key: cord-0046193-vik7hq2m
authors: Weniger, Bruce G.; Papania, Mark J.
title: Alternative vaccine delivery methods
date: 2020-06-22
journal: Vaccines
DOI: 10.1016/b978-1-4160-3611-1.50065-9
sha: ce1f8f6dcb35bdfc0e713889aee08bf65a638bfa
doc_id: 46193
cord_uid: vik7hq2m

nan

The earliest known route of vaccination was intranasal, by insuffl ation of scab material containing variola virus from smallpox patients, described in China around the fi rst millennium AD (see Chapters 1 [history] and 30 [smallpox]). 1 The cutaneous route for such variolation involved breaking the skin with a sharp instrument and was used in India perhaps as early as in China, but not documented until the 16th century. 2 Variolation was supplanted by safer cutaneous vaccination using material from cowpox lesions, a method known in the 18th century and fi rst published by Edward Jenner.

After 15th century experiments with hypodermic injection, 3 the introduction of the needle and syringe (N-S) in the mid 19th century by Pravaz, 4,5 Rynd 6 and Wood, 7 began a new era in medicine. Pasteur used a Pravaz syringe to inoculate sheep in the famed controlled challenge experiment demonstrating anthrax 'vaccination,' a term henceforth broadened to the administration of immunizing agents for various diseases, not just smallpox. 8 Upon acceptance of the germ theory and resulting sterilization by the early 20th century, 9 and with mass production of needles and glass (later plastic) syringes by mid century, hypodermic injection became the norm for convenient, accurate, and certain administration of most vaccines and many drugs. Regrettably, aseptic practice was ignored in many developing countries, 10 and among non-medical intravenous drug users everywhere, 11 leading to recognition of widespread iatrogenic and self-infl icted disease transmission during that era recently decried as the 'Injection Century.' 12 Other drawbacks of N-S include needlestick injuries to health care workers, 13, 14 needle-phobia and discomfort for patients facing increasingly crowded immunization schedules, 15, 16 and the costs and complexity of safe disposal of sharps in the medical waste stream. 17 In the early 21st century, preparedness efforts for threatened pandemics and bioterrorism, as well as new targets for disease control or eradication have rekindled an earlier interest in mass vaccination campaigns, 18 and stimulated research on vaccine delivery not requiring N-S. [19] [20] [21] [22] [23] [24] Existing and potential alternatives to conventional intramuscular (IM) and subcutaneous (SC) vaccination by N-S are classifi ed here into three major categories: cutaneous, jet injection and respiratory. The cutaneous route may be subdivided into intradermal (ID) via conventional needle; passive diffusion with or without chemical enhancers or adjuvants, and disruption or penetration of the stratum corneum by mechanical contact, heat, electricity, or light. Jet injection involves pressurizing liquid into high-velocity streams. Respiratory vaccination delivers airborne particles via the nose or mouth for deposition onto the mucosal surfaces of the upper or lower airways. between them, correlates with smaller molecules (<500 Da), lower melting points, increased lipophilicity (and correspondingly lower water solubility), higher (saturated) concentrations, and the paucity of pendant groups that form hydrogen bonds that slow diffusion. 22, 27 The specifi c mechanisms which produce the resulting immune response when vaccine antigen is introduced into the skin are not entirely clear. Upon stimulation, keratinocytes can produce pro-infl ammatory cytokines (interleukin 1) and can themselves function as antigen-presenting cells by displaying major histocompatibility complex (MHC) class II antigens (HLA-DR), as well as intercellular adhesion molecules (ICAM-1). 28 Epidermal Langerhans cells are believed to play a key role in cutaneous immunization, although other well-known immune system players also circulate or reside in the epidermis or dermis, such as CD8 + and CD4 + T lymphocytes, mast cells, macrophages, and dermal dendritic cells. [29] [30] [31] [32] The immature Langerhans cells reside like sentinels among the keratinocytes in the epidermis, comprising about a quarter of the skin surface area, 33 where they effi ciently capture foreign antigen by phagocytosis or endocytosis. As with similar dendritic cells in other tissues (see Chapter 5 [immunologic adjuvants]), upon activation ( Fig. 61-1 ) these professional antigen-presenting cells (APC) process the antigen as they migrate to draining lymph nodes. There, now mature, they express high levels of class II MHC molecules, and present the antigen brought from the skin to T helper (Th) lymphocytes, a critical step for the subsequent immune responses orchestrated by the latter cells.

During the more than 200 years of cutaneous vaccination against smallpox (see Chapter 30 [smallpox]), a variety of sharp instruments have been used to cut, scratch, poke and otherwise penetrate into the epidermis (and unnecessarily deeper into the dermis), for inoculation of cowpox or vaccinia virus (see Fig.  61 -2). 1 In the 18th and 19th centuries, the scarifi cation method involved scratching one or more lines into the skin with a needle, scalpel (lancet), or knife and rubbing vaccine into the resulting lesion. A rotary lancet fi rst described in the 1870s consisted of a shaft attached to the center of a small disk, the opposite 'patient's side' of which contained a central tine surrounded by multiple smaller tines. The twirling of the disk in a drop of vaccine on the skin produced much abrasion of the skin and often severe reactions from both vaccine and common bac-terial contaminants. In the less traumatic multiple pressure method introduced in the early 1900s, liquid vaccine was placed onto the skin and a straight surgical needle, held tangentially to the skin with its tip in the drop, was repeatedly and fi rmly pressed sideways into the limb 10 times for primary vaccination and 30 for revaccination. 34 Multi-tines devices have also been used. 35, 36 Tuberculosis vaccination

The Bacille Calmette-Guérin (BCG) vaccine for the prevention of disease from Mycobacterium tuberculosis was originally administered orally in the 1920s (see Chapter 33 [tuberculosis]). Safety concerns prompted a shift to cutaneous administration by ID needle injection (1927), 37 and later multiple puncture (1939), 38-41 scarifi cation (1947) , and multi-tine devices, 36 as described above for smallpox vaccine. BCG has also been delivered cutaneously by bifurcated needles 42 and jet injectors. 43

The ID needle technique used for BCG was originally developed by Felix Mendel 44 and Charles Mantoux 45 in the early 20th century for the administration of tuberculin (now replaced by purifi ed protein derivative) for the diagnosis of tuberculosis infection. It is now called the Mantoux method. This procedure has become the common route for ID injection of various antigens ( Fig. 61-2E) . A short-bevel, fi ne-gauge needle, usually 27 gauge (0.016 inch, 0.406 mm diameter), is inserted, bevel up, almost parallel at a 5-15 degree angle into slightly-stretched skin, often the volar surface of the forearm. 46 The tip is advanced about 3 mm until the entire bevel is covered. Upon injection of fl uid, proper location of the bevel in the dermis creates a bleb or wheal as the basement membrane and epidermis above are stretched by the fl uid. Leakage onto the skin indicates insufficient penetration to cover the bevel. Failure to produce a bleb indicates improperly deep location of the fl uid in the subcutaneous tissue. Drawbacks to the Mantoux method for mass vaccination campaigns are the training, skill, and extra time needed to accomplish it correctly.

The potential dose-sparing effect of ID vaccination, reducing needed antigen by up to 80 percent in reducing dose volume to 0.1 mL from the common 0.5 mL, has prompted renewed attention to this route because of concern for emerging threats like pandemic infl uenza, SARS, and bioterrorism that may leave populations vulnerable due to insuffi cient vaccine supply. Both old and new techniques can more easily achieve the effect of the Mantoux method in depositing the injectate into the skin to produce a visible wheal of temporary induration. Since the 1960s, multi-use-nozzle jet injectors (discussed in more detail below) delivered smallpox, BCG, and other vaccines ID by use of specialized nozzles (Fig. 61-2G ). 47-49 Modern disposable-cartridge injectors are being adapted with spacers to achieve that same route (Fig. 61-2H) . [50] [51] [52] Requiring less skill than the Mantoux method, a new investigational ID syringe with a 30-gauge needle (outer diameter [OD] ∼0.305 mm) that projects only 1.5 mm beyond its depth-limiting hub is inserted perpendicularly to deposit the dose into the skin (Fig. 61-2F) . 53,53a A 34-gauge equivalent (OD ∼0.178 mm) for animal models produced good immune responses to recombinant protective antigen (rPA) for anthrax, 54,54a conventional hemagglutinin (HA) and plasmid DNA antigens for infl uenza, 55 and live recombinant yellow fever vector for Japanese encephalitis vaccines. 56 ID-immunized rabbits challenged with ∼100 LD50 of Bacillus anthracis spores had identical survival rates (no adjuvant: 100%, aluminum salt adjuvant [alum]: 100%, CpG: 83%) as IM-immunized controls. 54 In clinical trials of conventional infl uenza HA antigen, the 30gauge ID syringe proved feasible and immunogenic. 57

In addition to smallpox and BCG, mentioned above, as well as combined BCG-smallpox vaccine, 58,59 over a dozen other vaccine types have been administered ID.

There is a substantial literature, since the 1930s, starting with Thomas Francis (of Salk polio vaccine trial fame), 60 documenting the equivalence and occasionally improved immunogenicity of ID infl uenza vaccination by needle-syringe compared to larger doses by the SC and IM routes. 57,61-79 On the other hand, a few studies found ID infl uenza responses less then IM or SC on some or all of the antigens that were studied. [80] [81] [82] [83] [84] [85] When identical amounts of reduced antigen were compared between the ID and IM or SC routes, there were confl icting results from mid-century trials using the whole-cell products of that era. Bruyn et al found GMTs in children receiving 0.2 mL intradermally of infl uenza vaccine to be higher than those receiving the same dose SC, 64 as did Davies et al 86 (Bioject, Inc. 50 ) used for subcutaneous injections; spacer creates a 2 cm air gap to weaken stream, leaving injectate in the skin. Items A, B, C, D, and G were used for smallpox vaccination; D is currently recommended.

by ID one-tenth (0.1 mL) the SC dose (1.0 mL) in varying dilutions below the labelled dosage of 800 chick cell agglutinating (CCA) units per mL, Stille et al also found greater ID responses, but only when the SC dose was low, at 8 or 0.08 CCA (ID dose: 0.8 and 0.008, respectively). 70 Conversely, SC responses exceeded ID ones when the standard SC dose was used or reduced by only one log (80 CCA, ID: 80 and 8 CC, respectively). This suggested a linear ID dose-response curve, but a sigmoid SC one, which favored the ID route at the lower-dose end. On the other hand, when identical reduced doses for a new shifted 'Asian' strain were given by the two routes (80, 40, or 20 CCA, compared to 200 per full 1.0 mL), both McCarroll et al, 87 studying hospital employees 18 to 65 years of age, and Klein et al, 88 studying infants 2 months to 5 years of age, found little difference in responses between the ID and SC routes. McCarroll speculated the ID superiority in earlier studies was due to an anamnestic effect not present that season. Klein simply doubted any ID superiority when equal volumes are used.

Regarding systemic reactions, among 101 infants from 2 months to 2 years of age receiving 0.1 mL of infl uenza vaccine in the Klein et al study, febrile reactions were reported among 34.7% (17/49) in the intradermal group and only 19.2% (10/52) in the subcutaneous group getting the same reduced dose. 88 Similarly, local reactions of small areas of erythema and induration with 2 to 3 days of slight tenderness and itching were described for 'all' intradermal participants (ages 2 month to 5 years, n = 96), while only 2 of 94 children vaccinated subcutaneously had local pain and induration. Considering the entire reduced-dose, ID infl uenza literature, one might conclude that this route may be considered when antigen shortages and distributive equity demand the use of the lower end of the doseresponse curve, where ID may outperform the SC/IM routes. The increased reactions described in these whole-virus studies may be mitigated by the modern use of less reactogenic splitvirus products.

The ID route was used extensively for the live, attenuated yellow fever French neurotropic vaccine (FNV), which was given by ID scarifi cation in the 1940s and 1950s in Francophone Africa (see Chapter 36 [yellow fever]). 89 The 17D strain showed both good 90 and poor 91 immune responses when jet-injected ID. The ID route also yielded mixed results for live measles vaccines. [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] Inactivated vaccines with good immune responses after ID injection include typhoid 105 and rabies, 106-113 the latter of which has been used widely for dose-sparing purposes in the developing world. 114 

ID injection-as well as IM-led to the serendipitous discovery in an infl uenza model 148 that viral genes encoded into bacterial DNA would somehow get expressed in vivo into their protein antigens, a seminal event in the modern era of recombinant nucleic acid vaccinology. 149 Gene proto-antigens to prevent infl uenza, 150 HIV/AIDS, 151,152 smallpox 153 and many other diseases are being inserted into both 'naked' DNA/RNA 154 and various vectors such as modifi ed vaccinia Ankara (MVA) virus, for delivery by the ID route. ID jet injection has been used for immunomodulators like interferon. 155

Novel methods to deliver antigen past the stratum corneum Various commercial patch delivery systems developed since 1981 have demonstrated the ability of certain therapeutic agents (e.g., scopolamine, nitroglycerin, clonidine, estradiol, fentanyl, nicotine and testosterone) to diffuse passively into bare, untreated skin without the use of the active technologies or enhancers described below. 27 But such passive diffusion usually works only for small molecules of certain physical characteristics. Thus, there are but a few animal models of immunization onto bare, untreated skin. 156-158 Newer methods to facilitate antigen delivery to the epidermis involve painlessly stripping, abrading, scraping, piercing, vaporizing, shocking, vibrating, bombarding and otherwise permeabilizing the barrier of the stratum corneum. 20, 22, 23, 27, 159, 160 Some methods combine several processes.

Tape and friction A variety of simple tools have been used to remove the stratum corneum. Common cellophane adhesive tape may be applied to the skin and pulled away, carrying away dead keratinocytes with each repetition. Such tape-stripping has been shown to enhance cytotoxic T cell and cytokine immune responses upon subsequent application of various antigens and adjuvants to the skin in mice. [161] [162] [163] [164] [165] [166] [167] Similarly, rubbing gauze, emery paper, EKG pads, or pumice on the skin removes cells by their abrasive effects, and have been found to enhance immune responses in humans. 168

The razor and the brush work as well. In a clinical trial of adenovirus vectors encoded to express infl uenza HA antigen, the abdominal skin of 24 adults was shaved with a disposable, twin-blade razor, followed by 'gentle brushing with a softbristle toothbrush for 30 strokes' and application of the antigen with an occlusive Tegaderm TM patch. 169 Two doses 28 days apart at the highest dose level produced 4-fold rises in HI titer in 67% of the cutaneous vaccinees. Occasional mild erythema at the abdominal site was reported in 61% and rash/itching in 39% of patients. This same research team, 170 studying mice, substituted an electric trimmer for shaving but otherwise used similar brushing to demonstrate that topical application of non-replicating Escherichia coli vectors overproducing antigens for Clostridium tetani and B. anthracis were immunogenic. 171,172 Control animals demonstrated that depilation alone had little effect; what made the difference was the mild brushing that produced minimal irritation (Draize scores = 1). 173

Other methods to abrade the stratum corneum take advantage of low-cost fabrication techniques adapted from the microelectronics industry to produce arrays of large numbers of submicron-to millimeter-sized tines (sometimes referred to as solid microneedles) of silicon, metal, or other material. 22, 174 One technology that abrades the skin before or after topical application of the antigen or therapeutic agent is named a microenhancer array (MEA) and consists of a square or round chip of about 1 cm 2 area of silicon or plastic microprojections that are mounted on a hand-held applicator (OnVax TM53 , Fig.  61-3A) . 175 Preclinical studies of the MEA device in mice inoculated with DNA plasmids encoding fi refl y luciferase and HBsAg found similar or greater light emission and immune responses, A clinical trial of the MEA measured transepidermal water loss (TEWL) as a surrogate indicator for removal of the stratum corneum following each of fi ve consecutive swipes across the same site on the volar forearm of volunteers. Projection heights of 100, 150 and 200 µm showed steadily increasing rates of TEWL, with the tallest projections producing the greatest water loss. Control swipes with fi brous and sandpaper EKG pads showed little or no TEWL. 175

Another method to carry antigen across the stratum corneum is by coating it onto solid microscopic projections or microtines, from which it dissolves and diffuses while held for variable periods of time in the epidermal layer. 176 But their suitability for human vaccination has not yet been fully demonstrated. 21, 177 One example of microtines is the investigational Macrofl ux ® microprojection array, 178 whose projections vary from 225 to 600 µm height and are packed into an area of 1 to 2 cm 2 at densities from 140 to 650 tines per cm 2 . They are inserted by a spring-mounted applicator and held in place by an adhesive patch ( Fig. 61-3B) . In a hairless guinea pig model, ovalbumin as a representative large antigenic protein was applied to the tines and administered in two doses 4 weeks apart. 179, 180 Post-booster titers for the device were comparable to control IM, SC and ID Mantoux method injections at higher doses, and surpassed IM and SC routes at lower doses. Other preclinical studies of the Macrofl ux have demonstrated delivery of oligonucleotides 181 and the peptide hormone desmopressin. 182 Another array of microtines is termed a Mictrostructured Transdermal System (MTS), 183 and consists of drug-coated pyramidal projections of 250 µm height, in a density of 1,300 projections per cm 2 , again mounted on an adhesive patch and applied with a spring-powered applicator ( Fig. 61-3C ). 184-187 In a rabbit model, several formulations in various ratios of tetanus toxoid and alum adjuvant coated onto the microtines induced antibody levels an order of magnitude higher than the presumed protective threshold (>0.2 IU), using just a fraction of the standard IM dose. 188 Experimental placement of the device on human volunteers found it to be 'well-tolerated,' 'nonintimidating and not painful.' 186 Among others working with microtines, Coulman et al studied nanoparticles and DNA plasmids expressing βgalactosidase and fl uorescent proteins applied to the epidermal surface of ex vivo human breast skin donated at mastectomy. 189 After applying the microtines to the skin for 10 seconds, they were able to verify epidermal penetration and gene expression by a variety of histologic and photometric means. Kwon et al developed biodegradable microtines made by dissolving drug in carboxymethylcellulose and casting into a solid by centrifugation in a mold and air drying (DrugMAT TM , VaxMAT TM ). [190] [191] [192] Others conducting work with microtines (solid microneedles) include Corium 194, 195 and Valeritas (Micro-Trans TM ). 197 

Hollow projections termed microneedles, produced by similar techniques as for the solid microtines described above, are designed to inject therapeutic agents through their tiny cannulae ( Fig. 61-3D) . 20, 176, 198 Although harder to manufacture and more easily broken and clogged, 174,176 fl ow rates of microneedles have been measured up to a remarkable 1 mL per minute per cannula. 176a Common lengths are 0.2 to 0.5 mm, short enough to be painless since their depth does not reach nerve endings in the dermis 22, 198, 199 Among those working on such microneedles are the Georgia Institute of Technology, 198, 200 Norwood Abbey, 201 NanoPass (MicroPyramid TM , MicronJet TM ), 202 SpectRx (SimpleChoice™), 196 and Valeritas. 197 

The use of light or electricity, or the heat or radiation they produce, has been pursued to facilitate entry of drug into the skin, either during a brief or constant application of energy, or through the pathways created after a short pulse.

Laser light has been used in two ways to breach the stratum corneum. In one, a brief pulse of laser light 'ablates' this layer, after which drugs are applied directly onto the exposed epidermis, often with an occlusive patch, for the few hours until the stratum regenerates. 20, 27, [203] [204] [205] [206] One device, the LAD (laser assisted drug delivery, Norwood Abbey) 201 generates an erbium-doped yttrium-aluminum-garnet (YAG) laser beam whose energy is highly absorbed by skin ( Fig. 61-3E) . 205 It was shown in adult volunteers to facilitate the anesthetic effect of the topical application of lidocaine, 205 and is licensed in the U.S. and Australia for that purpose. In another method, a high-power pulsed laser creates a photomechanical wave that drives particles representing drug carriers through the stratum corneum. [207] [208] [209] Preclinical or clinical studies for intended vaccination using such laser methods have not yet been reported.

Iontophoresis-fi rst demonstrated a century ago in rabbits 210 uses an electric current to drive charged molecules from an electrode of the same charge towards another of opposite charge located elsewhere on the body. 22,27,211-215 Among licensed devices applying this technique for skin anesthesia are the LidoSite TM (Actyve TM technology) 216 and the IONSYS TM (E-TRANS ® technology). 217 A related method is electro-osmosis, which induces a fl ow of solvent to carry non-charged molecules. 159,218 Voltages above 1 volt in themselves increase skin permeability, perhaps by opening up pathways along hair follicles. But these techniques do not work well at higher molecular sizes, which characterize many vaccine antigen proteins.

Thermoporation, also termed microporation, uses the heat of electrical resistance to vaporize tiny openings in the stratum corneum. 22,27, 219 In the PassPort TM system, 220 a disposable array of metallic fi laments is held momentarily against the skin by a device the size of a computer mouse which, upon activation, induces electric pulses in the fi laments ( Fig. 61-3F ). An adhesive patch containing vaccine or therapeutic agent is then applied over the micropores just created. In a hairless mouse model, this technique elicited 10-100-fold greater cellular and humoral responses to an adenovirus vaccine compared to intact skin, as well as 100 percent protection to surrogate tumor challenge (27 percent for intact skin). 219 In the same model, adenovirus-vectored melanoma antigen applied to the micropores roughly doubled the average onset time of tumors by challenge, and protected 1 of 6 mice compared to 0 of 8 vaccinated controls with intact skin. Microporated recombinant infl uenza H5 hemagglutinin protected BALB/c mice from challenge with a lethal H5N1 strain. 220a Skin micropores also permitted the passage of insulin in pharmacokinetic human trials with historical controls, 221, 222 and in the other direction allowed interstitial fl uid to be extracted for potential glucose monitoring. 223 Another method generates micropores with heat induced by radiofrequency waves (ViaDerm TM ). 224 Electroporation uses very short electrical pulses to produce in the intercellular lipid matrix of the stratum corneum temporary pores of nanometer range diameters, which remain open and permeable for hours. 22, [225] [226] [227] [228] [229] [230] In vitro and in vivo preclinical studies of this technique demonstrated entry into or through the cells of larger molecules, such as heparin (12 kDa), peptides and proteins (such as luteinizing-hormone-releasing hormone), and oligonucleotides (up to 24-mer), which hold promise for 61 Chapter 61 6 polysaccharides, proteins, nucleic acids, and even adenovirus vectors as vaccine antigens. 212, 219, [231] [232] [233] IM electroporation is also being pursued to enhance vaccination with DNA antigens. 230, 234, 235 A hollow needle injects the drug conventionally into muscle while parallel solid needles surrounding the injected dose create the current to generate pores in the target muscle tissue. Investigational or marketed products are CythorLab TM , 236 Easy Vax™, 193 Electrokinetic TM Device (EKD), 237 ECM, 238 MedPulser ® , 234, 235, 239 and TriGrid TM , 240, 241 among others.

The connection between keratinocytes can be solubilized to facilitate drug or antigen delivery by ultrasonic waves and short-duration shock waves. 20, 22, 159, [242] [243] [244] These are theorized to induce cavitation-the formation and collapse of microbubbles -which disrupts the intercellular bilayers within the stratum corneum. Low frequencies (<100 Khz) appear to work better than the higher frequencies used in therapeutic ultrasound (>1 MHz). Transdermal tetanus toxoid immunization of mice was enhancd 10-fold compared to the subcutaneous route when subjected to 20 kHz ultrasound. 245 High-molecular weight molecules delivered include insulin, erythropoeitin, interferon and low molecular weight heparin. 22, 243, 246, 247 Various groups are pursuing ultrasound for enhanced drug delivery. 201, 248, 249 

The transfection of cells by use of kinetic methods to deposit DNA-coated gold particles into them was pioneered in the 1980s. 250 The Helios ® or PDS 1000/HE 'gene guns' 251 and the Accell injector 252 have become standard bench tools for 'biolistic' delivery of nucleic acid plasmids into a wide variety of plants and animals to tranfect them to express the coded genes. 253, 254 Delivery of DNA into the skin overcomes the usual polarized Th1 response when DNA is delivered into muscle. 21,255,256 These devices are unavailable for human vaccination (patent rights are held by PowderMed 257 ). Documenting the safety of DNA as antigen by any route remains a major regulatory obstacle for such a paradigm shift in human vaccination. 21

The proprietary terms epidermal powder immunization (EPI) and particle-mediated epidermal delivery (PMED) refer to the use of helium gas to blow into the epidermis at supersonic speeds powdered proteins, polysaccharides, or inactivated pathogens, or DNA-coated particles, respectively. 258 This unique method of vaccination was developed in the early 1990s by Oxford BioSciences, which over the years was renamed PowderJect, acquired by Chiron, 259 spun off as PowderMed, 257 and acquired by Pfi zer 260 in 2006. Delivery is by either reusable (XR series) or single-use disposable (ND series) devices ( Fig. 61-3G) , with the latter targeted for commercialization.

Conventional protein antigens for delivery by EPI are spraydried into powders of suitable density and size (20-70 µm), 261, 262 but the economics of manufacturing such formulations may be an obstacle. 21 For DNA vaccines delivered by PMED, plasmids coding for desired antigens are coated onto gold beads (1-3 µm in diameter) and upon their deposition into epidermal antigenpresenting cells are eluted and transcribed. 263 Human trials of DNA vaccines containing up to one order of magnitude less antigen than used for IM routes have induced humoral and cellular immune responses for hepatitis B in subjects both naive and previously vaccinated with conventional vaccine. 264-267 PMED vaccination has also been studied for DNA priming in trials of malaria vaccine, 268,269 and produced the fi rst seroprotective immune responses by a DNA vaccine for seasonal infl uenza. 150,270 Clinical trials still ongoing or unpublished studied antigens for H5 avian infl uenza (DNA), 271 herpes simplex virus 2, 272 HIV and non-small cell lung cancer. 273, 274 In the hepatitis B and infl uenza trials cited above, there were no severe local reactions, but erythema, swelling, and fl aking or crust formation occurred in nearly all subjects, albeit resolving by day 28. Skin discoloration, however, persisted through day 56 in 29 (97%) of 30 subjects, 267 through day 180 in 21 (25%) of 84 injection sites 150 and beyond 12 months in 5 (25%) of 20 patients with long-term followup. 267 No anti-double-stranded DNA antibodies were detected. The disposition of the gold particles was studied in pigs, in whom most particles were deposited in the stratum corneum and epidermis, and eventually sloughed by exfoliation by 28 days. 275 At days 56 and 141 after administration, a few particles remained in the basal epidermal layer and in macrophages in the dermis and regional lymph nodes. Preclinical studies of EPI or PMED in murine, porcine, and primate models have shown immunogenicity or protection for either powdered or DNA plasmid antigens for various other pathogens, including Eurasian encephalitic viruses, 276 hantaviruses, 277 HIV, 278 malaria, 279 SARS coronavirus 280 and smallpox. 281

Microscission involves a stream of gas containing tiny crystals of inert aluminum oxide to bombard small areas of the skin. A mask on the skin limits the 'sandblasting' effect to narrow areas where channels are created in the stratum corneum, to which drug is then applied. 282 Another method employs a fast and powerful contractile fi ber-activated pump to fi re drug at the skin with suffi cient velocity to penetrate the epidermis. 201 A miniaturized form of traditional jet injection uses piezoelectric transducers to propel liquid microjets into the skin. 282a

As bathers notice in their fi ngertips, prolonged wetting of the skin, or occluding it to hold in body moisture, produces fl uid accumulation in intercellular spaces and swelling of the keratinocytes, which permits enhanced passage of applied agents. 168 Rubbing the skin with acetone also enhances antigen passage by extracting epidermal lipids. 163

Discovery of the remarkable adjuvant effect of bacterial ADPribosylating exotoxins, such as the B (binding) subunits of cholera toxin (CT) and the structurally-similar, heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC), has prompted much interest and work (see Chapter 9 [Cholera]). 158,283-288 For safety reasons, these toxins have been engineered, or mutants selected, to reduce toxicity while retaining adjuvanticity. [288] [289] [290] [291] Nevertheless, one such use as adjuvant in a licensed intranasal infl uenza vaccine was hypothesized as the cause of temporary paralysis of the 7th cranial nerve, prompting market withdrawal. 292

Skin vaccination using CT or LT as adjuvants and antigens has been advanced principally by Iomai, 293 which calls the process transcutaneous immunization, 294-296 although others have also studied this technique. 297 Such toxins may be administered by themselves as antigen to induce immunity against ETEC causing traveler's diarrhea or against Vibrio cholera, either with 298 or without 299,300 ETEC colonization factor ( Fig. 61-1) . A randomized, blinded fi eld trial among travelers to Central America found 75% effi cacy for the LT patch in protecting from moderate/severe diarrhea. 300a Their adjuvant effect has been explored for infl uenza vaccines, which have generally the lowest rates of immune response and effi cacy among licensed vaccines, particularly in the very young and old. Applying an LT patch near the site of injection of conventional parenteral infl uenza vaccine was found to improve HI titers in the serum and mucosa of both young and aged mice 301,302 and to increase or show an improving trend for adult volunteers over 60 years. 303 The use of CT or LT as cutaneous adjuvant has resulted in improved immune responses or challenge protection in animal models for tetanus, 304 anthrax, 305,306 malaria 307 and Helicobacter pylori. 308 Clinical trials found no serious reactions, 299 but pruritis and maculopapular rash at the patch site, were found in 13%, 303 74% 298 and 100% 300 of patients exposed to LTcontaining patches for 6 hours; 17% progressed to vesicle formation. 300 Delayed type hypersensitivity contact dermatitis was observed when using recombinant colonization factor. 298

Chemical penetration enhancers under consideration as skin adjuvants, alone or in conjuction with iontophoresis, ultrasound, and electroporation methods, include oleic and retinoic acids, 167 dimethylsulfoxide (DMSO), ethanol, limonene and polysorbate, among others. 22 Flagellin, a bacterial surface component protein, was engineered to express infl uenza nucleoprotein epitope and applied to the bare skin of mice, inducing virus-specifi c interferon-γ T cells. 158 Certain colloids may serve as antigen carriers. 23 Deformable lipid vesicles ('transfersomes') containing tetanus toxoid applied to animal skin yielded comparable immune responses with alum-adjuvanted tetanus toxoid given IM. 309 

Other novel methods of delivery include the use of short needles to poke an initial opening into the skin, followed immediately by SC or IM jet injection with much lower pressures than otherwise would be needed. 310, 311 Another method is termed a needle-free solid dose injector (Glide TM ). 312 It uses a spring-loaded device to push a sharp, pointed, biodegradable 'pioneer tip' and the solid or semisolid medication behind it in the chamberboth about the width of a grain of rice-into subcutaneous tissues.

Jet injectors (JIs) squirt liquid under high pressure to deliver medication needle-free into targeted tissues. [313] [314] [315] [316] [317] [318] Invented in France in the 1860s ( Fig. 61-4A ), 313,319,320 the technology was fi led for patent in 1936, 321 and reintroduced in the 1940s as the Hypospray ® 322,323 for patient self-injection with insulin ( Fig. 61 -4B; Table 61-1). In the 1950s, the U.S. military developed highspeed models (once referred to as 'jet guns') for mass vaccination programs ( Fig. 61-4C ). 371 18, [394] [395] [396] [397] During the swine infl uenza mass campaign of 1976-1977 in the U.S., a substantial proportion of the approximately 80 million doses distributed that season were administered by JIs (CDC, unpublished data). 398 JIs have also been used for a wide variety of therapeutic drugs, including local 399,400 and pre-general 401, 402 anesthetics, antibiotics, 403 

Common features of all JIs include a dose chamber of suffi cient strength to hold the liquid when pressurized, a moving piston at the proximal end to compress the liquid, and a tiny orifi ce (commonly ∼0.12 mm in diameter, ranging from 0.05 to 0.36 mm) 316, 368 at the distal end to focus the exiting stream for delivery into the patient. The pistons of the majority of modern JIs are pushed by the sudden release of energy stored in a compressed metal spring, while some use compressed gas such as carbon dioxide (CO 2 ) or nitrogen (N 2 ) (Table 61-1) . Two investigational ones are powered by the expanding pressure of chemical combustion. 197, 334 The source of energy to compress the spring is usually supplied manually or pedally through an integral or separate tool to apply mechanical advantage and/or hydraulic pressure. A few use electrical power from batteries or wall (main) electrical current.

Although devices vary, peak pressures within the dose chambers range from 14-35 MPa (∼2,000-5,000 psi) and occur quite early in order that the stream can puncture the skin. After the peak, pressures drop about one-third to two-thirds during a descending plateau phase until rapid tailoff at the end of the piston's stroke. The velocity of the jet stream exceeds 100 meters per second. 417 Complete injection lasts about 1 / 3 to 1 / 2 second, depending on volume delivered, orifi ce cross-section, and other variables.

JIs may be classifi ed in various ways: by their energy storage and sources described above, by intended market (human vs. veterinary), by intended usage (e.g., repeated self-administration of insulin by the same patient vs. use to vaccinate consecutive patients), by how the dose chamber is fi lled (medication vial attached 'on tool' vs. fi lled 'off tool'), by reusability of the entire device (single-use disposable vs. reusable), and by reusability of the fl uid pathway and patient-contact components (multi-use vs. disposable). This last criterion results in a key distinction between multi-use-nozzle jet injectors (MUNJIs) and disposable-cartridges jet injectors (DCJIs), with major implications for immunization safety (discussed below).

In vivo imaging indicated jet-injected medication tends to spread along paths of least resistance in a generally conical distribution. 328, [418] [419] [420] [421] [422] [423] The depth achieved depends primarily on the power imparted to the liquid and variables such as orifi ce diameter, viscosity of the dose, tautness and thickness of the skin and fat layer, and angle of injection, among other factors. 316, 317, 322, 417, 418, 424, 425 The SC compartment is the only one accessible by most marketed DCJIs, as well as by MUNJIs used in dental anesthesia 345, 346 and self-administration of insulin, hormones, and other drugs. Most MUNJIs developed for mass vaccination campaigns are powered to reach IM tissues, e.g., the Ped-O-Jet and Med-E-Jet, as is one DCJI, the Biojector ® 2000, which varies the orifi ce of different cartridges to deliver either IM or SC. 50 Given great patient variation, it is no surprise that imaging studies suggest JIs often miss the intended IM or SC compartment. 426 But this may have little clinical relevance, and be no different than needle injections for which fat pad thickness is often underestimated in selecting needle length, or which is not fully inserted. 427, 428 As mentioned in the cutaneous immunization section above, jet injectors are capable of classical ID delivery by use of specialized nozzles (Fig. 61-2G ). The most widely used Ped-O-Jet ® administered tens of millions of smallpox vaccine doses for the fi rst half of the WHO Smallpox Eradication Programme in South America and West Africa in the late 1960s to early 1970s, until invention of the simpler and swifter bifurcated needle. 1, 49, 381 Jet injectors also delivered ID the BCG vaccine [429] [430] [431] [432] [433] [434] and various tuberculosis skin testing antigens (TST). [435] [436] [437] [438] [439] [440] [441] [442] [443] However, variations in consequent TST reaction sizes 43,444 led WHO to discourage JI use for BCG and TST. 445, 446 In the absence of an ID nozzle, many have attached spacers or tubing to a regular nozzle, creating a gap between orifi ce and skin, which weakens the jet and provides space for a bleb that leaves the dose in the skin. 97 investigationally for local anesthesia 448 and DNA vaccines ( Fig.  61-2H) . 51,52,330 Intrapulmonary injections (between the ribs) of antibiotics, bronchodilators, and steroids were performed in Russia. 333 

A large clinical literature documents the immunogenicity of JIs to be usually equal to and sometimes better than that induced by conventional needle and syringe for a wide variety of vaccines. 314 

Comparisons of immediate pain between JIs and needles used to deliver IM and SC injections depend on the medication involved. Insulin, other non-irritating drugs, and non-adjuvanted vaccines are reported to result in either reduced or equivalent pain compared to needles, 322, 377, 389, 401, 415, 416, 424, 467 but not always. 461 True double-blinded, needle-controlled studies for such subjective criteria are nearly impossible to design and thus lacking.

Vaccines with alum adjuvants or other irritating components tend to result in higher frequencies of delayed local reactions (e.g., soreness, edema, erythema) when jet-injected, probably because small amounts remain in the track through skin and superfi cial tissue. These include vaccines for diphtheria-tetanuspertussis (whole-cell), 139 394 and typhoid. 452, 464, 489, 522 In most cases, local reactions were mild, resolved within days, and were not reported to compromise clinical tolerance and safety. A chronic granuloma was reported following JI vaccination with tetanus toxoid adsorbed to alum, 490 and pigmented macules persisted in a few hepatitis B vaccinees. 456 Other adverse events Bleeding, and less often ecchymosis, are reported to occur at the jet injection site more frequently than with needle injections. 78, 322, 348, 371, 373, 374, 385, 389, 401, 405, 414, 416, 424, 444, 452, 462, [491] [492] [493] Rarely, the jet stream may cause a laceration if the health care worker has not properly immobilized the limb and injector in relation to each other during injection. 322, 373, 389, 416, 452 Rare case reports of other adverse events include transient neuropathy 494, 495 and hematoma. 409, 496 Safety of multi-use-nozzle jet injectors (MUNJIs)

Beginning in the 1960s, concerns arose for potential iatrogenic transmission of bloodborne pathogens by multi-use-nozzle jet injectors (MUNJIs), which use the same nozzle to inject consecutive patients without intervening sterilization. 488, 492, 493, 497 Unpublished bench and chimpanzee studies indicated hepatitis B contamination could occur because blood or HBsAg remained in nozzle orifi ces despite recommended alcohol swabbing between injections. 498 Others, however, reported negative results in bench or animal testing to try to detect contamination, 372, 405, 499, 500 or pointed to the lack of epidemiologic evidence of a problem. 394, 499, 501, 502 Then in 1985, Brink et al described a careful animal model in which a Med-E-Jet transmitted lactic dehydrogenase (LDH) virus between mice in 16 (33%) of 49 animals. 503 A few months later, fact superseded theory when a Med-E-Jet caused an outbreak of several dozen cases of hepatitis B among patients in a California clinic. [504] [505] [506] Subsequent clinical, 507 fi eld, 508,509 bench, 510 animal 511,512 and epidemiologic, 513,514 studies added more evidence that MUNJIs could transmit pathogens between patients. This led to warnings and discontinuation of their use by public health authorities, 515, 516 and market withdrawal of the Ped-O-Jet and discontinuation of its U.S. military use in 1997. 318, 517 There have been efforts in the 2000s to reengineer MUNJIs with disposable caps or washers with a central hole for the jet stream to prevent blood or tissue fl uid from reaching the nozzle. 342 However, clinical studies revealed the caps were unable to prevent HBV contamination of subsequent in vitro injections assayed by PCR after injections of high-titer HBVcarrier volunteers. 518,518a MUNJIs also face doubts raised by highspeed microcinematography revealing extensive splashback, 317 and the challenge of proving that contamination does not occur and of convincing policymakers to set any level of acceptable risk. Despite the withdrawal of MUNJIs for vaccination, models such as the MadaJet 346 and SyriJet 345 continue to be used in dentistry and medicine for delivery of local anesthetics.

MUNJIs allowed a single health worker to vaccinate 600 or more patients per hour. 315, 373, 375, 389 Their withdrawal poses challenges for conducting mass immunization campaigns for disease control programs and in response to pandemic or bioterror threat. Indeed, while the Soviet biological warfare effort was underway in secret, 519 numerous clinical trials were published of high-speed Russian MUNJIs capable of rapidly protecting soldiers or civilians against potential biowarfare agents such as anthrax, botulism, plague, smallpox and tularemia. 314, 449, 450, 470, 489, [520] [521] [522] [523] 

To overcome concerns over MUNJIs and their withdrawal, since the early 1990s, a new generation of safer, disposable-cartridge jet injectors (DCJIs) have appeared on the market (Table  61-1) . 318 Each cartridge has its own sterile orifi ce and nozzle and is discarded between patients. Most are used for self-administration of insulin and other hormones. An exception is the Biojector ® 2000 ( Fig. 61-2H ) 50 which was designed for vaccination and delivers approximately one million doses per year at private, public, and U.S. Navy and Coast Guard immunization clinics. Another DCJI for SC delivery only, the Injex ® 50 (Fig.  61-4I) , 339 produced satisfactory immune responses to measlesmumps-rubella vaccine boosters. 467 To meet developing world needs for needle-free vaccination systems that are economical, autodisabling to prevent reuse, and suitable for both mass campaigns and routine immunization, DCJIs such as the PharmaJet 358 and the investigational LectraJet ® 24,335 and the Vitavax TM 50 are in research and development (Fig. 61-4 K, L, M, N) . Financial support for DCJI R&D has been provided by private sources, by the U.S. Government (CDC), and by the Program for Appropriate Technology in Health (PATH) 353 under a grant from the Bill and Melinda Gates Foundation.

Since early in the history of immunization, the respiratory tract has been considered a highly promising route for vaccine deliv- major advantages of respiratory immunization are that it avoids needles and generally provides stronger mucosal immunity than parenteral immunization.

The great majority of human pathogens gain access across mucosal surfaces in the gastrointestinal, respiratory, or genitourinary tracts. Mucosal immunity includes humoral and cellular components and prevents infection at these portals of entry. In contrast, systemic (humoral and cellular) immunity clears infection only after invasion by limiting replication and destroying the pathogens. Ideally, both mucosal and systemic immunity should be raised against targeted pathogens. Strong mucosal immunity may enhance the benefi ts of immunization for some diseases. For example, by preventing the initial infection, mucosal immunity reduces the risk of transmission to others, in addition to preventing clinical disease. Prevention of infection at the mucosal surface may be especially important for diseases in which effective systemic immunity has been diffi cult to achieve, such as for tuberculosis and AIDS.

Every mucosal surface for administering vaccines has been studied with a variety of antigens in animal models, including the oral, conjunctival, rectal and vaginal routes. Several human vaccines are already licensed and in use for delivery by oral ingestion, including vaccines for polio, cholera, rotavirus, typhoid and adenovirus, which are described in detail in other chapters. This chapter, however, will focus only on vaccines and technologies for respiratory tract immunization, including devices for depositing vaccines in the target area, delivery systems to optimize presentation of antigen to the respiratory immune tissues, and adjuvants to enhance the immune response.

Pathogens and vaccine antigens enter the respiratory tract in airborne particles through oral or nasal inhalation and deposit on respiratory surfaces. Air inspired through the nose is effectively fi ltered by the nasal hairs, by the external nasal valves which restrict the airfl ow from the nares into the internal nasal passages and by the convolutions of the turbinates. For example, Djupesland et al showed only 25% of large, high speed droplets (average 43 µm) of a traditional nasal spray traversed the external nasal valve. 524 Particles that deposit on nasal mucus join the fl ow of mucus which is swept by ciliated epithelia toward the pharynx, where it is swallowed. Immune surveillance of antigens in the mucus fl ow occurs by uptake into epithelial cells, intraepithelial dendritic cells, surface macrophages and microfold (M) cells. 525, 526 M cells are specialized epithelial cells which take up macromolecules, viruses and bacteria by endocytosis, and then present them to lymphocytes and dendritic cells that congregate in special pockets in the M cells. The predominant organized lymphoid tissue of the human respiratory tract is located in the pharynx, where the adenoids and other tonsils (collectively known as Waldeyer's ring) surround the nasal and oral passages. The epithelium overlying these tissues is rich with M cells. 527 Increased deposition of vaccine antigen in the posterior nasal passages and nasopharynx near Waldeyer's ring may be desirable to maximize the immune response. Breath actuation of a nasal spray and nasal inhalation of smaller aerosol particles (5-20 µm) are two methods to increase nasopharyngeal deposition (Fig. 61-5A,B) . 524, 528 The nasal fi ltration system is bypassed by mouth breathing (e.g., for vaccine delivery, through a mask or oral prong). In such case, particles impact in the oropharynx, larynx, or trachea. The bifurcation of the trachea into the right and left bronchi starts a series of bifurcations which trap airborne particles. Only very small, light, and slow-moving particles inhaled via either nose or mouth succeed in navigating the tortuous pulmonary passages to deposit in the lower airways. The smallest particles (<3 µm) may reach the alveoli, where they can be rapidly absorbed into systemic circulation. The complex branching of the lung passages also results in an astonishing alveolar surface area exceeding 100 square meters in a human adult male, compared with an average of about 150 square centimeters (0.015 m 2 ) in the nasal airways. 529 The lower airways in humans do not typically have organized lymphoid tissues, but they do have abundant numbers of intraepithelial dendritic cells and alveolar macrophages which process antigens. 530 Antigen presenting cells from the respiratory tract drain to regional lymph nodes where the B cells preferentially switch to IgA plasmablasts. These plasmablasts 'home' back to the airway epithelium to provide antigen specifi c IgA protection. 531 T cells also play a major role in mucosal immunological memory responses. Some lymphocytes exposed to antigen in the respiratory tract migrate to provide protection at remote mucosal sites, such as the vagina. This integrated network of immune cells and tissues is known as the common mucosal immune system. 532, 533 Because the respiratory tract is exposed to a myriad of non-pathogenic macromolecules, there are mechanisms for down-regulating the immune response to antigenic exposure. This is known as immunological tolerance and must be considered when developing respiratory immunization strategies. 534 

The fi rst challenge in respiratory immunization is to identify the appropriate target tissue. Most respiratory drugs traditionally target two areas. The nasal passages are the desired site of action for decongestants, while the lower airways are targeted by asthma medications. The optimal target tissue is not yet determined for most potential respiratory vaccines and may be different for different vaccines. The pharyngeal tonsils are likely candidates because of their key role in immunologic priming, however, some vaccines may require deposition in the lower airways. Scientifi c methods for evaluating and comparing different vaccine target tissues areas are not yet well developed. Interspecies differences in respiratory immunologic tissue organization makes it diffi cult to use animal models to determine optimal vaccine target tissues. Moreover, the relative size and anatomy of the respiratory tract of common research animals differ greatly from humans. For example, in small animals such as rodents, the use of nose drops may result in deposition to the entire respiratory tract which would not be the case in humans. Balmelli, et al estimated that 30% of 20 µL of vaccine given to mice as IN drops deposited into the lungs. 535 A second challenge to research is the lack of susceptibility in many animal models to many human diseases of interest. This makes it diffi cult to use live vectors as vaccines or to do challenge studies to determine vaccine protection. Such limitations impede the translation of promising results from animal research into safe and effective vaccines for human use.

A third challenge for respiratory immunization is the diffi culty in delivering a consistent dose. The mass or volume of the dose delivered depends on many factors, including variability in performance by the respiratory delivery device, the behavior and technique of the person administering the vaccine, and differences in anatomy and physiology in the vaccinates (animals) or vaccinees (humans). 536 Fortunately, for many vaccines there is a wide margin between the dose necessary to induce protection and the dose at which the risk of adverse events increases. The licensure in 2006 in the United States and Europe of the fi rst inhalable insulin (Exubera TM ), a drug for which dose accuracy and consistency is critical, suggests that this challenge can be overcome for respiratory vaccines. 537 A fourth major challenge is that accepted 'correlates of protection' for mucosal immune responses have yet to be determined. In contrast, for many diseases there are wellestablished laboratory assays of systemic immunity-such as antibody titers above certain cutoffs-that have served for many years as indicators of protection from disease.

Several immunization safety issues represent further challenges for respiratory vaccines. One is the risk that vaccine viruses, antigen, or adjuvant might affect nearby cranial nerves, 292 or travel along the olfactory nerve through the cribiform plate into the brain with resulting adverse central nervous system effects. Another risk that must be addressed is cross-contamination, in which respiratory pathogens from one patient may contaminate the respiratory immunization device, with the risk of their spread to subsequent patients using the device. Other safety issues for vaccines targeting lower airways include the possible induction or exacerbation of bronchospasm and/or pulmonary infl ammation, which can be life-threatening. Also, respiratory vaccine aerosols may spread beyond the intended vaccinee to other persons in the vicinity. Finally, certain live virus or bacterial vaccines might have a pathogenic effect on persons immunocompromised by HIV or other conditions.

Remaining challenges relate to the delivery devices. Although many devices already exist for delivering drugs to the respiratory tract, very few of them are designed for vaccine delivery. Most respiratory drug devices deliver repetitive doses to a single patient. In contrast, the expected usage for vaccination devices is to deliver single doses to multiple patients, which raises the cross-contamination issue mentioned above. Although single- 818 ). The nebulizer utilizes battery-powered piezoelectric energy to drive an aerosol from a perforated mesh plate to a disposable patient interface (nasal prong, oral prong or mask). Droplet size can be tailored for upper or lower airway delivery. (E) Classic Mexican Device (investigational); a non-medical electric compressor (not shown) delivers roughly 9 liters of air per minute at a pressure of 30-40 pounds per square inch to a jet nebulizer which is kept in crushed ice to maintain vaccine potency. The vaccine aerosol (roughly 0.15 cc) is delivered through a disposable paper cone held close to the patient's face for 30 seconds. 538-541 (F) AccuSpray TM nasal spray syringe (Becton, Dickinson and Co. 53 ); licensed to deliver FluMist TM infl uenza vaccine. Prefi lled and stored frozen for single patient use after thawing. The total volume is 0.5 mL, a dose separator stops delivery at 0.25 mL, and the remaining 0.25 mL is delivered to the opposite nostril. 61 Chapter 61 6 use, disposable devices might solve this, they may be costly. Some aerosol drug delivery devices require patient education to obtain the needed cooperation for adequate dose delivery, which is diffi cult in the brief time typical for vaccination. Some respiratory delivery methods are not effective for young children, who receive many vaccines. Although current respiratory drug delivery devices typically target the anterior nasal passages or the lower airway, respiratory vaccination may work best by deposition in the quite different area of the pharyngeal tonsils. New delivery technologies to meet the requirements of respiratory immunization are required if this route is to become practical and accepted. As a young fi eld, published research on devices used in respiratory vaccination of humans or animals is limited. In most reported animal studies, the IN delivery device is not mentioned at all, or a laboratory pipette unsuitable for humans is used for instillation.

The only device currently licensed and in use in the United States for respiratory delivery of a vaccine is the AccuSpray TM (Becton, Dickinson and Company (BD)), 53 which is used to deliver FluMist TM infl uenza vaccine. The AccuSpray TM is a nasal spray syringe preloaded for single patient use (Fig. 61-5F ). It produces particles with a mean aerosol diameter of 70 microns in a total dose of 0.5 mL, with 0.25 mL delivered consecutively through each nostril. Key advantages of this device are that it is simple to use, inexpensive, disposable and very diffi cult to refi ll and reuse. The large particle size minimizes deposition to the lower airways which reduces the risk of pulmonary adverse events.

Another respiratory immunization device that has been used in humans is the jet nebulizer system known as the Classic Mexican Device (CMD, Fig. 61-5E ). With slight modifi cations, this nebulizer delivered live attenuated measles vaccines in multiple clinical trials in Mexico and South Africa, and in a mass campaign which vaccinated over 3 million Mexican children against measles. 538,539,540,541 The system consists of a general-use (non-medical) compressor which delivers air to a jet nebulizer (IPI TM ) which holds the vaccine in crushed ice to maintain potency. The vaccine aerosol is delivered through a disposable cone (modifi ed paper cup) which is held close to the patient's face for 30 seconds. Typically, the aerosolized vaccine dose is roughly 0.15 mL, and the mass median aerosol diameter of the emitted particles is 4.3 µm. 542 The OptiMist TM is a breath-actuated nasal spray device for liquid or powders which delivers only during oral exhalation. 543 Because oral exhalation closes the connection between nose and throat, pulmonary deposition is avoided and delivery to the posterior nasal segments is increased (Fig. 61-5A,B) . 524 In a human study, inactivated infl uenza vaccine self-administered using the OptiMist TM resulted in signifi cant increases in virus-specifi c IgA in nasal secretions and protective levels of virus-specifi c serum antibodies after two doses in >80% of subjects. 544 A Combitips-plus syringe (Eppendorf) was used to deliver a dry powder Neisseria menigitidis vaccine IN to human subjects. IN-vaccinated subjects had serum bactericidal antibody titers comparable to those vaccinated by conventional injection, and 92% of IN vaccinees had protective titers after the second dose. One-third of IN vaccinees reported mild side effects, compared to two-thirds of injection vaccinees reporting mild injection pain. 545 BD 53 has demonstrated the utility of a novel device for delivery of vaccine powder (Fig. 61-5C ). Air from a syringe barrel ruptures the membranes of a capsule containing the vaccine and delivers the powder to the nasal tract. The device was effective in nasal delivery of infl uenza vaccine to rats and of anthrax vaccine to rabbits. 54,546

The Centers for Disease Control and Prevention (CDC) developed a nebulizer for vaccine delivery which utilizes a disposable aerosol-generating element and disposable patient interface to prevent cross contamination (Fig. 61-5D) . The aerosol it generates can provide either 10-25 µm droplets for upper airway delivery or <5 µm droplets for lower airway delivery, and can be used with a disposable nasal prong, oral prong or mask. Delivery of live attenuated measles vaccine with this device through a nasal prong was shown to be safe and immunogenic in macaques. 547 Ongoing research focuses on maximizing delivery to the nasopharynx. The AerovectRx TM company 548 has acquired the rights to manufacture and distribute this technology.

Non-replicating antigens delivered via the respiratory tract are typically poorly immunogenic and may require adjuvants to stimulate an appropriate immune response. Adjuvants which have been studied for respiratory delivery of vaccines include bacterial toxins and their derivatives, other bacterial components, bacterial DNA motifs, cytokines and chemokines, plant derivatives and other adjuvants (Table 61 -2). 549-553 Cholera toxin (CT) and E. coli heat labile toxin (LT) are potent respiratory immunization adjuvants but are considered too toxic for use in humans. 551,554-559 LT was an adjuvant in a commercially available IN infl uenza vaccine in Switzerland which was withdrawn from the market in 2001 due to an increased risk of Bell's palsy among vaccinees. 292,560 Although the pathogenesis of Bell's palsy has not been clearly defi ned, CT and LT have been shown to accumulate in the olfactory bulbs of Balb/c mice following nasal administration, sometimes with concurrent infl ammation, which suggests a risk for adverse neurological effects. 561 As a result, recent adjuvant research has focused on alternative subunits and variants of CT and LT. 562-580 Several of these, such as CTA1-DD, do not accumulate in the olfactory bulb of BALB/c mice. 581 Other bacterial products which induce potent activation of the innate immune system include bacterial lipopolysaccharide (LPS) and its derivative, monophosphoryl lipid A (MPL), as well as bacterial outer membrane protein proteosomes, fl agellins, lipopeptides and fi lamentous hemagglutinins [582] [583] [584] [585] [586] [587] [588] [589] [590] [591] [592] [593] (Table 61-2 ). An IN, proteosome-based, inactivated infl uenza vaccine produced serum and mucosal antibodies in human subjects. 583 CpG oligodeoxynucleotides (CpG ODNs) are short segments of synthetically constructed single stranded deoxynucleotides which contain CpG motifs found in bacterial DNA. These motifs are recognized as pathogen associated molecular patterns (PAMPs) by the innate immune system and are potent adjuvants. [594] [595] [596] [597] Abe et al found that a non-typeable Haemophilus infl uenzae (NTHi) vaccine, delivered IN with CPG ODNs, produced similar mucosal IgA and serum IgG responses as vaccine delivered with CT. Enhanced clearance of NTHi from the nasopharynx following challenge was shown equally in both groups. 598 However, in another study, daily injection of high dose (60 µg) CpG resulted in lymphoid follicle destruction and immunosuppression with liver necrosis after 20 days. 599 Therefore, potential adverse effects of CpG ODNs should be carefully monitored.

Because many adjuvants induce enhanced immune responses through the activation of chemokines and cytokines, investigators have studied these molecules themselves as adjuvants that 

+ Denotes a respiratory vaccination study in which an immune response was demonstrated using the adjuvant, but unadjuvanted vaccine was not studied as a control.

∧ Denotes a respiratory vaccination study in which the immune response was increased with the adjuvant compared to vaccination without the adjuvant.

Chapter 61 6 might minimize any adjuvant toxicity (Table 61 -2). [600] [601] [602] [603] [604] [605] Chemokines and cytokines have been added directly to the vaccine, or encoded for expression by a live vector or DNA vaccine. 606 Bracci and colleagues found a single IN dose of an inactivated infl uenza vaccine provided full protection against virus challenge in mice when type 1 IFN was included as an adjuvant. The same vaccine dose was only partially effective (40%) without it. 607 Chitin is a natural polysaccharide found in crustaceans. Its partial deacetylation yields chitosan, which is widely used in food products, as an excipient in drugs, and as a nutritional supplement. 608 Chitin and chitosan have mucoadhesive properties and stimulate the innate immune system. 609 In humans, the addition of chitosan to an IN vaccine based on CRM-197 diphtheria antigen signifi cantly increased toxinneutralizing antibody levels. 610 The saponins of the Quillaja saponaria tree are potent adjuvants with high toxicity. Quil A, QS-21 and ISCOPREP 703 are subcomponents with less toxicity. 552 As adjuvant to an IN DNA HIV-1 vaccine studied in mice, QS-21 consistently increased antigen-specifi c serum IgG and mucosal IgA compared to vaccine without adjuvant. 611 Quil A and ISCOPREP 703 are commonly used as components of immunostimulating complexes (ISCOMs), antigen delivery vehicles described in more detail in the next section. Combining adjuvants may synergistically enhance immune protection with respiratory immunization. For example, IN immunization of mice with a recombinant infl uenza HA (rHA) antigen, with a combination of proteosomes and LPS adjuvants, enhanced serum IgG and mucosal IgA antibodies up to 250-fold compared to vaccine alone. 587

Once the device has delivered vaccine to the appropriate region of the respiratory tract, suffi cient quantities of the antigen (and adjuvant) must penetrate mucosal barriers to gain access to appropriate cells to activate the immune system. The vehicles or vectors which may be used for this purpose include live attenuated viruses (including those acting as vectors for exogenous antigen), live attenuated bacteria (including vectors), commensal bacterial vectors, virosomes, virus-like particles (VLPs), liposomes, lipopeptides, ISCOMS, microparticles and nanoparticles (Table 61-3) . [612] [613] [614] [615] [616] 

Viruses are prototypical antigen delivery vehicles because they enter and commandeer cells to replicate themselves, thus multiplying the available antigen which they encode. Also, viruses can induce a natural adjuvant effect through activation of chemokines and cytokines. The most widely studied respiratory delivery vehicles are live attenuated strains of pathogenic viruses. 591, [617] [618] [619] [620] [621] [622] [624] [625] [626] [628] [629] [630] [631] [632] [633] [634] [635] [636] Their major risks are possible reversion to virulence, potential neurotoxicity via the olfactory route, and the risk of pathogenic effects in immunocompromised persons.

Live, attenuated cold adapted infl uenza vaccine (CAIV, FluMist ® ) 637 is the only vaccine currently licensed for delivery by the respiratory tract. Its development, testing and licensure are reviewed in detail in Chapter 16 [infl uenza, live]. As a model respiratory immunization, IN CAIV demonstrates several potential benefi ts of live virus respiratory immunization. It produces both mucosal and systemic immunity and provides higher protective effi cacy than injected inactivated vaccine. 638-641c It also provides heterotypic immunity against infl uenza strains that had antigenically drifted from the vaccine strains. 642 Finally, it may reduce the risk of infl uenza transmission because it reduces respiratory shedding among children challenged with a vaccine virus. 642 Also, modest coverage with CAIV among school children reduced infl uenza-related illness rates in unvaccinated adults in a community. 643 Apart from infl uenza, measles has been the disease for which vaccine delivery via the respiratory tract has been most thoroughly studied. In a review by Cutts et al through 1997, 104 and in more recent studies, three basic immune response patterns were revealed upon measles vaccine delivery. First, drops or sprays delivered to the conjunctiva, oral or nasal mucosa produced inconsistent immune responses. 101, [644] [645] [646] [647] [648] [649] [650] [651] [652] Second, among older children (>12 months), delivery of smallparticle aerosols via inhalation typically produced immune responses in very high proportions of subjects. Immune responses to aerosol vaccinees were usually equivalent to or greater than to injected vaccines. 540, 541, 644, 645, 649, 650, [653] [654] [655] [656] [657] [658] [659] [660] [661] [662] [663] [664] [665] For example, Dilraj et al found that 96.4%, 94% and 86% of schoolchildren who received aerosol measles vaccine had antibody titers >300 IU/L at 1, 2 and 6 years after vaccination, respectively, compared to 91.4%, 87% and 73% among injected vaccinees. 541, 664, 665 In addition to the clinical trials, de Castro reported >3.7 million children in Mexico were vaccinated by aerosol with no serious adverse events noted. 666 A subsequent outbreak investigation showed measles attack rates of 0.8% among aerosol-vaccinated children compared to 14.6% among injection vaccinees and 26.2% among the unvaccinated. The third pattern noted is that the aerosol route among children ≤12 months of age usually produced an immune response lower than that by injection when the two routes are compared directly. 538, 539, 648, [655] [656] [657] [658] [659] 662, 667, 668 For example, Wong-Chew et al found vaccination by injection provided immunity in 100% of 12-month-old and 9-month-old infants, while the rates among aerosol recipients were only 86% and 23%, respectively. 538, 539 No severe adverse events following aerosol measles vaccination have been reported in any of the studies. Rates of minor adverse events, when reported, have typically been less than or the same as vaccination by injection. 538,539,541,661,663,669 Based on the encouraging results of prior trials, the World Health Organization (WHO), in partnership with CDC and the American Red Cross, leads the Measles Aerosol Project. Its goal is licensure in the developing world of at least one live, attenuated aerosol measles vaccine consisting of the delivery device and the associated vaccine. The project has already documented immunogenicity, and safety (the lack of local or systemic toxicity) in animal studies. 547 Three devices were selected for Phase I clinical trials based on the criteria of 1) critical performance data, 2) usability under fi eld conditions, 3) vaccine potency during nebulization and 4) existing licensure for other uses. As of December, 2006, phase I clinical trials are in progress in India.

IN delivery of live attenuated rubella vaccine was investigated during the 1970s in multiple clinical trials. [670] [671] [672] [673] [674] [675] [676] [677] Ganguly et al demonstrated that drops or spray produced mucosal IgA antibody, equivalent serum IgG antibody, and better protection against reinfection by IN challenge of vaccine virus compared to subcutaneous vaccination. 672 The IN subjects, however, had higher rates of mild adverse events, usually rhinitis and sore throat. More recently, Sepulveda et al found aerosolized measles-rubella combination vaccine in school-age children not previously vaccinated against rubella produced high levels of rubella immunity, equivalent to subcutaneous administration. Fewer adverse events were reported in the aerosol group. 661 Recombinant viruses acting as vectors by incorporation of a gene expressing a heterologous antigen have similar advantages as conventional attenuated live virus vaccines. They deliver the antigen code into cells and get it replicated to activate the immune system. Viruses used as vaccine vectors ideally should have very low pathogenic potential, even in the immunocompromised, and the capacity to hold the necessary foreign genes expressing the desired antigens, promoters and adjuvants. Viruses which naturally infect or grow in respiratory tissues are especially well suited as vectors for respiratory immunization. Some viruses studied as vaccine vectors in animal models include adenoviruses, poxviruses, vesicular stomatitis virus and adeno-associated virus. [678] [679] [680] [681] [682] [683] [684] [685] [686] [687] [688] [689] IN adenovirus vectors produced immune responses against many diseases in several animal models (Table 61-2) . 169,171,690-706 For example, a replication defective adenovirus expressing M. tuberculosis antigen delivered IN to mice provided better protection against respiratory challenge than BCG vaccine. 697 Vaccinia strains, such as modifi ed vaccinia Ankara (MVA), have also been used as effective vectors for respiratory immunization. 603, [707] [708] [709] For example, an IN MVA vector expressing an HIV-1 antigen induced antigen-specifi c mucosal CD8( + ) T-cells in genital tissue and draining lymph nodes of mice, along with serum and vaginal antibodies. 710 One caveat to vectored vaccines is that pre-existing immunity in the population to the vector virus, either by natural exposure or by previous use in another vaccine, may reduce its effectiveness.

Bacteria have a major advantage over viruses as vaccine vectors because of their higher capacity for insertion of the heterologous genes expressing antigens, adjuvants, or plasmids for DNA vaccination (described in the next section). 613 Animal models of respiratory immunization have been used to study attenuated respiratory pathogens such as Mycobacterium bovis bacille Calmette-Guérin (BCG) and attenuated Bordetella pertussis, as well as non-respiratory pathogens such as salmonella and shigella (Table 61 -2). [711] [712] [713] Commensal bacteria such as food grade strains of lactococcus, lactobacillus and Streptococcus gordonii have also been explored as vaccine vectors. [714] [715] [716] [717] Bacterial expression of adjuvants such as CTB, IL-6 and IL-12 has been shown to increase the respiratory vaccine immune response. 718, 719 A potential risk of administering live microbes was revealed in mice who developed dose-dependent granulomatous BCG infi ltration of the lungs after IN but not subcutaneous vaccination. 720 As with viruses, pre-existing immunity to the bacterial vector may diminish the immune response. 721 Several studies in mice have demonstrated an improved immune response to conventional BCG vaccine delivered IN or by aerosol inhalation, compared to injection. 708, 718, 720, [722] [723] [724] [725] [726] The studies that also included a challenge found superior protection of the respiratory route over injection. Attenuated M. tuberculosis has also been immunogenic by the respiratory route. 727 Recombinant BCG has been used to express various heterologous antigens, including simian immunodefi ciency virus, Borrelia burgdorferi and Streptococcus pneumoniae. 728 [736] [737] [738] [739] Attenuated recombinant salmonella vaccines produced strong immune responses against a wide variety of pathogens when delivered IN in rodents. [740] [741] [742] [743] [744] [745] [746] [747] [748] [749] Similar results were reported for IN shigella vectors against enterotoxigenic E. coli and tetanus. 750, 751 DNA vaccines DNA vaccination involves the delivery of eponymous plasmids directly into host cells to express the desired antigens. 752 Delivery of 'naked' DNA to the respiratory tract as a vaccine has been studied in animal models for many diseases. [753] [754] [755] [756] [757] [758] [759] [760] [761] [762] [763] [764] [765] [766] [767] [768] [769] [770] [771] For example, Kuklin found nasal delivery of a herpes simplex DNA vaccine generated higher levels of vaginal IgA than by the IM route, although the IM vaccine produced stronger serum antibodies and better protection against challenge. 772 Live attenuated bacteria, especially salmonella and shigella, have been vectored to produce DNA for IN vaccination. 750, [773] [774] [775] [776] For example, cotton rats vaccinated with attenuated salmonella vaccine expressing DNA encoding for measles antigens resulted in signifi cant reduction in measles virus titers in lung tissues following challenge. 777 Virosomes, liposomes and microparticles-discussed next-have also delivered respiratory DNA vaccines. [778] [779] [780] [781] Non-replicating vaccine delivery systems Non-replicating vaccine delivery systems, including ISCOMs, liposomes, microparticles, nanoparticles, virosomes and viruslike particles (VLP), mimic live viruses in how they deliver antigen and adjuvant. They are particles about the same size as viruses, allowing similar uptake by antigen presenting cells. Many include a lipid component to increase cell membrane permeability, as well as viral or bacterial proteins to activate the immune system. Liposomes are vesicles composed of a phospholipid bilayer membrane. Antigen can be packaged in its aqueous core, inside the lipid bilayer, or on the outside of the membrane. 782-784 A liposomal HIV-1 delivered IN to mice resulted in strong IgG and IgA responses in serum and vaginal washes. 785 VLPs are aggregates of viral proteins that may include a lipid component. 786 IN immunization of mice with a VLP infl uenza vaccine demonstrated a higher antibody response than injection of the same vaccine, and provided 100% protection to challenge by 5 LD50. 787 Virosomes have lipid bilayer membranes with embedded viral proteins and resemble viruses except they lack the genetic material needed to replicate. 788, 789 An IN virosomal anti-cancer vaccine enhanced the immunologic and protective activity of the vaccine in mice. 790 ISCOMs are cage-like structures roughly 40 nm size composed of 12 subunits of saponin (such as Quil A) and cholesterol. Several antigens administered IN in ISCOM-based vaccines produced strong systemic and mucosal immune responses. 575, [791] [792] [793] [794] [795] For example, an IN respiratory syncytial virus ISCOM vaccine induced high levels of serum IgG and IgA in the respiratory tract which persisted for 22 weeks. 791 Respiratory delivery can also be enhanced by packaging antigens and adjuvants into microparticles or nanoparticles composed of polymers of biodegradable materials such as polylactide (PLA) and polylactide co-glycolide (PLGA), or into biopolymers such as chitin or chitosan. [796] [797] [798] [799] [800] [801] [802] Microparticles can be designed to slowly release antigens to increase the duration of antigen presentation. Carcaboso et al reported that mice immunized IN with a synthetic malaria vaccine encapsulated into 1.5 micron microparticles of PLGA had signifi cantly higher antigenspecifi c serum IgG titers than control mice vaccinated subcutaneously with alum adjuvant. 803 IN immunization of mice with an infl uenza vaccine in chitin microparticles yielded protection against virus challenge, even against a non-vaccine strain. 804 

Vaccines based on any of the above delivery systems could potentially be produced as dry powders with particle sizes suitable for delivery to the respiratory tract. [805] [806] [807] With appropriate formulation, powders can be highly thermostable which reduces the need for the cold chain. Powders can be prepackaged in inexpensive, single use respiratory delivery devices and delivered dry without aqueous reconstitution. Dry powder delivery to the lung typically requires active inhalation and thus may be diffi cult with small children. However, two potential delivery solutions for this age group are direct nasal delivery and dis-pensing the powder into a reservoir or 'spacer' from which the child can breathe normally. An IN infl uenza dry powder vaccine elicited high titers of nasal anti-infl uenza IgA as well as serum antibody titers equivalent to injected vaccine when administered to rats. 546 The powder formulation showed no loss of potency when stored at 25ºC and 25% relative humidity (RH) for up to 12 weeks. In one experiment it maintained full potency for 2 weeks at 40ºC and 75% RH. Impermeable packaging which maintains powders dry at very low humidity may maintain potency to substantially increase their shelf life. IN dry powder formulations of an anthrax vaccine have provided complete protection against inhalational anthrax challenge (103 LD50) in rabbits while providing superior stability compared to liquid formulations. 54, 807a,807b Dry powder formulations have also been tested for measles vaccines. Early formulations milled to a fi ne powder retained adequate potency, but immune responses were poor when delivered to the respiratory tract of macaques. 805,807 AKTIV-DRY 808 used a novel spray-drying system to manufacture and test powder formulations of live attenuated measles vaccines. Measles virus plaque assays demonstrated potency losses in the drying process of 0 to 22%, which is comparable to losses seen with lyophilization. 809 AKTIV-DRY is working with key partners including the Serum Institute of India(SII), CDC and the University of Colorado on a fi ve-year project funded at over $19 million under the Grand Challenges in Global Public Health program to refi ne the formulation, complete animal and clinical testing, license the vaccine and establish dry powder measles vaccine production capacity at SII. 810 

The respiratory route is common in veterinary medicine. 811 Aerosol vaccines for the IN route or pulmonary inhalation are commercially available for cows (bovine herpes virus-1, parainfl uenza virus-3), pigs (Salmonella), horses (infl uenza, Streptococcus equi), dogs (Bordetella bronchiseptica), cats (feline calcivirus, feline herpesvirus-1) and chickens (infectious bronchitis virus, infectious laryngotracheitis virus, Newcastle disease virus). Almost all of the respiratory veterinary vaccines use live attenuated pathogens.

Many bioterror or biowarfare agents cause life-threatening respiratory infections, and could be dispensed as aerosols. Thus, vaccine-induced mucosal immunity may be very useful. Compared to the parenteral route, respiratory vaccination increased survival following aerosol exposure of deadly agents in animal studies. For example, a microsphere-based liquid anthrax vaccine delivered IN to mice completely protected against aerosol challenge with anthrax spores. 812 Two doses of human parainfl uenza virus vectored Ebola vaccine were highly immunogenic in macaques and protected all animals against lethal Ebola virus challenge. 812a A powdered formulation anthrax vaccine with CPG ODNs administered IN to rabbits also provided full protection. 54 Other bioterror agents for which respiratory vaccines have shown increased protection against aerosol challenge include Francisella tularensis, staphylococcal enterotoxin B (SEB), Burkholderia mallei (glanders) and Yersinia pestis (plague). [813] [814] [815] [816] [817] The threat of a global pandemic of respiratory disease such as infl uenza or severe acute respiratory syndrome (SARS) is a major public health concern. Respiratory vaccination may be useful in a pandemic setting because of the ease of administration for mass vaccination and the potential for enhanced mucosal immunity resulting in decreased disease transmission. Simple respiratory vaccination devices, such as single use dry powder inhalers, could be widely distributed to avoid the need to congregate for mass vaccination. IN delivery of salmonella vectored vaccine against the SARS coronavirus resulted in higher production of specifi c IgG and IgA than orogastric, intraperitoneal, or intravenous administration and provided high levels of specifi c cytotoxic T lymphocytes in Balb/c mice. 817a Two doses of IN, live attenuated, H5N1 infl uenza A vaccine fully protected mice and ferrets against pulmonary replication of homologous and heterologous wild type H5N1 strains. 817b Protection against antigenically diverse strains is highly desirable for a pandemic vaccine because of rapid changes in the infl uenza surface antigens.

Cutaneous, jet-injected, respiratory and other novel delivery methods may overcome the drawbacks of the traditional needle and syringe. However, demonstrating non-inferiority to the traditional route for existing vaccines will require expensive clinical data not yet generated for some of these methods. 21 Economic analysis that recognizes the hidden costs of needles and syringes may justify the necessary R&D investment. For diseases not yet vaccine-preventable-such as gonorrhea, herpes simplex, HIV, Chlamydia, respiratory syncytial virus, parainfl uenza and SARS-these alternate routes, taking advantage of the cutaneous or respiratory immune systems and their novel adjuvants and immunopotentiators, may fi nally provide vaccines to conquer them. 

53 Figure 61-3E, Norwood Abbey. 201 Figure 61-3F, Altea Therapeutics. 220 Figure 61-3G, PowderMed. 257 Figure 61-4E, Mada. 346 Figure 61-4F, Activa Brand Products

University of Pittsburgh) for lending vaccinostyle and rotary lancet (Fig. 61-2A,B), Robert H. Thrun (Anchor Products Company) for surgical needle (Fig. 61-2C) and to the following organizations and individuals for photographs, pre-publication manuscripts, reference material, fact-checking and other assistance

OptiNose 543 (Per Gisle Djupesland), PATH (Darin Zehrung)

Development of the global smallpox eradication programme

The Greatest Killer: Smallpox in History

Un prestigieux centenaire polytechnicien Charles-Gabriel Pravaz

Description of an instrument for the subcutaneous introduction of fl uids in affections of the nerves

New method of treating neuralgia by the direct application of opiates to the painful points

Compte rendu sommaire des expériences faites à Pouilly-le-Fort, près Melun, sur la vaccination charbonneuse (avec la collaboration de MM

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Safe management of wastes from healthcare activities

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Mass vaccination programs in developing countries

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Acne treatment by a dissolvable micro-needle patch

microtine technology acquired from Proctor & Gamble Company)

The Proctor & Gamble Company)

United States Patent and Trademark Offi ce

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Lack of pain associated with microfabricated microneedles

Mid-infrared laser ablation of stratum corneum enhances in vitro percutaneous transport of drugs

Transdermal drug delivery enhanced and controlled by erbium:YAG laser: a comparative study of lipophilic and hydrophilic drugs

Laserassisted penetration of topical anesthetic in adults

Laser-assisted anesthesia reduces the pain of venous cannulation in children and adults: a randomized controlled trial

Photomechanical transcutaneous delivery of macromolecules

Topical drug delivery in humans with a single photomechanical wave

Photomechanical delivery of 100-nm microspheres through the stratum corneum: implications for transdermal drug delivery

Electric Ions and Their Use in Medicine. London: Robman

Transdermal iontophoretic delivery of ketoprofen through human cadaver skin and in humans

Electrically Assisted Transdermal and Topical Drug Delivery

Transdermal drug delivery: overcoming the skin's barrier function

Synergistic effects of iontophoresis and jet injector pretreatment on the in-vitro skin permeation of diclofenac and angiotensin II

Iontophoretic drug delivery

New Jersey 07410

ALZA Corporation

Iontophoresis: electrorepulsion and electroosmosis

Enabling topical immunization via microporation: a novel method for pain-free and needlefree delivery of adenovirus-based vaccines

Needlefree skin patch delivery of a vaccine for a potentially pandemic infl uenza virus provides protection against lethal challenge in mice

Transdermal insulin infusion through thermally created micropores in humans

Transdermal basal insulin delivery through micropores

Fluorescein kinetics in interstitial fl uid harvested from diabetic skin during fl uorescein angiography: Implications for glucose monitoring

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A practical assessment of transdermal drug delivery by skin electroporation

In vivo effi cacy and safety of skin electroporation

Transdermal delivery of macromolecules using skin electroporation

Radiofrequency-driven skin microchanneling as a new way for electrically assisted transdermal delivery of hydrophilic drugs

Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases

Enhanced delivery of naked DNA to the skin by non-invasive in vivo electroporation

Needle-free topical electroporation improves gene expression from plasmids administered in porcine skin

DNA electrotransfer: its principles and an updated review of its therapeutic applications

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DNA electroporation prime and protein boost strategy enhances humoral immunity of tuberculosis DNA vaccines in mice and non-human primates

SE-227 36 Lund

The Woodlands, Texas 77381

Enhancement of immune responses to an HBV DNA vaccine by electroporation

Ultrasound-mediated transdermal protein delivery

Low-frequency sonophoresis: a review

Ultrasound and transdermal drug delivery

Lowfrequency ultrasound as a transcutaneous immunization adjuvant

Transdermal delivery of insulin by ultrasonic vibration

Ultrasoundenhanced transdermal transport

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Tucson, AZ 85719

High velocity microprojectiles for delivering nucleic acids into living cells

Delivery of DNA to skin by particle bombardment

Nonviral Gene Transfer Techniques

The use of Th1 cytokines, IL-12 and IL-23, to modulate the immune response raised to a DNA vaccine delivered by gene gun

Route and method of delivery of DNA vaccine infl uence immune responses in mice and nonhuman primates

Gene gun bombardment with gold particles displays a particular Th2-promoting signal that overrules the Th1-inducing effect of immunostimulatory CpG motifs in DNA vaccines

UK (subsidiary of Pfi zer)

Needle-free epidermal powder immunization

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Epidermal powder immunization of mice and monkeys with an infl uenza vaccine

Powder and particle-mediated approaches for delivery of

Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection

Tetanus toxoid-loaded transfersomes for topical immunization

Hypodermic fl uid dispenser

Needle assisted jet injector

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19446-4520, USA; amojet@aol.com (the Am-O-Jet™ is an exact design of the out-of-patent Ped-O-Jet ® device)

USA (successor of Medi-Ject, Daystrol-Scientifi c, and Derata corporations; Vaccijet™ technology acquired in 2001 from Endos Pharma, Laons, France)

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Comparative evaluation of three different intramuscular delivery methods for DNA immunization in a nonhuman primate animal model

Voronezh, Russia; www. chimavtomatika.ru/ (technology developed initially at All-Union Scientifi c Research Institute of Surgical Equipment and Tools -VNIIKHAI

Lechenie zabolevanii legkikh vnutrilegochnym ugol'no-struinym vvedeniem lekarstvennykh veshchestv [Treatment of lung diseases by intrapulmonary jet injection of drugs

Optimization of DNA-based vaccination in cows using green fl uorescent protein and protein A as a prelude to immunization against staphylococcal mastitis

Available at: www.emsmedical.com (EMS/MPM device from

In vivo gene transfer by low-volume jet injection

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Shawnee Mission, KS 66214, USA. Online. Available at: www

Ped-O-Jet previously manufactured by Scientifi c Equipment Manufacturing Corporation (SEMCO), Lodi, NJ and Larchmont, NY, and developed by Medicinal Equipment Development Laboratory

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Ministry of Defense

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CA 92618-1605, USA. Online. Available at

Online. Available at: www.path.org (MEDIVAX™ project in partnership with

Available at: www.sanofi pasteur.com (jet injection technology developed under corporate predecessors: Institut Mérieux

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Un progrès dans le domaine de l'injection sans aiguille

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France (formerly Béarn Mécanique Aviation SA, F-64143

com (absorbed in 1998 into drug delivery unit of Cardinal Health. Online. Available at: www.cardinal.com/pts/content/ delivery). K3 model was manufactured by Messer Griesheim GmbH (subsequently BIT Analytical Instruments GmbH

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Needle-free jet injection of a mixture of Japanese encephalitis DNA and protein vaccines: A strategy to effectively enhance immunogenicity of the DNA vaccine in a murine model

Medical Jet s.r.l

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com); marketed by Scientifi c Equipment Manufacturing Corporation (SEMCO)

USA (technology originated by Weston Medical, plc and then further developed by Aradigm Corporation)

The penetration of a soft solid by a liquid jet, with application to the administration of a needle-free injection

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Pan American Health Organization

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Clinical evaluation of the effi cacy of anesthesia and patient preference using the needle-less jet syringe in a pediatric dental practice

A comparison of a needle-free injection system for local anesthesia versus EMLA for intravenous catheter insertion in the pediatric patient

Preanesthetic medication of children with midazolam using the Biojector jet injector

A new route, jet injection for anesthetic induction in children: I. midazolam dose-range fi nding studies

Hypospray administration of penicillin in the treatment of gonorrhea

Administration of penicillin and streptomycin by means of the Hypospray apparatus

Prophylactic low-dose heparin by jet injection

Effectiveness of a jet injection system in administering morphine and heparin to healthy adults

Enfuvirtide plasma levels and injection site reactions using a needle-free gas-powered injection system (Biojector)

Comparison of two steroid preparations used to treat tennis elbow, using the Hypospray

Medijector-A new method of corticosteroid-anesthetic delivery

Jet injection of drugs into malignant neoplasms

The treatment of palmar and plantar warts using natural alpha interferon and a needleless injector

The application of insulin using the jet injector DG-77

Reduction of variability in the anovulatory period following medroxyprogesterone acetate injection by using jet injectors

Growth hormone treatment without a needle using the Preci-Jet 50 transjector

Are needle-free injections a useful alternative for growth hormone therapy in children? Safety and pharmacokinetics of growth hormone delivered by a new needle-free injection device compared to a fi ne gauge needle

A comparative evaluation of the jet injection technique (Hypospray) and the hypodermic needle for the parenteral administration of drugs: a controlled study

Transdermal drug delivery by jet injectors: energetics of jet formation and penetration

Anatomic evaluation of a jet injection instrument designed to minimize pain and inconvenience of parenteral therapy

Jet injection of insulin vs. the syringe-and-needle method

Rontgenologische Darstellung der Gelenks-und Weichteilinfi ltration mit dem 'Hypospray Jet Injector'

Porton Jet injector

Studies on tissue penetration characteristics produced by jet injection

Visualization of injection depot after subcutaneous administration by syringe and needle-free device (Medi-Jector): fi rst results with magnetic resonance imaging

Jet injection in pediatric practice

Needle-free jet injections: dependence of jet penetration and dispersion in the skin on jet power

Guide to selection and use of Biojector syringes

Intramuscular or intralipomatous injections?

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A comparison of intradermal BCG vaccination by jet injection and by syringe and needle. A report from the Research Committee of the British Thoracic and Tuberculosis Assocation

Ped-o-jet et viabilité du BCG

Infl uence du Ped-o-jet sur la viabilité du vaccin BCG intradermique lyophilisé: étude au laboratoire

Jet gun or syringe? A trial of alternative methods of BCG vaccination

Comparison of BCG inoculation by conventional intradermal and jet methods

A comparison of jet injection with the mantoux test in mass skin testing with tuberculin

A new method of administering the tuberculin skin test

Consideraciones sobre el tuberculino-diagnóstico. Estudio comparativo del Mantoux y la jeringuilla Dermo-Jet

Mantoux tuberculin testing-Standard method vs. jet injection

Comparative tuberculin testing. Intradermal gun versus intradermal needle

Jet injector tuberculin skin testing: Methodology and results

Jet injector tuberculin skin testing: a comparative evaluation. Quantitative aspects

Intradermoréaction tuberculinique et vaccination B.C.G. intradermique par injecteur à jet sous pression

Viabilidade da aplicação do teste tuberculínico com o Dermo-jet

Skin testing: A comparison of the jet injector with the mantoux method

Jet-injectors in BCG vaccination

Module 5: Tuberculosis. Geneva: World Health Organization, Global Programme For Vaccines And Immunization, Expanded Programme On Immunization

Titration of live measles and smallpox vaccines by jet inoculation of susceptible children

Painless intravenous catheterization by intradermal jet injection of lidocaine: A randomized trial

Immunizatsiia assotsiirovannymi di-i trivaktsinami protiv chumy, tuliaremii i sibirskoi iazvy pri pomoshchi bezygol'nogo in'ektora

Russian: Plague, tularemia and anthrax immunization with associated di-and trivaccines using a jet injector

Bezygol'naia immunizatsiia assotsiirovannoi vaktsinoi protiv chumy, tuliaremii i sibirskoi iazvy

Vaccination anticholérique par voie intradermique au Pedojet. Réponse clinique et immunologique (d'après une expérience sénégalaise)

Clinical immunogenicity and tolerance studies of liquid vaccines delivered by jet-injector and a new single-use cartridge (Imule ® ): comparison with standard syringe injection

Administration of hepatitis A vaccine to a military population by needle and jet injector and with hepatitis B vaccine

Immunogenicity and safety of a new inactivated hepatitis A vaccine: a clinical trials with comparison of administration route

Hepatitis A vaccine administration: comparison between jet-injector and needle injection

Subcutaneous administration of inactivated hepatitis B vaccine by automatic jet injection

Hepatitis B vaccine administration: comparison between jetgun and syringe and needle

Effi cacité comparée de deux techniques de vaccination contre la grippe. Taux sérologique obtenus après administration du vaccine par le Porton Jet et la seringue

Intradermal infl uenza vaccination

Response of normal children to infl uenza A/New Jersey/76 virus vaccine administered by jet injector

Safety and immunogenicity of varying doses of trivalent inactivated infl uenza vaccine administered by needle-free jet injectors

Antibody response to poliomyelitis vaccine administered by jet injection

Essais de primo-vaccination antitétanique en un temps avec une anatoxine concentrée inoculée par injecteurs sans aiguille (Note préliminaire)

Reactions and serologic responses to monovalent acetone-inactivated typhoid vaccine and heat-killed TAB when given by jet injection

An evaluation of measles and smallpox vaccines simultaneously administered

Measles vaccination with reduced dosage

Clinical immunogenicity of measles, mumps and rubella vaccine delivered by the Injex jet injector: comparison with standard syringe injection

Vaccination against smallpox. II. Jet injection of chorioallantoic membrane vaccine

Smallpox vaccination by intradermal jet injection. C. Cutaneous and serological responses to primary vaccination in children

Immunologicheskaia effektivnost' privivok protiv ospy i tuliaremii bezygol'nym metodom [Russian: Immunological effectiveness of immunization against smallpox and tularemia by the jet injection method

Comparison of antibody response and patient tolerance of yellow fever vaccine administered by the Biojector ® needle-free injection system versus conventional needle/syringe injection

Meningococcal infections. 3. Studies of group A polysaccharide vaccines

The immunological responses observed in fi eld studies in Africa with group A meningococcal vaccines

Control of meningococcal infection in the African meningitis belt by selective vaccination

Control of epidemic meningococcal meningitis by mass vaccination

Epidemic of meningococcal meningitis in Bamako, Mali: epidemiological features and analysis of vaccine effi cacy

Évaluation sérologique d'une campagne de vaccination antiméningococcique de masse au Niger

Serologic response to serogroup C meningococcal vaccine in Brazilian preschool children

Disease prevention by meningococcal serogroup C polysaccharide vaccine in preschool children: Results after eleven months in Sao Paulo

Avaliação do efeito protector de vacina polissacaridica antimeningococica da grupa C em crianças de 6 a 36 meses

Genetic immunization by jet injection of targeted pDNA-coated nanoparticles

Phase I study in advanced cancer patients of a diversifi ed prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anticarcinoembryonic antigen immune responses

Microsphere encapsulation or Biojector™ delivery enhances adjuvanted DNA vaccines in rhesus macaques

Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with B-cell lymphoma

DNA mucosal HIV vaccine in humans

Evidence for antigen production in muscles by dengue and Japanese encephalitis DNA vaccines and a relation to their immunogenicity in mice

Hepatitis A in the US Army: epidemiology and vaccine development

Investigations into the risk of infection by the use of jet injectors

Effektivnost' kompleksnoi immunizatsii briushnotifoznoi vaktsinoi s poliana-toksinom v sochetanii s chumnym i ospennym antigenam [Effectiveness of comprehensive immunization with typhoid fever vaccine and polyanatoxin in combination with plague and small pox antigens

Foreign body granuloma caused by jet injection of tetanus toxoid

Painfulness of needle and jet injection in children with diabetes mellitus

Infl uenza Virus Vaccine Polyvalent (Types A and B)

Traumatic ulnar neuropathy from jet injection: Case Report

Transient neuropathy after needle-free injection outside of recommended sites

Side effect of the jet injector for the production of local anesthesia

Transference of blood by various inoculation devices

Special Investigations Section, Hepatitis Laboratories Division)

Jet vaccination

The risk of transmission of hepatitis B virus using jet injection in inoculation

Sterilität von Impfpistolen [Letter: Sterility of vaccination guns

Hepatitisubertragung durch Hochdruckinjektion?

Virus transmission by subcutaneous jet injection

Hepatitis B associated with jet gun injection-California

World Health Organization, Expanded Programme on Immunization. Transmission of hepatitis B associated with jet gun injection

An outbreak of hepatitis B associated with jet injections in a weight reduction clinic

Risk of virus transmission by jet injection

The risk of transmission of HIV and other blood-born diseases via jet injectors during immunization mass campaigns in Brazil

Vaccines in the Military: a Department of Defense-wide Review of Vaccine Policy and Practice. A Report for the Armed Forces Epidemiological Board

Potential for cross-contamination from use of a needleless injector

Bezygol'nye in'ektsii i 'shpritsevye' infektsii

A model to assess the infection potential of jet injectors used in mass immunisation

Outbreak of Mycobacterium chelonae infection associated with use of jet injectors

Prevalância e fatores associados a marcadores do virus da hepatite B em população rural do Brasil central. Rev Panam Salud Pública

Expanded Programme on Immunization, Global Advisory Group. IV. Injection equipment and sterilization practices

Centers for Disease Control and Prevention. General Recommendations on Immunization: Recommendations of the Advisory Committee on Immunization Practices (ACIP)

Subj: MMQC-97-1169 Automatic jet hypodermic injection units/ withdrawal (DPSC 970147)

Pilot safety study to determine the ability of the protector cap jet injector to prevent cross-contamination. Online. Available at: www. clinicaltrials.gov/ct/show/NCT00219453. Accessed November 22, 2006. 518a. Program for Appropriate Technology in Health

The Chilling True Story of the Largest Covert Biological Weapons Program in the World-Told from Inside by the Man Who Ran It

Immunizatsiia zhivoi sibireiazvennoi vaktsinoi STI pri pomoshchi bezygol'nogo in″ektora v eksperimente

Immunizatsiia liudei protiv sibirskoi iazvy bezygol'nym metodom [Russian: Immunization against anthrax by a needleless method

Bezygol'nyi metod immunizatsii assotsiirovannymi sorbirovannymi vaktsinami [Russian: Jet method of immunizing with associated adsorbed vaccines

Kliniko-immunologicheskoe obosnovanie assotsiirovannoi immunizatsii [Russian: The clinico-immunological validation of associated immunization

Breath actuated device improves delivery to target sites beyond the nasal valve

Role of epithelium in antigen presentation

Antigen handling and presentation by mucosal dendritic cells and macrophages

Inductive and effector tissues and cells of the mucosal immune system

Comparison of nasal deposition and clearance of aerosol generated by a nebulizer and an aqueous spray pump

Intranasal drug delivery-potential advantages and limitations from a clinical pharmacokinetic perspective

Mucosal and other mechanisms of resistance in the respiratory tract: An overview

The human mucosal B-cell system

Mucosal vaccines: the promise and the challenge

The common mucosal immune system and current strategies for induction of immune responses in external secretions

Immune response versus mucosal tolerance to mucosally administered antigens

Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice

Intranasal immunization of mice with group B streptococcal protein Rib and cholera toxin B subunit confers protection against lethal infection

Effectiveness of the quillaja saponin semisynthetic analog GPI-0100 in potentiating mucosal and systemic responses to recombinant HagB from Porphyromonas gingivalis

Mucosal immunization with virus-like particles of simian immunodefi ciency virus conjugated with cholera toxin subunit B

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigenspecifi c serum and mucosal immune responses in the presence of anti-rCTB antibodies

Mucosal immunization with recombinant MOMP genetically linked with modifi ed cholera toxin confers protection against Chlamydia trachomatis infection

A novel adjuvant for mucosal immunity to HIV-1 gp120 in nonhuman primates

A comparative evaluation of nasal and parenteral vaccine adjuvants to elicit systemic and mucosal HIV-1 peptide-specifi c humoral immune responses in cynomolgus macaques

Improved design and intranasal delivery of an M2e-based human infl uenza A vaccine

The combined CTA1-DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specifi c targeting of B cells

The nontoxic CTA1-DD adjuvant enhances protective immunity against Helicobacter pylori infection following mucosal immunization

Phase I evaluation of intranasal trivalent inactivated infl uenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform

Enhancement of intranasal vaccination in mice with deglycosylated chain A ricin by LTR72, a novel mucosal adjuvant

The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and effi cacy of intranasally administered vaccines

The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause infl ammation or accumulate in the nervous tissues

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge

Intranasal administration of a proteosome-infl uenza vaccine is well-tolerated and induces serum and nasal secretion infl uenza antibodies in healthy human subjects

Outer membrane vesicles of Neisseria lactamica as a potential mucosal adjuvant

A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern

Protollin: a novel adjuvant for intranasal vaccines

An intranasal vaccine targeting both the Bacillus anthracis toxin and bacterium provides protection against aerosol spore challenge in rabbits

Enhanced immunity and protective effi cacy against SIVmac251 intrarectal challenge following ad-SIV priming by multiple mucosal routes and gp120 boosting in MPL-SE

Effi cient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant

Improved intranasal immunization with liveattenuated measles virus after co-inoculation of the lipopeptide MALP-2

Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis

A bacterial fl agellin, Vibrio vulnifi cus FlaB, has a strong mucosal adjuvant activity to induce protective immunity

Nasal immunization studies using liposomes loaded with tetanus toxoid and CpG-ODN

Protective immunity to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant system IC31

Safety and effi cacy of nasal application of CpG oligodeoxynucleotide as a mucosal adjuvant

Intranasal CpGoligodeoxynucleotide is a potent adjuvant of vaccine against Helicobacter pylori, and T helper 1 type response and interferon-gamma correlate with the protection

Nasal vaccination with CpG oligodeoxynucleotide induces protective immunity against nontypeable Haemophilus infl uenzae in the nasopharynx

Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration

Plasmid interleukin-23 (IL-23), but not plasmid IL-27, enhances the protective effi cacy of a DNA vaccine against Mycobacterium tuberculosis infection

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors

Mucosal application of plasmid-encoded IL-15 sustains a highly protective anti-Herpes simplex virus immunity

Control of simian/human immunodefi ciency virus viremia and disease progression after IL-2-augmented DNA-modifi ed vaccinia virus Ankara nasal vaccination in nonhuman primates

Increased protection against pneumococcal disease by mucosal administration of conjugate vaccine plus interleukin-12

Infl uence of DNA encoding cytokines on systemic and mucosal immunity following genetic vaccination against herpes simplex virus

Cytokines as adjuvants for the induction of mucosal immunity

Type I IFN as a vaccine adjuvant for both systemic and mucosal vaccination against infl uenza virus

Chitosan and its use as a pharmaceutical excipient

Chitosan as a novel nasal delivery system for vaccines

Intranasal immunization with genetically detoxifi ed diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan

Induction of systemic and mucosal immune responses to human immunodefi ciency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes

Antigen delivery systems I: Non-living microparticles, liposomes, and Immune Stimulating Complexes (ISCOMS)

Antigen delivery systems II: Development of live recombinant attenuated bacterial antigen and DNA vaccine delivery vector vaccines

Recombinant live viral vectors as vaccines for mucosal immunity

Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans

Antigen delivery systems

Comparative study of the immune response in mice immunized with four live attenuated strains of mumps virus by intranasal or intramuscular route

The specifi c activity and immunological safety of a live mumps vaccine from the Leningrad-3 strain in intranasally revaccinated adult subjects

An intranasal method of revaccination against mumps

Vaccination by non-parenteral routes: characteristics of immune response

Responses of varicella zoster virus (VZV)-specifi c

Humoral immunoresponse to varicella-zoster virus pernasally coadministered with Escherichia coli enterotoxin in mice

Genetically engineered herpes simplex viruses that express IL-12 or GM-CSF as vaccine candidates

Genetic stability determinants of temperature sensitive, live attenuated respiratory syncytial virus vaccine candidates

Live-attenuated intranasal parainfl uenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses

Coffi n SE. Induction of mucosal B-cell memory by intranasal immunization of mice with respiratory syncytial virus

Evaluation of combined live, attenuated respiratory syncytial virus and parainfl uenza 3 virus vaccines in infants and young children

Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses

Protection by intranasal immunization of a nef-deleted, nonpathogenic SHIV against intravaginal challenge with a heterologous pathogenic SHIV

Immune responses and protection against vaginal infection after nasal or vaginal immunization with attenuated herpes simplex virus type-2

Intra-nasal infection of macaques with Yellow Fever (YF) vaccine strain 17D: a novel and economical approach for YF vaccination in man

Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective effi cacy of a modifi ed vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-defi cient host

Comparative effi cacy of modifi ed vaccinia Ankara (MVA) as a potential replacement smallpox vaccine

Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modifi ed vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model

Available at: www.medimmune.com

Correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal infl uenza virus vaccine

Effectiveness of live attenuated intranasal infl uenza virus vaccine in healthy working adults: a randomized trial

Superior relative effi cacy of live attenuated infl uenza vaccine compared with inactivated infl uenza vaccine in young children with recurrent respiratory tract infections

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

Live attenuated versus inactivated infl uenza vaccine in infants and young children

Safety, effi cacy, and effectiveness of cold-adapted infl uenza vaccine-trivalent against communityacquired, culture-confi rmed infl uenza in young children attending day care

Effi cacy and safety of a live attenuated, cold-adapted infl uenza vaccine, trivalent against cultureconfi rmed infl uenza in young children in Asia

Effi cacy of vaccination with live attenuated, coldadapted, trivalent, intranasal infl uenza virus vaccine against a variant (A/Sydney) not contained in the vaccine

Herd immunity in adults against infl uenza-related illnesses with use of the trivalent-live attenuated infl uenza vaccine (CAIV-T) in children

Studies with live attenuated measles virus vaccine: clinical and immunologic responses in institutionalized children

Clinical trials with living attenuated measles virus vaccines

Studies on an attenuated measles virus vaccine

Antimeasles vaccination by natural routes-experimental background and practical consequences

Failure of measles vaccine sprayed into the oropharynx of infants

Theoretical substantiation and effectiveness of immunization with aerosols of liquid measles vaccine

Studies with live attenuated measles virus vaccine

Measles vaccination of Thai infants by intranasal and subcutaneous routes: possible interference from respiratory infections

Immune response to Edmonston-Zagreb measles virus strain in monovalent and combined MMR vaccine

Studies on the combined use of killed and live measles vaccine. II. Advantages of the inhalation method

Studies on the combined use of killed and live measles vaccine. III. Conditions for the 'take' of live vaccine

Clinical trials of inhaled aerosol of human diploid and chick embryo measles vaccine

Successful immunization of children with and without maternal antibody by aerosolized measles vaccine. I. Different results with undiluted human diploid cell and chick embryo fi broblast vaccines

Successful immunization of infants with and without maternal antibody by aerosolized measles vaccine. II. Vaccine comparisons and evidence for multiple antibody response

High effectiveness of aerosolized chick embryo fi broblast measles vaccine in seven month old and older infants

Diploid cell measles vaccine

Measles in Ghana: A trial of an alternative means of administration of measles vaccine

A randomized trial demonstrating successful boosting responses following simultaneous aerosols of measles and rubella vaccines in school age children

Aerosolized measles and measles-rubella vaccines induce better measles antibody booster responses than injected vaccines: randomized trials in Mexican schoolchildren

Immunologic studies of specifi c mucosal and systemic immune responses in Mexican school children after booster aerosol or subcutaneous immunization with measles vaccine

Persistence of measles antibody two years after revaccination by aerosol or subcutaneous routes

Aerosol and subcutaneous measles vaccine: measles antibody responses 6 years after re-vaccination

La vacunación antisarampionosa en Mexico por el metodo de aerosol

Comparison of Edmonston-Zagreb and Schwarz strains of measles vaccine given by aerosol or subcutaneous injection

Immunization by inhalation of aerosolized measles vaccine

Reacciones tempranas en escolares vacunados con antisarampionosa en aerosol

Risk of contact infection after intranasal rubella vaccination

Rubellaspecifi c serum and nasopharyngeal immunoglobulin responses following naturally acquired and vaccine-induced infection. Prolonged persistence of virus-specifi c IgM

Rubella immunization of volunteers via the respiratory tract

Studies with rubella vaccine (RA 27-3) using the subcutaneous and intranasal routes

Intranasal vaccination with RA 27-3 attenuated rubella virus

Immunisation of women with rubella (RA27-3) vaccine administered intranasally

Immunisation of schoolchildren with rubella (RA27-3) vaccine. Intranasal and subcutaneous administration

Subcutaneous and intranasal administration of RA 27-3 rubella vaccine. Alone and in conjunction with live attenuated measles vaccine

Intranasal vaccination with recombinant adeno-associated virus type 5 against human papillomavirus type 16 L1

A novel recombinant adeno-associated virus vaccine reduces behavioral impairment and beta-amyloid plaques in a mouse model of Alzheimer's disease

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodefi ciency virus

Use of recombinant virusvectored tuberculosis vaccines for respiratory mucosal immunization

An optimized vaccine vector based on recombinant vesicular stomatitis virus gives high-level, longterm protection against Yersinia pestis challenge

Elicitation of neutralizing antibodies by intranasal administration of recombinant vesicular stomatitis virus expressing human immunodefi ciency virus type 1 gp120

Priming with plasmid DNAs expressing interleukin-12 and simian immunodefi ciency virus gag enhances the immunogenicity and effi cacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus

Strong cellular and humoral anti-HIV Env immune responses induced by a heterologous rhabdoviral prime-boost approach

Successful mucosal immunization of cotton rats in the presence of measles virus-specifi c antibodies depends on degree of attenuation of vaccine vector and virus dose

Attenuated vesicular stomatitis viruses as vaccine vectors

High-level primary CD8(+) T-cell response to human immunodefi ciency virus type 1 gag and env generated by vaccination with recombinant vesicular stomatitis viruses

Vaccination with a recombinant vesicular stomatitis virus expressing an infl uenza virus hemagglutinin provides complete protection from infl uenza virus challenge

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus

Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen

Effi cient intranasal immunization of newborn mice with recombinant adenovirus expressing rotavirus protein VP4 against oral rotavirus infection

Intranasal immunisation with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus

Intranasal immunization with a replication-defi cient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity

Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis

Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodefi ciency virus vaccine and localization in the central nervous system

Mucosal immunization with a replication-defi cient adenovirus vector expressing murine cytomegalovirus glycoprotein B induces mucosal and systemic immunity

Induction of mucosal immunity with a replication-defective adenoviral recombinant

Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization

Induction of mucosal immunity in cotton rats to haemagglutinin-esterase glycoprotein of bovine coronavirus by recombinant adenovirus

Immunogenicity of recombinant adenovirushuman immunodefi ciency virus vaccines in chimpanzees following intranasal administration

Effi cacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model

Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B

Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice

Enhanced immunogenicity and protective effi cacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting with a recombinant modifi ed vaccinia virus Ankara

The immunogenicity and effi cacy of intranasally or parenterally administered replicationdefi cient vaccinia-parainfl uenza virus type 3 recombinants in rhesus monkeys

Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: Enhanced immunogenicity after DNA prime-Modifi ed Vaccinia Virus Ankara boost immunization schedule

Live bacterial vectors for intranasal delivery of protective antigens

Intranasal vaccination with a defi ned attenuated Francisella novicida strain induces gamma interferon-dependent antibody-mediated protection against tularemia

Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A

Nasal vaccination using live bacterial vectors

The use of live attenuated bacteria as a delivery system for heterologous antigens

A new attenuated Mycobacterium bovis vaccine protects brushtail possums (Trichosurus vulpecula) against experimental tuberculosis infection

Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations

Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum

Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice

Lactic acid bacteria as live vaccines

Immune response induced by recombinant Mycobacterium bovis BCG producing the cholera toxin B subunit

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis co-expressing antigen and cytokine

Intranasal bacille Calmette-Guerin (BCG) vaccine dosage needs balancing between protection and lung pathology

Prior immunity to homologous and heterologous Salmonella serotypes suppresses local and systemic anti-fragment C antibody responses and protection from tetanus toxin in mice immunized with Salmonella strains expressing fragment C

Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis

Intranasal BCG vaccination protects BALB/ c mice against virulent Mycobacterium bovis and accelerates production of IFN-gamma in their lungs

Intranasal vaccination of mice against infection with Mycobacterium tuberculosis

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG

Aerosol vaccination of the brushtail possum (Trichosurus vulpecula) with bacilli Calmette-Guerin: the duration of protection

A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential

A cocktail of Mycobacterium bovis BCG recombinants expressing the SIV Nef, Env, and Gag antigens induces antibody and cytotoxic responses in mice vaccinated by different mucosal routes

Safety and immunogenicity of recombinant Bacille Calmette-Guerin (rBCG) expressing Borrelia burgdorferi outer surface protein A (OspA

Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi

Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guerin vaccines expressing pneumococcal surface protein A

Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough

Attenuated Bordetella pertussis: new live vaccines for intranasal immunisation

Bordetella pertussis from functional genomics to intranasal vaccination

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis

Tetanus toxin fragment C-specifi c priming by intranasal infection with recombinant Bordetella pertussis

Production of non-typeable Haemophilus infl uenzae HtrA by recombinant Bordetella pertussis with the use of fi lamentous hemagglutinin as a carrier

Production of Neisseria meningitides transferrin-binding protein B by recombinant Bordetella pertussis

Induction of mucosal immune responses against a heterologous antigen fused to fi lamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis

Salmonella enterica serovar Typhi live vector vaccines delivered intranasally elicit regional and systemic specifi c CD8+ major histocompatibility class I-restricted cytotoxic T lymphocytes

Novel bacterial delivery system with attenuated Salmonella typhimurium carrying plasmid encoding Mtb antigen 85A for mucosal immunization: Establishment of proof of principle in TB mouse model

Recombinant Salmonella enterica serovar Typhi in a prime-boost strategy

Mucosally delivered Salmonella live vector vaccines elicit potent immune responses against a foreign antigen in neonatal mice born to naive and immune mothers

A Salmonella enterica serovar typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice

Nasal immunisation with Salmonella typhimurium producing rotavirus VP2 and VP6 antigens stimulates specifi c antibody response in serum and milk but fails to protect offspring

Nasal vaccination with attenuated Salmonella typhimurium strains expressing the Hepatitis b nucleocapsid: dose response analysis

Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium diffi cile toxin A

A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization

Intranasal immunogenicity of a Delta cya Delta crp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse

DeltaguaBA attenuated Shigella fl exneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin

Further characterization of delta aroA delta virG Shigella fl exneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli

DNA vaccines for mucosal immunity to infectious diseases

DNA vaccines: protective immunizations by parenteral, mucosal and gene gun inoculations

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model

Enhanced immunization after intranasal coadministration of Escherichia coli heat-labile enterotoxin B subunit and human papillomavirus 16-L1 DNA vaccine

Mucosallyadministered human-simian immunodefi ciency virus DNA and fowlpoxvirus-based recombinant vaccines reduce acute phase viral replication in macaques following vaginal challenge with CCR5-tropic SHIVSF162P3

Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in longterm HIV-1 neutralizing humoral mucosal and systemic immunity

Intranasal delivery of chitosan-DNA vaccine generates mucosal SIgA and anti-CVB3 protection

Nasal immunization of mice with a rotavirus DNA vaccine that induces protective intestinal IgA antibodies

Pulmonary DNA vaccination: concepts, possibilities and perspectives

Human immunodefi ciency virus type 2 DNA vaccine provides partial protection from acute baboon infection

Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of a DNA vaccine encoding HLA-A*0201-restricted Tcell epitopes of Mycobacterium tuberculosis

Intranasal administration of human immunodefi ciency virus type-1 (HIV-1) DNA vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against HIV-1

Intranasal immunization of a DNA vaccine with IL-12-and granulocyte-macrophage colony-stimulating factor (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cellmediated immune responses against HIV-1 antigens

Rapid and wide-reaching delivery of HIV-1 env DNA vaccine by intranasal administration

Protective immunity against infl uenza A virus induced by immunization with DNA plasmid containing infl uenza M gene

Protective DNA immunization against Chlamydia pneumoniae

M cell DNA vaccination for CTL immunity to HIV

Improved tuberculosis DNA vaccines by formulation in cationic lipids

Route and method of delivery of DNA vaccine infl uence immune responses in mice and non-human primates

Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization

Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for t cells against HIV

Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella fl exneri vector

Vaccination with a Shigella DNA vaccine vector induces antigen-specifi c CD8(+) T cells and antiviral protective immunity

Immunogenicity of an HIV-1 gag DNA vaccine carried by attenuated Shigella

Attenuated Salmonella enterica serovar Typhi and Shigella fl exneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specifi c immune responses and protection in cotton rats

Chitin Micro-Particles (CMP): a useful adjuvant for inducing viral specifi c immunity when delivered intranasally with an HIV-DNA vaccine

Mucosal immunization with HIV-1 gag DNA on cationic microparticles prolongs gene expression and enhances local and systemic immunity

Intranasal immunization with mumps virus DNA vaccine delivered by infl uenza virosomes elicits mucosal and systemic immunity

DNA vaccination against respiratory infl uenza virus infection

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

A controlled clinical study of the effect of nasal immunization with a Streptococcus mutans antigen alone or incorporated into liposomes on induction of immune responses

Intranasal immunisation of mice with liposomes containing recombinant meningococcal OpaB and OpaJ proteins

HIV-1 neutralizing antibodies in the genital and respiratory tracts of mice intranasally immunized with oligomeric gp160

Enhancement of mucosal immune responses by chimeric infl uenza HA/SHIV viruslike particles

Virus-like particle vaccine conferred complete protection against a lethal infl uenza virus challenge

Adjuvant and antigen delivery properties of virosomes

The virosome concept for infl uenza vaccines

Immune-reconstituted infl uenza virosome containing CD40L gene enhances the immunological and protective activity of a carcinoembryonic antigen anticancer vaccine

Induction of antibody responses in the common mucosal immune system by respiratory syncytial virus immunostimulating complexes

ISCOM is an effi cient mucosal delivery system for Mycoplasma mycoides subsp. Mycoides (MmmSC) antigens including high mucosal and systemic antibody responses

The combined CTA1-DD/ISCOMs vector is an effective intranasal adjuvant for boosting prior BCG immunity to Mycobacterium tuberculosis

Induction of protective and mucosal immunity against diphtheria by a immune stimulating complex (ISCOMS) based vaccine

Bovine respiratory syncytial virus ISCOMs-protection in the presence of maternal antibodies

Absorption enhancers for nasal drug delivery

Nanoparticles as carriers for nasal vaccine delivery

Microparticles for intranasal immunization

Chitosan for mucosal vaccination

Strong systemic and mucosal immune responses to surface-modifi ed PLGA microspheres containing recombinant hepatitis b antigen administered intranasally

Intranasal immunization of BALB/c mice with enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in biodegradable poly(DL-lactide-co-glycolide) microspheres

In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT

Potent, long lasting systemic antibody levels and mixed Th1/Th2 immune response after nasal immunization with malaria antigen loaded PLGA microparticles

Protection against infl uenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

A powder formulation of measles vaccine for aerosol delivery

Evaluation of novel aerosol formulations designed for mucosal vaccination against infl uenza virus

Measles vaccination of macaques by dry powder inhalation

Anthrax vaccine powder formulations for nasal mucosal delivery

Intranasal administration of dry powder anthrax vaccine provides protection against lethal aerosol spore challenge

Available at: www.aktiv-dry

Nearcritical Fluid Micronization of Stabilized Vaccines

Global Health. Needle-Free Delivery: Finding New Ways to Give Immunizations. Online. Available at: www.gcgh. org/subcontent.aspx?SecID=419. Accessed

Mucosal veterinary vaccines: Comparative vaccinology

Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice

Successful topical respiratory tract immunization of primates against Ebola virus

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice

Intranasal and intramuscular proteosomestaphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and effi cacy against lethal SEB intoxication in mice

Inactivated Francisella tularensis live vaccine strain protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-dependent fashion

Aerogenic immunization of man with live tularemia vaccine

Intranasal protollin/F1-V vaccine elicits respiratory and serum antibody responses and protects mice against lethal aerosolized plague infection

Type IVB pilus operon promoter controlling expression of the Severe Acute Respiratory Syndrome-associated coronavirus nucleocapsid gene in Salmonella enterica serovar Typhi elicits full immune response by intranasal vaccination

Live, attenuated infl uenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets

Available at: www.creare.com

Credits for previously unpublished photography: Figure 61 -