Pulmonary Vaccine Delivery: A Realistic Approach?

Abstract

Pulmonary vaccine delivery has gained increasing attention during the last decade because this vaccination method combines potential advantages such as the fact that it omits the use of needles and may elicit immunity at the port of entry for many pathogens. In this review the current status of pulmonary vaccination, the potential advantages of pulmonary vaccine delivery, the hurdles to overcome in the future, and the overall perspectives of this vaccination strategy are described.

Introduction

Vaccination often is the most cost-effective approach to prevent and to control many infectious diseases. The traditional way of vaccine administration is by subcutaneous or intramuscular injection using syringes and needles. This invasive route of administration has several drawbacks like limited acceptance (needle phobia), transmission of diseases by needle stick injuries, and the need for trained healthcare workers.^(1,2) In addition, the introduction of new vaccines will require the development of a new combination vaccine, if the number of injections in a vaccination program is not to be increased. This may lead to potential problems related to formulation design and/or efficacy in humans. As a result, there is a need for needle-free vaccine delivery strategies as an alternative for administration of vaccines by injection. In this review, the current status of pulmonary vaccination, the potential advantages of pulmonary vaccine delivery, the hurdles to overcome, and the possible future directions are described.

Pulmonary vaccination

Pulmonary vaccination makes use of the unique physiology, especially the immune system, of the respiratory tract. As such, it has several advantages over conventional vaccination by needles and syringes. The respiratory tract is the port of entry for pathogens that may cause pulmonary diseases. To deal with these pathogens, the lungs are equipped with all the prerequisites in order to confer immunity to pulmonary delivered antigens. In the lung, there is a very large surface area available for interaction with the antigens. From a physiological point of view, the lung can be divided into two parts: the conducting and transitional airways and the lung parenchyma. The conducting airways have a dense mucus layer with dendritic cells (DCs) that facilitates trapping of pathogens or vaccines. Underneath, local antibodies can be produced by the lamina propria. The lung parenchyma is covered with epithelial cells and contains antigen-presenting cells (APCs) like DCs and alveolar macrophages.⁽³⁾ When antigens enter the lung, they can be taken up by these macrophages or DCs. After antigen uptake, the DCs migrate to draining lymph nodes where antigens are presented to naive T cells. These T cells can differentiate into memory or effector T cells. Via efferent lymphatics, T cells can provide help to B cells in the systemic germinal centers resulting in production of systemic antibodies.⁽⁴⁾ Next to this systemic immune response, pulmonary delivery of vaccines is thought to induce local immunity more effectively than conventional vaccination. Local immunity, provided by secreted IgA (sIgA), is induced by the bronchoalveolar lymphoid tissue (BALT). It provides protection at the site of infection^(5,6) and sIgA is thought to be more crossprotective against different subtypes of a pathogen.^(7,8) However, to date, little is known about the role of BALT in immunological memory and cellular immunity.

Pulmonary pathogens and the need for improved vaccines

As described in the previous section, the respiratory tract is a common port of entry for many pathogens that may cause pulmonary diseases (see Table 1). Pneumonia is the most common infection in children and the leading cause of mortality in children in developing and industrialized countries.⁽⁹⁾ Although most pathogens that invade via the pulmonary route, some of them can cause diseases other than pulmonary diseases. The majority of pulmonary infections is of viral origin and accounts for 50% of pneumonia in children younger than 5 years. Among the bacterial pathogens, the most common organism in children younger than 5 years is Streptococcus pneumonia, whereas Mycoplasma is most frequently causing pneumonia in children older than 5 years.⁽⁹⁾ As shown in Table 1, many new vaccines against pathogens, that cause pulmonary diseases, are under investigation. Depending on the pathogen, the vaccine is developed for a particular target. Many of the vaccines in Table 1 are developed to be administered by i.m. or s.c. injection. However, several vaccines, like those against influenza, pertussis, and tuberculosis, have limited efficacy. Consequently, improved vaccines against those diseases are needed. The development of pulmonary vaccines might fulfill some of these needs. Pulmonary vaccination against microorganisms that are transmitted via the pulmonary route has the potential to induce broader/other antibody and T cell (memory) responses and induce immunity at the site of entry of the respective pathogen, resulting in more efficient protection.

Table 1.

Selection of Viral and Bacterial Pathogens That Cause Pulmonary Diseases

Pathogen	Vaccine	Target population	Route of administration	Comment
Viral
Human Metapneumovirus	None	Children and elderly	—	Pathogen for acute respiratory infection
Human bocavirus	None	Mostly children	—	—
Adenovirus	Oral live attenuated vaccine⁽¹⁰⁾ against types 4 and 7	Military trainees	Oral (tablet)	Production stopped in 1996 based on economical reasons and the requirement to upgrade plants to modern standards⁽¹¹⁾
Human parainfluenza virus	Live attenuated virus vaccine, clinically tested⁽¹²⁾	Children and adults	intranasal	Second most common etiology of acute respiratory illness (after RSV)⁽¹²⁾
Influenza	Subunit/split influenza vaccine	Patient groups at risk	i.m. injection	IgG in lungs by transudation (transfer of IgG antibodies from blood to lung)
Influenza	Live attenuated vaccine	Registered for individuals 2–49 years of age	intranasal	Improved cellular immunity (CTL) compared to i.m. injection, IgA in respiratory tract. Not registered for children and elderly.
RSV	Some live attenuated vaccines and adjuvanted subunit vaccine in preclinical and clinical development⁽¹³⁾	Children⁽¹⁴⁾and elderly⁽¹³⁾	i.m. injection and intranasal (only some of the live attenuated vaccines)	—Every newborn will be infected during the first year of life. —RSV is causative agent not only of pneumonia but also of asthma and COPD exacerbations⁽¹⁵⁾ —First attempts in the 1960s employed formalin-inactivated whole virus adjuvanted with alum, this vaccine led to enhanced disease upon natural infection⁽¹³⁾
Measles	Edmonsten-Zagreb	Children	i.m. injection, pulmonary administration	Linked to community acquired pneumonia⁽¹⁶⁾
Bacterial
Streptococcus pneumoniae	Pneumococcal polysaccharide vaccine (PPV) 4 vaccines available, different amount of serotypes (7,10, 13, and 23) ⁽¹⁷⁾	Adults	i.m. injection	Effective in developing countries. Benefit is not seen in industrialized populations⁽¹⁷⁾
Mycoplasma pneumonia	Inactivated M. pneumonia vaccine, clinically tested, did not reach the market.	Patient groups at risk	i.m. injection	—Vaccine may reduce total rates of both pneumonia and respiratory infections by 40%⁽¹⁸⁾ —Live attenuated vaccines never reached the stage of clinical trials for efficacy evaluation due to the fear that the attenuated strains were still virulent
Pseudomonas aeruginosa	2 vaccines studied⁽¹⁹⁾: — LPS vaccine (outer membrane compound having TLR4 activity) — Flagellin vaccine (protein having intrinsic TLR5 activity)	Patient groups at risk, including CF patients	i.m. injection	Conjugate vaccines containing the polysaccharide alginate are tested preclinically, no results on clinical follow up are disclosed yet
Heamophilus influenza B (HiB)	Hib conjugate vaccine⁽¹⁴⁾	Children	i.m. injection	Also causing meningitis.
Bordetella pertussis (whooping cough)	Acellular pertusis or Cellular pertussis vaccine	Children	i.m. injection	Currently pertussis seem to cause more new infections, probably caused by mutants that escape vaccine-induced immunity and/or impaired long-term immunity⁽²⁰⁾
Tuberculosis (Mycobacterium tuberculosis)	Live attenuated vaccine (like BCG)	Children	i.m. injection	Only used in a limited number of countries. Efficacy of BCG vaccines is limited, due to pathogen escape mechanisms (like genetic evolution)⁽²¹⁾
Coxiella burnetii (Q-fever disease)	Formalin-inactivated phase 1 Henzerling strain (CSL Limited)	Abattoir workers, sheep shearers, dairy and beef farmers (including family members)	injection	Requires screening to exclude people with preexisting immunity in order to minimize risk of developing systemic and severe local reactions (especially immune abscesses) following vaccination⁽²²⁾

RSV, respiratory syncytial virus; BCG, bacille Calmette-Guerin.

Current Research

In the past decades, a lot of preclinical and also some clinical research has been done on pulmonary administration of vaccines (Tables 2 and 3). The first vaccinations known to be administered via the pulmonary route were given to poultry in the 1950s. Since then, many different types of vaccines have been tested in other animal models, such as mice, rats, guinea pigs, and macaques and later in humans.

Table 2.

Preclinical Development of Pulmonary Vaccines

Antigen	Formulation	Delivery and device	Adjuvant	Species	Comment	Ref
Whole inactivated influenza virus and split-subunit vaccine	Lipid microparticles by spray drying	Insufflator (Penn-Century, Inc.)	Lipid microparticle	Mice and rats	Effect of spray dried microparticles on local and systemic immune response Result: local and systemic immunity induced (IgG in BAL and sera). More effective in triggering specific immunity. No IgA	⁽³⁹⁾
Influenza Whole inactivated influenza and subunit vaccine	Liquid	injected into lungs via trachea by micropipette and microsyringe	None	Mice	Study effect of deposition site on immune response.	⁽²³⁾
Influenza subunit vaccine	Dry powder, SFD, and Liquid	Insufflator and Microsprayer (Penn-Century, Inc.)	None	Mice	Comparison between pulmonary administration of liquid aerosol and powder aerosol and intramuscular injection. Result: High IgG and IgA in sera and BAL by dry powder. Liquid and intramuscular injection had equally lower immune responses.	⁽²⁴⁾
Influenza whole inactivated virus vaccine	Liquid, Dry powder (by SFD)	Insufflator and Microsprayer (Penn-Century, Inc.)	None	Mice	Result: no difference between liquid and dry powder in IgG titers, IgA titers higher after dry powder administration	⁽⁴⁰⁾
Edmonston-Zagreb Measles vaccine	Liquid aerosol	Different nebulizers, with and without mask	None	Macaques	—Comparison of aerosol and injection and different devices. —Safety study in immunocompromised animals	⁽²⁷⁾
Edmonston-Zagreb Measles vaccine	Dry powder, by milling and blending of lyophilized vaccine	Intratracheally by compressed air or dry powder insufflator	None	Macaques	Comparison between dry powder aerosol and subcutaneous injection. One group received a tracer for deposition study. Result: lower immune response in pulmonary groups than after injection	⁽²⁶⁾
Edmonston Zagreb Measles vaccine	Dry powder by supercritical drying	PuffHaler	None	Cotton rats	Comparison between pulmonary administration of dry powder and subcutaneous injection. Result: immune response after pulmonary administration comparable to injection	⁽⁴¹⁾
Edmonston Zagreb Measles vaccine	Dry powder by supercritical drying	PuffHaler and BD solovent	None	Macaques	To test the efficacy of two dry powder inhalers. Result: vaccination using these inhalers induced robust immune responses	⁽⁴²⁾
Ag85B tuberculosis antigen	Suspension of nanoparticles (30 nm) conjugated with Ag85B	Delivered through nostrils	Nanoparticle and CpG	mice	Comparing intradermal and pulmonary route of administration. Result: better protection against challenge, compared to soluble Ag and intradermal delivery	⁽³⁸⁾
Live attenuated tuberculosis vaccine bacille Calmette-Guérin (BCG)	Dry powder by spray drying, rod-shaped particles	Insufflator (Penn-Century, Inc.)	None	Guinea pigs	Comparing protection against Mycobacterium tuberculosis after subcutaneous or pulmonary administration	⁽⁴³⁾
Hepatits B vaccine (HBsAg)	Liquid, suspension of PLA and PLGA nanoparticle	Microsprayer (Penn-Century, Inc.)	Nanoparticle	Rats	Comparing antibodies after intramuscular injection or pulmonary administration	⁽³¹⁾
Hepatits B vaccine (HBsAg)	Dry powder by spray drying, nanoparticle	Insufflator and Microsprayer (Penn-Century, Inc.)	Nanoparticle	Guinea pigs	Comparing different pulmonary formulation with intramuscular injection. Result: lower IgG, high IgA.	⁽⁴⁴⁾
Hepatits B vaccine (HBsAg)	Liquid	Microsprayer (Penn-Century, Inc.)	Surfactant (TDM)	Rats	Influence of different amounts of TDM	⁽⁴⁵⁾
Hepatits B vaccine (HBsAg)	PLGA microspheres	Suspension of microspheres in PBS, Microsprayer (Penn-Century, Inc.)	Microsphere	Rats	Influence of particle size. Result: Immunogenicity increased as function of particle size.	⁽³⁰⁾
Diphteria CRM-197 antigen	Dry powder, by spray drying	Ìnsufflator (Penn Century, Inc.)	PLGA particle	Guinea pigs	Comparing different pulmonary formulations with intramuscular injection. Result: Lower IgG in sera but higher IgA in BAL, compared to intramuscular injection	⁽⁴⁶⁾

BAL, bronchoalveolar lavage.

Table 3.

Clinically Tested Pulmonary Vaccines

Vaccine	Formulation	Delivery and device	Adjuvant	Target group	Comment	Ref
Measles (Edmonston-Zagreb en Edmonston B)	Reconstituted lyophilized powder (Liquid aerosol)	Nebulizer with mask	None	Infants <9 months	Meta-analysis: four of six trials better responses in subcutaneous group compared to aerosol group. Several studies reported practical difficulties in aerosol administration in young infants. Older infants showed better serum responses.	⁽⁴⁷⁾
Edmonston-Zagreb Measles vaccine+rubella	Reconstituted lyophilized powder (Liquid aerosol)	Nebulizer	None	Children 6 years old	Comparison of antibody response after aerosolized and injected measles vaccine. Result: immunogenicity by aerosol superior to injection even when applied in 1/5th of the injected dose	⁽⁴⁸⁾
MMR (Measles, Mumps, Rubella)	Liquid aerosol	Nebulizer with paper cone (not real mask)	None	Adults	Pulmonary versus subcutaneous. Result: no statistical differences between groups. Both high serum antibody responses.	⁽⁴⁹⁾
Edmonston-Zagreb Measles vaccine	Reconstituted lyophilized powder (Liquid aerosol)	Nebulizer with cone (not real mask)	None	Infants <9 months	Comparison of antibody response after aerosolized and injected measles vaccine. Result: lower immunity in aerosol groups.	⁽⁵⁰⁾
Schwarz or Edmonston-Zagreb Measles vaccine	Reconstituted lyophilized powder (Liquid aerosol)	Nebulizer with mask	None	Children 5–14 years old	Comparison between subcutaneous and pulmonary administration. Result: Higher response in pulmonary group than after subcutaneous injection.	⁽⁵¹⁾
Human papiloma virus (HPV16)	Suspension, VLP (Liquid aerosol)	Nebulizer	None	Adults (women)	Comparing IgG and IgA after pulmonary, nasal or intramuscular administration. Result: aerosol vaccination comparable to i.m. vaccination	⁽⁵²⁾
23-valent pneumococcal capsular polysaccharide vaccine (23-PPV)	Liquid aerosol	Nebulizer	None	Adults	Comparing antibody titers after subcutaneous injection or pulmonary administration. Result: inhaled vaccine produced no response	⁽⁵³⁾

Preclinical research

Pulmonary vaccine concepts currently under development can be arranged by the manner of vaccine delivery:

1. vaccine delivery to a specific area of the lungs (site of deposition: main trachea, bronchus, bronchiole, and/or alveoli), as liquid (solution or suspension) or dry powder

2. vaccine delivery to and induction of specific immune cells by use of adjuvants: delivery systems (like: nanoparticles, liposomes) and/or immune potentiators (like: toll like receptor TLR agonists, mannosereceptor agonists, or chemokines).

Vaccines can be administered via the pulmonary route as liquid aerosols or dry powder aerosols. In both types of formulations, liquid or dry powder, the antigen can be presented as such (live attenuated, inactivated, subunit, toxoid) or in an adjuvanted form.

Deposition and dry powder aerosols

When an aerosol is administered to the lungs, different parts of the lung can be targeted by using the appropriate aerodynamic size distribution and inhalation maneuver. Research in mice showed the influence of the site of deposition of the antigen on the immune response. For example, Minne et al.⁽²³⁾ obtained better systemic, local, and cellular immune responses when the influenza split virus vaccine was delivered to the deeper areas of the lungs than when the vaccine was targeted to the upper or central airways. Until today, proof to which site of the lungs (alveoli, bronchus, bronchiole, or trachea) a vaccine should be targeted by studies in human is missing.

Amorij et al.⁽²⁴⁾ produced a dry powder of the subunit vaccine of influenza by spray-freeze drying. In a study in mice, pulmonary administration of this dry powder formulation was compared to pulmonary administration of an aqueous solution of the subunit vaccine. The aerodynamic particle size of the dry powder aerosols was smaller than that of the droplets in the liquid aerosol (aerodynamic diameters of 5.3 and 25 μm, respectively). Particles with a smaller aerodynamic particle size can lead to a deeper penetration into the lung.⁽²⁵⁾ Results showed higher serum IgG titers in the dry powder group. Furthermore, locally produced IgA titers were also higher after pulmonary administration of a dry powder. The difference in immune response could be due to the site of deposition, a result of differences in aerodynamic partcle size and difference in device, but could also be due to the particulate nature of the formulation or a combination of both.

In a study in macaques, the site of deposition of a dry powder measles vaccine was investigated. Results showed that the iron oxide labeled vaccine was mainly visible (optical microscopy of ex vivo lungs) in the upper airways (trachea and primary bronchi) and only traces were found in bronchioles and alveoli. Compared to intramuscular injection of the vaccine, levels of immunity were lower in animals vaccinated with a nebulized dry powder aerosol.⁽²⁶⁾ The same group also studied a liquid formulation for pulmonary vaccination in macaques. In this case, the pulmonary delivered formulation induced immunogenicity in macaques comparable to that induced by injection of the formulation.⁽²⁷⁾

Vaccine delivery and efficient induction of specific immune cells

To improve the immunogenicity of antigens, adjuvants can be added to the formulation. Adjuvants can be divided into two groups: delivery systems and immunostimulators.^(28,29) Traditionally, delivery systems provide multimeric presentation of antigen, target to APC, and protect antigens against premature degradation. Immunostimulators activate the innate immune system via recognition by danger sensors like Toll-like receptors on APC. However, adjuvants can combine both functions; meaning that adjuvants can act as both delivery system and immunostimulator.

In preclinical studies for pulmonary vaccine formulations, several adjuvant systems have been investigated/used (see Table 2). Delivery of hepatitis B vaccine, incorporated in poly (lactic-co-glycolic acid) (PLGA) microparticles, to the lungs of rats has been shown to induce significantly higher antibody titers than pulmonary delivery of plain antigen.⁽³⁰⁾ Moreover, pulmonary vaccination with hepatitis B surface antigen (HBsAg) encapsulated in 5-μm PLGA microspheres elicited a significantly higher immune response compared to 12-μm PLGA-HBsAg particles. Although the deposition pattern of the particles was not studied, the higher immune response of the 5-μm PLGA microspheres was related to the better uptake by rat macrophages as studied by uptake of PLGA microparticles by rat alveolar macrophages ex vivo.⁽³⁰⁾ In another study by the same group, confocal images showed most preferential uptake of 1–1.5-μm encapsulated fluorescein-labeled bovine serum albumin particles compared to smaller particles (around 700 nm).⁽³¹⁾ In addition, modifying the PLGA microparticles with polyethylenimine or stearylamine revealed that positive charged microparticles were more immunogenic than hydrophobic particles.⁽³⁰⁾ Similar, particles from PLGA and poly (lactic-acid)(PLA) have been compared for pulmonary delivery of HBsAg.⁽³¹⁾ The larger hydrophobic particles (>500 nm) induced a more increased sIgA response upon pulmonary delivery in rats and were more efficiently internalized by rat alveolar macrophages compared to the smaller hydrophilic particles (<500 nm). So several studies indicate that efficient immunity induced by the (sub)microparticle formulations might be related to an improved interaction with APCs by the large surface of particles with positively charged, hydrophobic, or receptor-interacting properties.^(4,31) Unfortunately, (combinations with) in vivo deposition studies have been missing to draw final conclusions.

Next to PLGA and PLA, various other particulate delivery systems have been investigated for pulmonary administration of vaccines, such as polyelectrolyte microcapsules, ISCOMATRIX™ and polypropylene sulfide nanoparticles.

Biodegradable polyelectrolyte microcapsules, based on the biopolymers dextran-sulfate and poly-L-arginine for pulmonary delivery have been studied by De Koker et al.⁽³²⁾ Pulmonary immunization of mice with the model antigen ovalbumine encapsulated in these microcapsules induced a strongly Th17-polarized immune response. This may be beneficial for the development of (therapeutic) vaccines against extracellular bacteria and fungi in the lungs, because Th17 responses have been linked to induction of protective immunity against these extracellular pathogens.^(33,34)

ISCOMATRIX™ is an adjuvant that is a particulate delivery system (40-nm sized structure) containing Quil A as immunostimulator. Upon parenteral administration, ISCOMATRIX™ has been shown to increase cellular and humoral immunity against codelivered antigens.⁽³⁵⁾ Pulmonary administration of ISCOMATRIX™ containing vaccines against influenza,⁽³⁶⁾ cytomegalovirus, or Helicobacter pylori showed antigen-specific mucosal and systemic immunity.⁽³⁷⁾

Polypropylene sulfide nanoparticles have been investigated for pulmonary delivery of tuberculosis antigen in order to increase T cell immunity against tuberculosis. In a study by Ballester et al.⁽³⁸⁾ it was shown that pulmonary administration of these nanoparticles coupled to Ag85B and mixed with CpG induced mucosal and systemic Th17 responses in mice that gave substantial reduction of lung bacterial burden upon challenge with Mycobacterium tuberculosis. However, the safety of adjuvants, like CpG, has still to be extensively addressed in further (pre-)clinical studies for pulmonary vaccination.

Clinical research

Only a limited number of clinical studies in humans has been performed on pulmonary vaccination (see Table 3). The selection of vaccines studied for pulmonary administration highlighted here are live attenuated measles vaccine, a virus-like particle vaccine against human papiloma virus (HPV), and a capsular polysaccharide vaccine against Streptococcus pneumonia.

Measles

The measles vaccine is the most extensively studied vaccine for pulmonary vaccination. The first clinical trials in infants below 9 months of age, in the 1980s in Mexico, demonstrated the feasibility of pulmonary administration of the nebulized measles vaccine.⁽⁴⁷⁾ Although compared to subcutaneous vaccination lower antibody responses were found upon pulmonary vaccination in the younger infants, probably related to difficulties with aerosol administration, pulmonary vaccination of the older infants showed comparable responses. In a study in 2002 the immune responses in 6-year-old children who received the vaccine by liquid aerosol administered to the lungs were significantly higher than those receiving the vaccine by injection.⁽⁴⁸⁾ In contrast, in a study by Wong-Chew, 9-month-old children who received the measles vaccine pulmonary via liquid aerosol showed lower cellular and humoral immunity to measles than those receiving the vaccine by subcutaneous injection.⁽⁵⁰⁾ The long-term persistence of measles antibodies after revaccination by the pulmonary route has been investigated by Dilraj et al.^(51,54) Six years after revaccination of 5- to 14-year-old South African schoolchildren, it was found that revaccination by liquid aerosol had evoked a stronger and longer lasting antibody response than injected vaccine. Despite the numerous clinical trials that have been performed on pulmonary vaccination with nebulized measles vaccine, no final conclusion can be drawn. The results of these clinical trials are often contradictory. Exact reasons are not known; however, some facts should be addressed. First of all, only a few studies have been performed in infants without preexisting (maternal) immunity against measles. The exact influence of this preexisting immunity on the study outcome is not known. In addition, measles vaccines are highly unstable and the potency losses due to the process of reconstitution and nebulization are not consequently monitored. As a result, proper comparison between and within the several studies is hard without knowing the exact pulmonary deposited dose. Despite these contradictory results, pulmonary vaccination with measles vaccines will probably be the pulmonary vaccination strategy with the highest potential to reach application in daily practice. Aerosol vaccination against measles might have advantages over subcutaneous administration with live-attenuated vaccine and thought to be especially effective as a booster vaccination.⁽⁵⁵⁾ New formulations (including dried formulations^(42,56)) and delivery devices that guarantee the delivery of the correct dose (reproducible), especially in the target population, as well as more insight in measles immunity may help pulmonary measles vaccination become a reality.

HPV

Pulmonary vaccination has also been investigated in humans for induction of mucosal immunity to more remote mucosa via the compartmentalized mucosal immune system. Depending on the site of mucosal vaccination, the inductive site (a.o. sublingual, nasal, intestinal, genital mucosa) certain effector sites including remote mucosa can be induced. However, not all mucosa are equally “connected” resulting in compartmentalization of the common mucosal immune system.⁽⁵⁷⁾ In the case of human papillomavirus, the port of entry in woman is the cervix. As a result, the efficacy of a vaccine against HPV may benefit from induction of local immunity in the cervix. Pulmonary HPV vaccination has been studied for this purpose in a limited (5–10 subjects per group) Phase I Clinical trial. Nardelli-Haefliger and colleagues⁽⁵²⁾ vaccinated 18–45-year-old women with an HPV16 virus-like particle (VLP) vaccine without adjuvant with a Systam^® nebulizer (aerosolation by sonification) in a prime boost regime (t=0 and 2 weeks). After administration of human papillomavirus (HPV) to the lung, mucosal immune responses, anti-HPV16 VLP IgA secreting cells in peripheral blood mononuclear cells (PBMCs) and sIgA in secretions of the cervix of the women, were found. In addition, local immune responses induced by pulmonary aerosol administration were significant higher than those induced by nasal administration of the same vaccine. Next to this local immune response, serum titers were comparable to the intramuscular injection group. A potential advantage of this aerosol vaccination under investigation is the potential to overcome the substantial variation in HPV16 antibodies at the cervix seen with the parenteral vaccine in ovulating women.

Streptococcus

Streptococcus pneumonia is the most common cause of pneumonia world wide as well as a major cause of invasive disease syndromes like meningitis. Although, vaccines like the 7-valent pneumococcal conjugate vaccine in the United States have proven to work against invasive disease, these conjugate vaccines are substantially less effective against pneumonia, the main feature of pneumococcal disease. As a result a pneumococcal vaccine that induces local immunity in the respiratory tract might decrease the disease burden of pneumococcal disease. Gordon et al.⁽⁵³⁾ evaluated the pulmonary mucosal immunoglobulin response of inhaled 23-valent pneumococcal polysaccharide vaccine in adults and compared the immune response to injected vaccine. Whereas the injected vaccine induced significant systemic immunity, the pulmonary delivered vaccine induced neither mucosal nor serum antibodies. The failure of the vaccine to induce an immune response could not be addressed to stability problems with formulation or inappropriate aerosol size, because potency (ELISA) and median mass aerosol diameter were determined and checked after nebulization. These findings may indicate that capsular polysaccharide vaccines are less suitable to induce immunity via the respiratory tract. Exact mechanism are not known yet; however, the absence of an adjuvant may have influenced the immunological processing in the lungs. In addition, pneumococci colonizing mucosa appear to have the phenotype to express relative little capsule and have prominent surface proteins, in contrast to bacteria in the bloodstream having a heavily capsulated phenotype (opaque phase).^(58,59) This indicates that pulmonary delivered antigens should be tailored to the pathogen-related immune mechanisms involved. As a result, a pulmonary pneumococcal vaccine containing the prominent surface proteins might be more effective in inducing mucosal immunity.

Aerosol Delivery and Formulation

Aerosol delivery devices for vaccines

Unlike inhaled medication in asthma or chronic obstructive pulmonary disease (COPD) therapy, vaccines are administered only a few times. Therefore, a highly reliable and reproducible delivered fine particle dose and deposition of this dose in the target area are needed to guarantee sufficient immunization efficacy. It also has the consequence that the delivery device should preferably be single use to avoid patient contamination and transmission of diseases. Different principles for the generation and administration of medical aerosols are known. They produce aerosols with different properties. Lu and Hickey⁽⁶⁰⁾ recently reviewed most of the currently marketed devices including (jet and ultrasonic) nebulizers, metered-dose liquid inhalers, pressurized metered-dose inhalers (MDIs), and dry powder inhalers (DPIs). They emphasized the relevance of knowing the particle size distribution of the aerosol delivered by the device as this influences the site of deposition. Larger particles are needed to prevent upper respiratory infections, whereas smaller particles more effectively penetrate the deep lung. The total size range for both sites may reach from 1 to 7.5 microns, depending also on the inhalation maneuver.

For effective vaccination, more aspects are to be considered next to particle size distribution and emitted fine particle mass fraction of the dose, however. From the extensive list of national and international immunization programs, it becomes clear that the majority of vaccinations take place in developing countries. For this reason, the aerosol administration devices have to be cheap. The climate and logistics in developing countries furthermore lead to a strong preference for the use of stabilized dry powder vaccines. Dry powders may have a lower potency loss over the same period in the absence of cold chain facilities. When they can be administered as such, reconstitution is not necessary. This reduces safety risks from using contaminated materials. On the basis of these considerations, dry powder inhalers seem most suitable for pulmonary vaccine administration. They are generally less complex and less expensive than nebulizers and metered-dose liquid inhalers, and therefore, more appropriate as single use devices. Additionally, powders have the advantage of being often more immunogenic compared to liquids.^(24,40) Recently Saluja et al.⁽⁶¹⁾ described the development of stable powder formulations with influenza vaccine in a dry powder inhaler.

Immunization programs of the World Health Organization focus particularly on children. For instance, measles vaccine is annually administered to millions of children in the age varying from 9 months to 15 years.⁽⁶²⁾ Pulmonary vaccine administration to special target groups, like children, raises specific problems and concerns. Although the most optimal pulmonary target area for vaccines in humans, part of the conducting airways or lung parenchyma is not known to date; it is expected that deeper lung deposition will be advantageous for efficient vaccination. To achieve effective drug deposition in the target area, particularly when this is in the deep lung, a good control of the inhalation maneuver is needed. To transport the aerosol into the alveoli with a single breathing stroke, a steady and deep inhalation to total lung capacity is necessary, following maximal exhalation to residual volume first. Next, a certain breath-hold period of minimally 5 s is desired to give particles sufficient time for sedimentation. The necessity to perform such a maneuver makes pulmonary vaccine delivery to the alveoli inappropriate for small children and some elderly. Elderly may have residual lung volumes being larger than their alveolar volumes, meaning that it is virtually impossible to achieve convective aerosol transport into this final airway generation. In addition, the total inhalation maneuver, taking more than 10 to 15 s including the breath-hold period, may be too long. This would be at the cost of a high sedimentation efficacy. Small children also have high breathing frequencies. Babies under 6 to 12 months are nose breathers only and first from the age of 4 to 6 years children may be able to comply with the inhalation instruction given. This includes the generation of a flow rate, which is sufficiently high for powder dispersion, although this may not be necessary when an active dry powder inhaler is used relying on a battery of pressurized air driven dispersion system. However, such devices are too expensive to be disposable. It also includes inhalation of sufficient volume to empty the dose system. Many inhalers, particularly those having capsules as doses system, require the inhalation of 1.5 to 2.5 liters of air to release the entire dose in the inhaled air stream, whereas children up to 6 years of age have vital capacities smaller than 1.5 L. Effective drug deposition in the alveoli requires that the bulk of the dose is aerosolized even within the first 0.5 to 1 L of inhaled air.

Table 4 shows various target groups, classified by age, with the arguments against the use of a dpi for vaccine delivery and what seems to be the most suitable alternative, providing that a formulation for the vaccine can be developed for the alternative administration system.

Table 4.

Target Groups and Arguments Against the Use of DPI

Target groups	Age (years)	Argument(s) against the use `of a dry powder inhaler	DPI compatible	Most suitable alternative ^a
Neonates	<0.5	Nose breathing only	no	Nebulizer with face mask
Infants	0.5–1.5	Frequently nose breathing up to 1 year, inhalation instruction impossible; insufficient vital capacity for dose emptying	no	Nebulizer with face mask; (MDI with VHC^b and face mask)
Toddlers	1.5–4	Possibly insufficient vital capacity for complete dose release; no to poor compliance with the inhalation instruction; possibly insufficient flow rate for powder dispersion	no^c	MDI with VHC^b (and face mask), DPI with (valved) aerosol chamber (and face mask)^c
School children	4–12	Possibly poor compliance with the inhalation instruction	possibly/yes	Breath triggered MDI. DPI with (valved) aerosol chamber^c
Teenagers	12–20	No arguments against DPI use^d	yes	Breath triggered MDI
Adults	20–60	No arguments against DPI use^d	yes	Breath triggered MDI
Elderly	>60	Possibly insufficient vital capacity for alveolar deposition, breath holding may be difficult	possibly	Soft mist inhaler

If compliant with the drug (vaccine) formulation.

Valved-holding chamber.

Possibly PuffHaler.⁽⁴²⁾

Except for subjects with severely reduced vital capacity.

Vaccine formulation for pulmonary vaccine delivery

Various dry powder formulations have been developed for pulmonary administration of vaccines (see Table 2). In the dry state, the molecular mobility is strongly reduced, which makes the vaccine compound less prone to physical and chemical degradation. Dry vaccine formulations for pulmonary delivery can be produced by several drying methods including jetmilling,⁽⁵⁶⁾ spray-drying,^{(43,44,46,61,63)} spray-freeze drying,^{(24,39,40,61)} and supercritical fluid drying.^(41,42,64) Current developments in the design of dry powders for pulmonary vaccine delivery are described in more detail by Sou et al.⁽³⁾

Dry powder formulations for pulmonary delivery are in general delivered to the lung by use of DPIs. However, as discussed in the previous section, not all target groups are capable of using a DPI, especially in case a deep lung deposition is needed. For these groups nebulizers could be used. Nebulizing vaccines, however, has certain disadvantages. First, for nebulization a liquid formulation is needed. However, as described earlier, vaccines in an aqueous environment are prone to degradation. Making of a stable dry powder formulation and reconstitute just before nebulization could be a solution to this problem. Second, during nebulization the vaccine is exposed to high shear forces, which could damage the vaccine. Although degradation of a vaccine during nebulization has not been demonstrated, protein degradation during nebulization has been demonstrated.⁽⁶⁵⁾ Therefore, proper formulation of liquid vaccines is needed for those vaccines that have to be delivered by nebulization.

Immunology and Safety

As of now, knowledge of pulmonary immunology is still very limited, especially for humans. Important characteristics of the human pulmonary immune system with special focus on specific respiratory tract DC (RTDC) subpopulation is highlighted in a review by Blank et al.⁽⁴⁾ Knowledge is required about which area of the lungs should be targeted for the best immunity, which immune cell subtypes [DC subtypes, mycosis fungoides (MFs)] are involved (and where to find them in the lungs) and what kind of immune responses can or should be induced (Th1, Th2, or Th17) via the lungs. In addition, for vaccination this has an extra dimension because the majority of vaccines are developed for children, and as such, additional insights in immunology of the child as function of age are needed. In addition to children, other groups are intended for vaccination that will have their own specific characteristic, for example, the elderly and persons at risk (e.g., COPD patients). These groups are, for example, the predominant target groups for influenza vaccinations. From this perspective pulmonary vaccination is a great challenge.

One of the predominant factors that determine the success of a vaccine is its safety profile. Compared to other interventions, the risk–benefit ratio for vaccination is very conservative. This lies in the fact that vaccines are predominantly applied in the healthy population. Therefore, only very limited toxicity is acceptable for vaccination. Moreover, currently regulatory authorities require besides extensive postmarketing surveillance relative large Phase III clinical trials (trials of 3000 subjects are no exception) in order to proof the efficacy (and therewith be capable to do risk–benefit analysis), because only a restricted part of the population will experience an infection.

Several clinical studies on pulmonary vaccination, have paid attention to safety in terms of side effects. In the clinical study of Wong-chew et al.,⁽⁵⁰⁾ tolerability of measles aerosols in vaccinated children of 9 months (n=46) was comparable to the tolerability of subcutaneous delivered measles (n=53) for rhinitis, cough, conjunctivitis, diarrhea, and arthralgias. Fever was the only adverse reaction presented more frequently for the aerosol group (39 vs. 21%). In another study in adults, no self-reported side effects between the injected (n=48) and aerosol (n=46) group receiving measles, mumps, rubella (MMR) vaccine were significant different.⁽⁴⁹⁾ In the dose-escalating study on pulmonary HPV16 vaccination in adult women (18–45 years old),⁽⁵²⁾ the only local symptom reported in the aerosol group (n=15) was mild pharyngeal discomfort. One volunteer reported a reversible systemic side effect of moderate intensity (high-dose group) that was probably related to the vaccine. After a booster vaccination she experienced dyspnea, chills (a side effect that is often seen upon pulmonary administration in humans, even for placebo formulations) and fever that were relieved by self-administered aspirin. In the study on pulmonary vaccination with the 23-valent pneumococcal vaccine (32±12 years, n=17),⁽⁵³⁾ only minor side effects were reported like dry mouth, headache, diarrhea, dizziness, and chills. However, it should be noted that almost no volunteer that had been pulmonary vaccinated developed measurable antibody titers. The main side effects in the control group receiving the vaccine subcutaneous were pain at the injection site, general malaise, and headache.

Overall, the data on safety of pulmonary delivered vaccines is very limited. Ultimately, clinical trials with higher numbers of subjects are to be performed to determine the real safety patterns of these pulmonary applied vaccines. In addition, attention should be paid to the site of delivery, especially delivery to the conducting airways or lung parenchyma. In the ideal situation, safety data as well as efficacy data are coupled to deposition studies. With regard to safety, a specific target group, like children and immunocompromised populations, require extra attention. Primate models may help to acquire preliminary safety data in these cases. For example, in a study by De Swart et al.⁽²⁷⁾ in immunocompetent and immunocompromised macaques, no evidence for a safety hazard (like vaccine virus-induced pneumonia or encephalitis) associated with the pulmonary route of measles vaccination was found. Although in this study vaccine potency was determined directly after vaccination, many studies give only limited attention to the integrity of the vaccine after vaccination (shelf-life; temperature impact) or even after aerosolization (shear stresses).

Future Directions

Pulmonary vaccination is still in its infancy, and several challenges have still to be addressed. As discussed in this review, preclinical and clinical research has proven that inducing an immune response after pulmonary administration of vaccines is definitely possible. However, certain fundamental questions have to be answered. For example, why does a particular vaccine induces a robust immune response and protects against infection (challenge studies) after pulmonary administration while another type of vaccine does not? This could be due to the fact that the different vaccines are formulated differently resulting in different aerosol properties, like particles size distribution. Furthermore, different inhalation devices have been used. The type of formulation and inhalation device will affect the deposition efficiency and site of deposition, which will influence the immune response. On the other hand, from an immunological point of view, one vaccine may be more immunogenic than the other. Most likely, it is a combination of both factors. Therefore, collaborations of immunologists and pulmonary drug delivery researchers would be ideal to optimize both the aerosol properties and the antigen processing in the lungs. In these experiments factors such as site of deposition, the intrinsic immunogenicity of the antigen to activate the innate immune system and what kind of adjuvants are suitable for pulmonary administration, should be addressed in order to obtain a proper immune response that is giving protection. Research should be done to elucidate which cells are involved in inducing an immune response after pulmonary administration of a vaccine and where they are localized. By answering these questions, the optimal site of deposition can be determined. Based on the site of deposition, proper formulations and aerosols can be developed in combination with an inhalation device suitable for the target populations. A proper understanding of how antigens are exactly processed after pulmonary administration and which aerosol and formulation characteristics are the most important for inducing protection could make pulmonary vaccination a realistic approach.

Footnotes

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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