation of intranasal trivalent inactivated infl uenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers. J Virol 2006; 80: 4962–70.

47 Joseph A, Itskovitz-Cooper N, Samira S, et al. A new intranasal infl uenza vaccine based on a novel polycationic lipid—ceramide carbamoyl-spermine (CCS) I: immunogenicity and effi cacy studies in mice. Vaccine 2006; 24: 3990–4006.

48 Ko SY, Ko HJ, Chang WS, Park SH, Kweon MN, Kang CY. alpha-Galactosylceramide can act as a nasal vaccine adjuvant inducing protective immune responses against viral infection and tumor. J Immunol 2005; 175: 3309–17.

49 Youn HJ, Ko SY, Lee KA, et al. A single intranasal immunization with inactivated infl uenza virus and alpha-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system. Vaccine 2007; 25: 5189–98.

50 Shim DH, Ko HJ, Volker G, et al. Effi cacy of poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) as mucosal adjuvant to induce protective immunity against respiratory pathogens. Vaccine; 28: 2311–17.

51 Guy B, Pascal N, Francon A, et al. Design, characterization and preclinical effi cacy of a cationic lipid adjuvant for infl uenza split vaccine. Vaccine 2001; 19: 1794–805.

52 Nishino M, Mizuno D, Kimoto T, et al. Infl uenza vaccine with Surfacten, a modifi ed pulmonary surfactant, induces systemic and mucosal immune responses without side eff ects in minipigs. Vaccine 2009; 27: 5620–27.

53 Bracci L, Canini I, Puzelli S, et al. Type I IFN is a powerful mucosal adjuvant for a selective intranasal vaccination against infl uenza virus in mice and aff ects antigen capture at mucosal level. Vaccine 2005; 23: 2994–3004.

54 Bracci L, Canini I, Venditti M, et al. Type I IFN as a vaccine adjuvant for both systemic and mucosal vaccination against infl uenza virus. Vaccine 2006; 24 (suppl 2): S2 56–57.

55 Arulanandam BP, O’Toole M, Metzger DW. Intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity. J Infect Dis 1999; 180: 940–49.

56 Joseph A, Louria-Hayon I, Plis-Finarov A, et al. Liposomal immunostimulatory DNA sequence (ISS-ODN): an effi cient parenteral and mucosal adjuvant for infl uenza and hepatitis B vaccines. Vaccine 2002; 20: 3342–54.

57 Ichinohe T, Kawaguchi A, Tamura S, et al. Intranasal immunization with H5N1 vaccine plus poly I: poly C12U, a toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect 2007; 9: 1333–40.

58 Song SK, Moldoveanu Z, Nguyen HH, et al. Intranasal immunization with infl uenza virus and Korean mistletoe lectin C (KML-C) induces heterosubtypic immunity in mice. Vaccine 2007; 25: 6359–66.

59 Zanvit P, Havlickova M, Tacner J, et al. Protective and cross-protective mucosal immunization of mice by infl uenza virus type A with bacterial adjuvant. Immunol Lett 2008; 115: 144–52.

60 Plante M, Jones T, Allard F, et al. Nasal immunization with subunit proteosome infl uenza vaccines induces serum HAI, mucosal IgA and protection against infl uenza challenge. Vaccine 2001; 20: 218–25.

61 Lovgren K, Kaberg H, Morein B. An experimental infl uenza subunit vaccine (iscom): induction of protective immunity to challenge infection in mice after intranasal or subcutaneous administration. Clin Exp Immunol 1990; 82: 435–39.

62 Sjolander S, Drane D, Davis R, Beezum L, Pearse M, Cox J. Intranasal immunisation with infl uenza-ISCOM induces strong mucosal as well as systemic antibody and cytotoxic T-lymphocyte responses. Vaccine 2001; 19: 4072–80.

63 Coulter A, Harris R, Davis R, et al. Intranasal vaccination with ISCOMATRIX adjuvanted infl uenza vaccine. Vaccine 2003; 21: 946–49.

64 Helgeby A, Robson NC, Donachie AM, et al. The combined CTA1-DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specifi c targeting of B cells. J Immunol 2006; 176: 3697–706.

65 Hu KF, Lovgren-Bengtsson K, Morein B. Immunostimulating complexes (ISCOMs) for nasal vaccination. Adv Drug Deliv Rev 2001; 51: 149–59.

66 Scheerlinck JP, Gekas S, Yen HH, et al. Local immune responses following nasal delivery of an adjuvanted infl uenza vaccine. Vaccine 2006; 24: 3929–36.

67 Read RC, Naylor SC, Potter CW, et al. Eff ective nasal infl uenza vaccine delivery using chitosan. Vaccine 2005; 23: 4367–74.

68 Bacon A, Makin J, Sizer PJ, et al. Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens. Infect Immun 2000; 68: 5764–70.

69 Amidi M, Romeijn SG, Verhoef JC, et al. N-Trimethyl chitosan (TMC) nanoparticles loaded with infl uenza subunit antigen for intranasal vaccination: Biological properties and immunogenicity in a mouse model. Vaccine 2007; 25: 144–153.

70 Illum L, Jabbal-Gill I, Hinchcliff e M, Fisher AN, Davis SS. Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev 2001; 51: 81–96.

71 Huang J, Garmise RJ, Crowder TM, et al. A novel dry powder infl uenza vaccine and intranasal delivery technology: induction of systemic and mucosal immune responses in rats. Vaccine 2004; 23: 794–801.

72 Garmise RJ, Staats HF, Hickey AJ. Novel dry powder preparations of whole inactivated infl uenza virus for nasal vaccination. AAPS PharmSciTech 2007; 8: e81.

73 Smith DJ, Bot S, Dellamary L, Bot A. Evaluation of novel aerosol formulations designed for mucosal vaccination against infl uenza virus. Vaccine 2003; 21: 2805–12.

74 Song JH, Nguyen HH, Cuburu N, et al. Sublingual vaccination with infl uenza virus protects mice against lethal viral infection. Proc Natl Acad Sci USA 2008; 105: 1644–49.

75 McCluskie MJ, Davis HL. Oral, intrarectal and intranasal immunizations using CpG and non-CpG oligodeoxynucleotides as adjuvants. Vaccine 2000; 19: 413–22.

76 Katz JM, Lu X, Young SA, Galphin JC. Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated infl uenza virus vaccine. J Infect Dis 1997; 175: 352–63.

77 Barackman JD, Ott G, Pine S, O’Hagan DT. Oral administration of infl uenza vaccine in combination with the adjuvants LT-K63 and LT-R72 induces potent immune responses comparable to or stronger than traditional intramuscular immunization. Clin Diagn Lab Immunol 2001; 8: 652–57.

78 Lu X, Clements JD, Katz JM. Mutant Escherichia coli heat-labile enterotoxin [LT(R192G)] enhances protective humoral and cellular immune responses to orally administered inactivated infl uenza vaccine. Vaccine 2002; 20: 1019–29.

79 Conacher M, Alexander J, Brewer JM. Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine 2001; 19: 2965–74.


Review

80 Kunzi V, Klap JM, Seiberling MK, et al. Immunogenicity and safety of low dose virosomal adjuvanted infl uenza vaccine administered intradermally compared to intramuscular full dose administration. Vaccine 2009; 27: 3561–67.

81 Skountzou I, Quan FS, Jacob J, Compans RW, Kang SM. Transcutaneous immunization with inactivated infl uenza virus induces protective immune responses. Vaccine 2006; 24: 6110–19.

82 Chen D, Endres RL, Erickson CA, Maa YF, Payne LG. Epidermal powder immunization using non-toxic bacterial enterotoxin adjuvants with infl uenza vaccine augments protective immunity. Vaccine 2002; 20: 2671–79.

83 Chen D, Endres R, Maa YF, et al. Epidermal powder immunization of mice and monkeys with an infl uenza vaccine. Vaccine 2003; 21: 2830–36.

84 Sesardic D, Rijpkema S, Patel BP. New adjuvants: EU regulatory developments. Expert Rev Vaccines 2007; 6: 849–61.

85 Kanerva M, Mannonen L, Piiparinen H, Peltomaa M, Vaheri A, Pitkaranta A. Search for Herpesviruses in cerebrospinal fl uid of facial palsy patients by PCR. Acta Otolaryngol 2007; 127: 775–79.

86 van Ginkel FW, Jackson RJ, Yoshino N, et al. Enterotoxin-based mucosal adjuvants alter antigen traffi cking and induce infl ammatory responses in the nasal tract. Infect Immun 2005; 73: 6892–902.

87 van der Laan JW, Herberts C, Lambkin-Williams R, Boyers A, Mann AJ, Oxford J. Animal models in infl uenza vaccine testing. Expert Rev Vaccines 2008; 7: 783–93.

88 Bodewes R, Rimmelzwaan GF, Osterhaus AD. Animal models for the preclinical evaluation of candidate infl uenza vaccines. Expert Rev Vaccines 2010; 9: 59–72.

89 The European agency for the evaluation of medicinal products (EMEA) committee for proprietary medicinal products (CPMP). Note for guidance on harmonization of requirements for infl uenza vaccines (1997). http://www.ema.europa.eu/docs/en_GB/document_library/Scientifi c_guideline/2009/09/WC500003945.pdf (accessed July 1, 2010).

90 Clements ML, Betts RF, Tierney EL, Murphy BR. Serum and nasal wash antibodies associated with resistance to experimental challenge with infl uenza A wild-type virus. J Clin Microbiol 1986; 24: 157–60.

91 Renegar KB, Small PA Jr. Immunoglobulin A mediation of murine nasal anti-infl uenza virus immunity. J Virol 1991; 65: 2146–48.

92 Mizuno D, Ide-Kurihara M, Ichinomiya T, Kubo I, Kido H. Modifi ed pulmonary surfactant is a potent adjuvant that stimulates the mucosal IgA production in response to the infl uenza virus antigen. J Immunol 2006; 176: 1122–30.

93 Rimmelzwaan GF, Fouchier RA, Osterhaus AD. Infl uenza virus-specifi c cytotoxic T lymphocytes: a correlate of protection and a basis for vaccine development. Curr Opin Biotechnol 2007; 18: 529–36.

94 McElhaney JE, Xie D, Hager WD, et al. T cell responses are better correlates of vaccine protection in the elderly. J Immunol 2006; 176: 6333–39.

95 He XS, Holmes TH, Zhang C, et al. Cellular immune responses in children and adults receiving inactivated or live attenuated infl uenza vaccines. J Virol 2006; 80: 11756–66.

96 Chen D, Weis KF, Chu Q, et al. Epidermal powder immunization induces both cytotoxic T-lymphocyte and antibody responses to protein antigens of infl uenza and hepatitis B viruses. J Virol 2001; 75: 11630–40.

97 Treanor J, Wright PF. Immune correlates of protection against infl uenza in the human challenge model. Dev Biol (Basel) 2003; 115: 97–104.

98 Wilschut J, de Jonge J, Huckriede A, Amorij JP, Hinrichs WL, Frijlink HW. Preservation of infl uenza virosome structure and function during freeze-drying and storage. J Liposome Res 2007; 17: 173–82.

99 Abramson JS. Intranasal, cold-adapted, live, attenuated infl uenza vaccine. Pediatr Infect Dis J 1999; 18: 1103–04.

100 Belshe R, Lee MS, Walker RE, Stoddard J, Mendelman PM. Safety, immunogenicity and effi cacy of intranasal, live attenuated infl uenza vaccine. Expert Rev Vaccines 2004; 3: 643–54.

101 Cox RJ, Brokstad KA, Ogra P. Infl uenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated infl uenza vaccines. Scand J Immunol 2004; 59: 1–15.

102 Beyer WE, Palache AM, de Jong JC, Osterhaus AD. Cold-adapted live infl uenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine effi cacy. A meta-analysis. Vaccine 2002; 20: 1340–53.

103 Glezen WP. Control of infl uenza. Tex Heart Inst J 2004; 31: 39–41.

104 Fleming DM, Crovari P, Wahn U, et al. Comparison of the effi cacy and safety of live attenuated cold-adapted infl uenza vaccine, trivalent, with trivalent inactivated infl uenza virus vaccine in children and adolescents with asthma. Pediatr Infect Dis J 2006; 25: 860–69.

105 Greenbaum E, Furst A, Kiderman A, et al. Mucosal [SIgA] and serum [IgG] immunologic responses in the community after a single intra-nasal immunization with a new inactivated trivalent infl uenza vaccine. Vaccine 2002; 20: 1232–39.

106 Greenbaum E, Engelhard D, Levy R, Schlezinger M, Morag A, Zakay-Rones Z. Mucosal (SIgA) and serum (IgG) immunologic responses in young adults following intranasal administration of one or two doses of inactivated, trivalent anti-infl uenza vaccine. Vaccine 2004; 22: 2566–77.

107 Samdal HH, Bakke H, Oftung F, et al. A non-living nasal infl uenza vaccine can induce major humoral and cellular immune responses in humans without the need for adjuvants. Hum Vaccin 2005; 1: 85–90.

108 Muszkat M, Greenbaum E, Ben-Yehuda A, et al. Local and systemic immune response in nursing-home elderly following intranasal or intramuscular immunization with inactivated infl uenza vaccine. Vaccine 2003; 21: 1180–86.

109 Atmar RL, Keitel WA, Cate TR, Munoz FM, Ruben F, Couch RB. A dose-response evaluation of inactivated infl uenza vaccine given intranasally and intramuscularly to healthy young adults. Vaccine 2007; 25: 5367–73.

110 Haan L, Verweij WR, Holtrop M, et al. Nasal or intramuscular immunization of mice with infl uenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity. Vaccine 2001; 19: 2898–907.

111 McCluskie MJ, Weeratna RD, Clements JD, Davis HL. Mucosal immunization of mice using CpG DNA and/or mutants of the heat-labile enterotoxin of Escherichia coli as adjuvants. Vaccine 2001; 19: 3759–68.

112 Hagiwar Y, Tsuji T, Iwasaki T, et al. Eff ectiveness and safety of mutant Escherichia coli heat-labile enterotoxin (LT H44A) as an adjuvant for nasal infl uenza vaccine. Vaccine 2001; 19: 2071–79.

113 Barchfeld GL, Hessler AL, Chen M, Pizza M, Rappuoli R, Van Nest GA. The adjuvants MF59 and LT-K63 enhance the mucosal and systemic immunogenicity of subunit infl uenza vaccine administered intranasally in mice. Vaccine 1999; 17: 695–704.

114 Halperin SA, Smith B, Clarke K, Treanor J, Mabrouk T, Germain M. Phase I, randomized, controlled trial to study the reactogenicity and immunogenicity of a nasal, inactivated trivalent infl uenza virus vaccine in healthy adults. Hum Vaccin 2005; 1: 37–42.

115 Ugwoke MI, Agu RU, Verbeke N, Kinget R. Nasal mucoadhesive drug delivery: background, applications, trends and future perspectives. Adv Drug Deliv Rev 2005; 57: 1640–65.

116 Robert Johnson. Interferon as a mucosal adjuvant for infl uenza vaccine given intranasally (NCT00436046). http://clinicaltrials.gov/ct2/show/study/NCT00436046?sect=X501 (accessed July 1, 2010).

117 Sullivan VJ, Mikszta JA, Laurent P, Huang J, Ford B. Noninvasive delivery technologies: respiratory delivery of vaccines. Expert Opin Drug Deliv 2006; 3: 87–95.

118 Jiang G, Joshi SB, Peek LJ, et al. Anthrax vaccine powder formulations for nasal mucosal delivery. J Pharm Sci 2006; 95: 80–96.

119 Huo Z, Sinha R, McNeela EA, et al. Induction of protective serum meningococcal bactericidal and diphtheria-neutralizing antibodies and mucosal immunoglobulin A in volunteers by nasal insuffl ations of the Neisseria meningitidis serogroup C polysaccharide-CRM197 conjugate vaccine mixed with chitosan. Infect Immun 2005; 73: 8256–65.

120 Garmise RJ, Mar K, Crowder TM, et al. Formulation of a dry powder infl uenza vaccine for nasal delivery. AAPS PharmSciTech 2006; 7: e19.

121 Soane RJ, Frier M, Perkins AC, Jones NS, Davis SS, Illum L. Evaluation of the clearance characteristics of bioadhesive systems in humans. Int J Pharm 1999; 178: 55–65.


Review

122 McInnes FJ, Thapa P, Baillie AJ, et al. In vivo evaluation of nicotine lyophilised nasal insert in sheep. Int J Pharm 2005; 304: 72–82.

123 Newman SP, Pitcairn GR, Dalby RN. Drug delivery to the nasal cavity: in vitro and in vivo assessment. Crit Rev Ther Drug Carrier Syst 2004; 21: 21–66.

124 Laube BL. Devices for aerosol delivery to treat sinusitis. J Aerosol Med 2007; 20 (suppl 1): s5–17.

125 Groneberg DA, Paul H, Welte T. Novel strategies of aerosolic pharmacotherapy. Exp Toxicol Pathol 2006; 57 (suppl 2): 49–53.

126 Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005; 11 (suppl 4): s45–53.

127 Lambrecht BN, Prins JB, Hoogsteden HC. Lung dendritic cells and host immunity to infection. Eur Respir J 2001; 18: 692–704.

128 von Garnier C, Filgueira L, Wikstrom M, et al. Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J Immunol 2005; 175: 1609–18.

129 Holt PG. Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract. Proc Am Thorac Soc 2005; 2: 116–20.

130 Holmgren J, Czerkinsky C, Lycke N, Svennerholm AM. Mucosal immunity: implications for vaccine development. Immunobiology 1992; 184: 157–79.

131 Mann JJ, Waldman RH, Togo Y, Heiner GG, Dawkins AT, Kasel JA. Antibody response in respiratory secretions of volunteers given live and dead infl uenza virus. J Immunol 1968; 100: 726–35.

132 Kasel JA, Fulk RV, Togo Y, et al. Infl uenza antibody in human respiratory secretions after subcutaneous or respiratory immunization with inactivated virus. Nature 1968; 218: 594–95.

133 Waldman RH, Mann JJ, Kasel JA. Infl uenza virus neutralizing antibody in human respiratory secretions. J Immunol 1968; 100: 80–85.

134 Waldman RH, Mann JJ, Small PA Jr. Immunization against infl uenza: prevention of illness in man by aerosolized inactivated vaccine. JAMA 1969; 207: 520–24.

135 Waldman RH, Jurgensen PF, Olsen GN, Ganguly R, Johnson JE 3rd. Immune response of the human respiratory tract—I: Immunoglobulin levels and infl uenza virus vaccine antibody response. J Immunol 1973; 111: 38–41.

136 Waldman RH, Wigley FMSPA Jr. Specifi city of respiratory secretion antibody against infl uenza virus. J Immunol 1970; 105: 1477–83.

137 Haigh W, Howell RW. The effi cacy of the A2/Aichi/68 strain in inhaled infl uenza immunisation against the A/England/42/72 variant. J Soc Occup Med 1973; 23: 125–7.

138 Haigh W, Howell RW, Meichen FW. A comparative trial of infl uenza immunization by inhalation and hypojet methods. Practitioner 1973; 211: 365–70.

139 Wee JL, Scheerlinck JP, Snibson KJ, et al. Pulmonary delivery of ISCOMATRIX infl uenza vaccine induces both systemic and mucosal immunity with antigen dose sparing. Mucosal Immunol 2008; 1: 489–96.

140 Amorij JP, Saluja V, Petersen AH, Hinrichs WL, Huckriede A, Frijlink HW. Pulmonary delivery of an inulin-stabilized infl uenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice. Vaccine 2007; 25: 8707–17.

141 Dalby R, Spallek M, Voshaar T. A review of the development of respimat soft mist inhaler. Int J Pharm 2004; 283: 1–9.

142 de Boer AH, Hagedoorn P, Westerman EM, Le Brun PP, Heijerman HG, Frijlink HW. Design and in vitro performance testing of multiple air classifi er technology in a new disposable inhaler concept (Twincer) for high powder doses. Eur J Pharm Sci 2006; 28: 171–78.

143 Lu D, Hickey AJ. Pulmonary vaccine delivery. Expert Rev Vaccines 2007; 6: 213–26.

144 Meyer P, Menzel M, Muellinger B, Weber N, Haeussinger K, Ziegler-Heitbrock L. Inhalative vaccination with pneumococcal polysaccharide in patients with chronic obstructive pulmonary disease. Vaccine 2006; 24: 5832–38.

145 Minne A, Huaux F, Jaworska J, Rha RD, Hamelmann E, Vanbever R. Safety evaluation of pulmonary infl uenza vaccination in healthy and asthmatic mice. Vaccine 2008; 26: 2360–68.

146 Senel S, Kremer M, Nagy K, Squier C. Delivery of bioactive peptides and proteins across oral (buccal) mucosa. Curr Pharm Biotechnol 2001; 2: 175–86.

147 Challacombe S, Coogan M, Williams D, Greenspan J. Overview and research agenda arising from the 5th World Workshop on Oral Health and Disease in AIDS. Adv Dent Res 2006; 19: 5–9.

148 Otten K, Wang HC, Wyde PR, Klein JR. Modulation of gamma delta T cells in mouse buccal epithelium following antigen priming. Biochem Biophys Res Commun 2002; 294: 626–29.

149 Gebert A, Pabst R. M cells at locations outside the gut. Semin Immunol 1999; 11: 165–70.

150 Childers NK, Powell WD, Tong G, Kirk K, Wiatrak B, Michalek SM. Human salivary immunoglobulin and antigen-specifi c antibody activity after tonsillectomy. Oral Microbiol Immunol 2001; 16: 265–69.

151 Bakke H, Samdal HH, Holst J, et al. Oral spray immunization may be an alternative to intranasal vaccine delivery to induce systemic antibodies but not nasal mucosal or cellular immunity. Scand J Immunol 2006; 63: 223–31.

152 Canonica GW, Passalacqua G. Noninjection routes for immunotherapy. J Allergy Clin Immunol 2003; 111: 437–48.

153 Cuburu N, Kweon MN, Song JH, et al. Sublingual immunization induces broad-based systemic and mucosal immune responses in mice. Vaccine 2007; 25: 8598–610.

154 Avtushenko SS, Sorokin EM, Zoschenkova NY, Zacharova NG, Naichin AN. Clinical and immunological characteristics of the emulsion form of inactivated infl uenza vaccine delivered by oral immunization. J Biotechnol 1996; 44: 21–28.

155 Moldoveanu Z, Clements ML, Prince SJ, Murphy BR, Mestecky J. Human immune responses to infl uenza virus vaccines administered by systemic or mucosal routes. Vaccine 1995; 13: 1006–12.

156 Lazzell V, Waldman RH, Rose C, Khakoo R, Jacknowitz A, Howard S. Immunization against infl uenza in humans using an oral enteric-coated killed virus vaccine. J Biol Stand 1984; 12: 315–21.

157 Bergmann KC, Waldman RH, Tischner H, Pohl WD. Antibody in tears, saliva and nasal secretions following oral immunization of humans with inactivated infl uenza virus vaccine. Int Arch Allergy Appl Immunol 1986; 80: 107–09.

158 Gazzaniga A, Maroni A, Sangalli ME, Zema L. Time-controlled oral delivery systems for colon targeting. Expert Opin Drug Deliv 2006; 3: 583–97.

159 Huang CM. Topical vaccination: the skin as a unique portal to adaptive immune responses. Semin Immunopathol 2007; 29: 71–80.

160 Yu RC, Abrams DC, Alaibac M, Chu AC. Morphological and quantitative analyses of normal epidermal Langerhans cells using confocal scanning laser microscopy. Br J Dermatol 1994; 131: 843–48.

161 Glenn GM, Kenney RT. Mass vaccination: solutions in the skin. Curr Top Microbiol Immunol 2006; 304: 247–68.

162 Chiu SS, Peiris JS, Chan KH, Wong WH, Lau YL. Immunogenicity and safety of intradermal infl uenza immunization at a reduced dose in healthy children. Pediatrics 2007; 119: 1076–82.

163 Belshe RB, Newman FK, Cannon J, et al. Serum antibody responses after intradermal vaccination against infl uenza. N Engl J Med 2004; 351: 2286–94.

164 Kenney RT, Frech SA, Muenz LR, Villar CP, Glenn GM. Dose sparing with intradermal injection of infl uenza vaccine. N Engl J Med 2004; 351: 2295–301.

165 Holland D, Booy R, De Looze F, et al. Intradermal infl uenza vaccine administered using a new microinjection system produces superior immunogenicity in elderly adults: a randomized controlled trial. J Infect Dis 2008; 198: 650–58.

166 Chi RC, Rock MT, Neuzil KM. Immunogenicity and safety of intradermal infl uenza vaccination in healthy older adults. Clin Infect Dis 2010; 50: 1331–38.

167 Nicholson KG, Thompson CI, Klap JM, et al. Safety and immunogenicity of whole-virus, alum-adjuvanted whole-virus, virosomal, and whole-virus intradermal infl uenza A/H9N2 vaccine formulations. Vaccine 2009; 28: 171–78.

168 Chiu SS, Chan KH, Tu W, Lau YL, Peiris JS. Immunogenicity and safety of intradermal versus intramuscular route of infl uenza immunization in infants less than 6 of age: a randomized controlled trial. Vaccine 2009; 27: 4834–39.


Review

169 Gelinck LB, van den Bemt BJ, Marijt WA, et al. Intradermal infl uenza vaccination in immunocompromized patients is immunogenic and feasible. Vaccine 2009; 27: 2469–74.

170 Jo YM, Song JY, Hwang IS, et al. Dose sparing strategy with intradermal infl uenza vaccination in patients with solid cancer. J Med Virol 2009; 81: 722–27.

171 Belshe RB, Newman FK, Wilkins K, et al. Comparative immunogenicity of trivalent infl uenza vaccine administered by intradermal or intramuscular route in healthy adults. Vaccine 2007; 25: 6755–63.

172 Auewarakul P, Kositanont U, Sornsathapornkul P, Tothong P, Kanyok R, Thongcharoen P. Antibody responses after dose-sparing intradermal infl uenza vaccination. Vaccine 2007; 25: 659–63.

173 Hunsaker BD, Perino LJ. Effi cacy of intradermal vaccination. Vet Immunol Immunopathol 2001; 79: 1–13.

174 Vandervoort J, Ludwig A. Microneedles for transdermal drug delivery: a minireview. Front Biosci 2008; 13: 1711–15.

175 Alarcon JB, Hartley AW, Harvey NG, Mikszta JA. Preclinical evaluation of microneedle technology for intradermal delivery of infl uenza vaccines. Clin Vaccine Immunol 2007; 14: 375–81.

176 Arnou R, Eavis P, Pardo JR, Ambrozaitis A, Kazek MP, Weber F. Immunogenicity, large scale safety and lot consistency of an intradermal infl uenza vaccine in adults aged 18-60 years: Randomized, controlled, phase III trial. Hum Vaccin 2010; 6: 346–54.

177 Arnou R, Icardi G, De Decker M, et al. Intradermal infl uenza vaccine for older adults: a randomized controlled multicenter phase III study. Vaccine 2009; 27: 7304–12.

178 The European Agency for the Evaluation of Medicinal Products (EMEA). http://www.ema.europa.eu/humandocs/Humans/EPAR/intanza/intanza.htm, 2010 (accessed July 1, 2010).

179 McAllister DV, Wang PM, Davis SP, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc Natl Acad Sci USA 2003; 100: 13755–60.

180 Mikszta JA, Alarcon JB, Brittingham JM, Sutter DE, Pettis RJ, Harvey NG. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery. Nat Med 2002; 8: 415–19.

181 Van Damme P, Oosterhuis-Kafeja F, Van der Wielen M, Almagor Y, Sharon O, Levin Y. Safety and effi cacy of a novel microneedle device for dose sparing intradermal infl uenza vaccination in healthy adults. Vaccine 2009; 27: 454–59.

182 Baxter J, Mitragotri S. Needle-free liquid jet injections: mechanisms and applications. Expert Rev Med Devices 2006; 3: 565–74.

183 Mitragotri S. Current status and future prospects of needle-free liquid jet injectors. Nat Rev Drug Discov 2006; 5: 543–48.

184 Jackson LA, Austin G, Chen RT, et al. Safety and immunogenicity of varying dosages of trivalent inactivated infl uenza vaccine administered by needle-free jet injectors. Vaccine 2001; 19: 4703–09.

185 Glenn GM, Flyer DC, Ellingsworth LR, et al. Transcutaneous immunization with heat-labile enterotoxin: development of a needle-free vaccine patch. Expert Rev Vaccines 2007; 6: 809–19.

186 Mahor S, Gupta PN, Rawat A, Vyas SP. A needle-free approach for topical immunization: antigen delivery via vesicular carrier system(s). Curr Med Chem 2007; 14: 2898–910.

187 Glenn GM, Kenney RT, Hammond SA, Ellingsworth LR. Transcutaneous immunization and immunostimulant strategies. Immunol Allergy Clin North Am 2003; 23: 787–813.

188 Vogt A, Mahe B, Costagliola D, et al. Transcutaneous anti-infl uenza vaccination promotes both CD4 and CD8 T cell immune responses in humans. J Immunol 2008; 180: 1482–89.

189 Garg S, Hoelscher M, Belser JA, et al. Needle-free skin patch delivery of a pandemic infl uenza vaccine protects mice from lethal viral challenge. Clin Vaccine Immunol 2007; 14: 926–28

190 Sarphie DF, Johnson B, Cormier M, Burkoth TL, Bellhouse BL. Bioavailabillity following transdermal powdered delivery (TDP) of radiolabeled insulin to hairless guinea pigs. J Control Release 1997; 47: 61–69.

191 Chen D, Endres RL, Erickson CA, et al. Epidermal immunization by a needle-free powder delivery technology: immunogenicity of infl uenza vaccine and protection in mice. Nat Med 2000; 6: 1187–90.

192 Dean HJ, Chen D. Epidermal powder immunization against infl uenza. Vaccine 2004; 23: 681–86.

193 Chen D, Periwal SB, Larrivee K, et al. Serum and mucosal immune responses to an inactivated infl uenza virus vaccine induced by epidermal powder immunization. J Virol 2001; 75: 7956–65.

194 Matriano JA, Cormier M, Johnson J, et al. Macrofl ux microprojection array patch technology: a new and effi cient approach for intracutaneous immunization. Pharm Res 2002; 19: 63–70.

195 Koutsonanos DG, del Pilar Martin M, Zarnitsyn VG, et al. Transdermal infl uenza immunization with vaccine-coated microneedle arrays. PLoS ONE 2009; 4: e4773.

196 Kim YC, Quan FS, Yoo DG, Compans RW, Kang SM, Prausnitz MR. Enhanced memory responses to seasonal H1N1 infl uenza vaccination of the skin with the use of vaccine-coated microneedles. J Infect Dis 2010; 201: 190–98.

197 Gill HS, Prausnitz MR. Coating formulations for microneedles. Pharm Res 2007; 24: 1369–80.

198 Lee JW, Park JH, Prausnitz MR. Dissolving microneedles for transdermal drug delivery. Biomaterials 2008; 29: 2113–24.

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