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Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences
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Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences Reinhard Pabst ∗ Institute of Immunomorphology Centre of Anatomy Medical School, Hannover, Germany
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 25 June 2015 Accepted 8 July 2015 Available online xxx Keywords: Intranasal vaccination Nose-associated lymphoid tissue (NALT) Nasal mucosa Species differences
a b s t r a c t The advantage of mucosal vaccination in viral and bacterial infections in different age groups is of enormous clinical relevance. The advantages and potential hazards of intranasal vaccination have always to be considered. The intranasal route for vaccination is very successful for some antigens. Specific adjuvants are necessary. In the nose of rodents there is a structured lymphoid tissue (nose-associated lymphoid tissue (NALT)). This abbreviation should not be used for nasopharynx-associated lymphoid tissue, as this includes parts of the tonsils. In children lymphoid tissue is more dispersed in the nose and not concentrated at the bottom of the dorsal nose ducts as in rodents. There are no data on organized lymphoid tissue in the nose of adults. In NALT of rodents there is a unique structure of adhesion molecule expression; the postnatal development and the different composition of T and B lymphocytes in comparison with Peyer’s patches document the uniqueness of this lymphoid organ. There is also a mucosa in the nose with antigen-presenting dendritic cells. Thus, it is often unclear whether intranasal vaccination is initiated via NALT or the diffuse nasal mucosa. There are still many open questions e. g., which adjuvant is necessary for a specific virus, bacterium or other allergen, how many doses are critical for an effective nasal vaccination. Species differences are of major importance when extrapolating results from rodents to humans. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction More than 60 years ago the oral polio vaccine was introduced. However, this needle free route has not resulted in many commercially available mucosa vaccines as summarized by Holmgren and Lycke and Fukuyama et al. [2]. There are still several unsolved aspects like safe adjuvants and live vectors [3]. There is an obvious lack of knowledge why mucosal immunization of an organ does not a result in secretory antibody production in all organs covered by a mucosa [4]. Therefore the often used term common mucosa-associated-lymphoid tissue [2] (cMALT) should be replaced by integrated MALT [5,6]. An excellent example for this
Abbreviations: BrdU, Bromodesoxy Uridine; CV, conventional; DC, dendritic cell; GF, germ free; HEV, high endothelial venule; MAdCAM-I, mucosal adhesion molecule; MALT, mucosa-associated lymphoid tissue; M cell, microfold cell; NALT, nose-associated lymphoid tissue; PNAd, peripheral node adhesion; SEB, staphylococcal enterotoxin B; Th1, T helper 1 cell; Th2, T helper 2 cell. ∗ Correspondence to: Immunomorphology, Medical School Hannover, CarlNeuberg-Str. 1, 30625 Hannover, Germany. Tel.: +49 511 532 6742; fax: +49 511 532 8256. E-mail address: [email protected]
compartmentalization is the substantial IgA and IgG antibody response in the vagina after nasal immunization [7]. Organized mucosal-associated lymphoid tissue/MALT has to meet certain criteria: follicular-like accumulation of lymphoid cells with typical B and T lymphocyte compartments (follicles and the interfollicular area with high endothelial venules (HEV), the epithelium to the lumen of the organs is mostly bulging into the lumen, infiltrated by lymphoid cells and covered by specialized epithelial cells—Microfold cell (M cells). These are effective in the uptake of particulate antigen, and can be used as an entry site for different antigens as reviewed by Neutra and Kozlowski [8]. MALT is the typical inductive site for mucosal immunity. The best known and characterized part of MALT are the Peyer’s patches in the small intestine. As antigen uptake is via the epithelium there is no need for afferent lymphatics, but all MALT organs have efferent lymphatics. NALT drains into the cervical lymph nodes. MALT should not be mixed up with the dispersed immune cells in the epithelium and lamina propria of the mucosal organs [6]. NALT stands for nose-associated lymphoid tissue and not for nasal as this would be linguistically wrong. About 20 years ago Kuper [9] reviewed the structure of NALT and Kraal focussed on NALT in rodents [10]. More recently Kiyono [11] summarized the nasopharyngeal immune system and compared it to the oral immune system.
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Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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Waldeyer’s ring of tonsils is well described for many species. It consists of the paired palatine tonsils, a single pharyngeal tonsil at the roof of the pharynx, the lingual tonsil and the paired tonsils at the back of the pharynx. In humans there is a typical sequence of hypertrophy, e.g., in small children at kindergarten age the pharyngeal tonsil is often hypertrophied and can obstruct the breathing through the nose. This is often an indication for an adenoidectomy. At primary school age the palatine tonsils are frequently hypertrophied after repetitive tonsillitis. It is debateable whether these tonsils are an indication for a tonsillectomy. Rodents do not have tonsils and it has often been argued that the NALT takes over the role of the tonsils in other species. The nose can be used for drug delivery such as protein hormones. The problems and possibilities for the route to the brain via the olfactory nerve have been cortically reviewed by Illum [12]. The pharmaceutical aspects of delivering vaccines and the role of particulate systems are extensively reviewed by Sharma et al. [13]. The nasal route is also effective in a mouse model for vaccination against Streptococcus suis, a bacterium of great relevance in pigs and also in humans [14]. Mucosal vaccinations against bacterial respiratory infections have been summarized by Baumann [15]. Commensal bacteria were of relevance in rat nasal vaccination against Mycoplasma pulmonis [16]. The current literature will be reviewed to answer these clinically relevant questions. Vaccination is very effective in preventing different infections. The parenteral route is often only effective against some viruses and bacteria, and needles or other expensive delivery devices are necessary. In a recently published review the relevance of upper and lower respiratory vaccination in contrast to the parenteral or oral route was outlined and species differences described [15–17]. The mucosa-lined organs function more or less independent of the systemic immune system or the skin. Thus, mucosal or topical immunization strategies are of outstanding relevance as reviewed in 2010 [18] and also in 2013 [1]. The airways and the digestive tract are exposed to different microbial agents and it has to be considered that there are not only a number of common structural and functional aspects, but also major differences between these two groups of mucosa-lined systems (for review see Kunisawa [19]). The intranasal route for vaccination against influenza has already been described in 2001 [20] and summarized recently [21]. Inactivate bacteria like menigococcus and pertussis have been reported as adjutants in inactivated influenza virus vaccination [21]. Pseudomonas aeruginosa is a further bacterium of great clinical relevance and different routes and techniques to enhance the immunogenicity have been tested in humans [21–26]. Furthermore, the intranasal deliveries of vaccines against HIV have been reported [27]. So far it is not known, why an intranasal vaccination preferentially stimulates immunoreactions in the female genital tract but not in the gut (for review see [1,2]). Upper respiratory tract infections are very common, in particular in young children. Pneumococci play an important role in respiratory tract infections. The impact of vaccines on the epidemiology and risk factors of Streptococcus pneumoniae have been critically discussed by Lynch and Zhanel [28]. Therefore, several groups have tested the intranasal route for vaccination. Effective immune responses in the nose depend on specific adjuvants ranging from interleukins to toxins. Some very basic aspects such as nasal clearance will also be of important in nasal vaccination, because bioadhesion and absorption enhancement are not only relevant in nasal drop application [30]. The critical questions, however, are: will the protective immune reaction be initiated in an organized lymphoid tissue such as NALT or by the diffusely distributed immune cells in the nasal mucosa? Are there species differences which are critical when extrapolating animal data to the situation in
humans? What is known about age effects of NALT in the different species?
2. Structure of the nose In humans the back bone of the nose consists of bone and cartilage. The base consists of the palate, the border to the oral cavity. There are three ducts: the superior, middle and lower meatus, the right and left part being separated from each other by the septum. It is of great clinical relevance that at the roof of the superior meatus the olfactory epithelium is found, as these are the endings of the first cranial nerve which directly connects the nose to the brain. This is a rather small area and about 10% of the nasal surface, in man but of enormous clinical relevance. There are major species differences such as in dogs this olfactory area is about 80% of the total nasal cavity [32]. Thus, viruses can adhere to these nerves and use the nerves as travel routes to the brain (Fig. 1). This was the reason to withdraw an anti-influenza vaccine from further testing. Mutsch et al. [31] documented an increased risk of Bell’s palsy after intranasal influenza vaccination in comparison to the parenteral route. The problems of nasal drug delivery have been discussed [32]. The olfactory nerve is quite unique because the olfactory surgery neurons undergo programmed cell death during development and also in the adult [33]. A further unique aspect is that there are olfactory ensheathing cells, which are able to impact the apoptotic neurons [33,34]. The olfactory ensheathing cells are therefore be suggested to be part of the innate immune reactions [35]. This is part of a strategy to prevent or minimize entry and spreading of neurotropic viruses [36]. It is very likely that the entry of a virus into the brain during intranasal immigration happens via this route. Therefore, it is recommended to test potential life virus vaccines in vitro, because the olfactory ensheathing cells can be cultured [37].
3. Confusion in terminology of NALT The term NALT had been introduced to describe organized lymphoid tissue in the nose of rodents [9,29]. However, other authors have used the same abbreviation [2,11] for nasopharynx-associated lymphoid tissue by including the pharyngeal tonsils, often also called “adenoids” (Fig. 2). Mucosa-associated lymphoid tissue “MALT” is found in different parts of the body [e.g., [1,5,6]]. There is the inductive site (organized tissue) and the effector site (by differently distributed immune cells). However, as there are dendritic cells (DC) in the mucosal layers which very effectively take up antigens and transport these to draining lymph nodes, this might also be called inductive site for immune reactions.
4. NALT in different species 4.1. Rat Organized lymphoid tissue was documented at the bottom of both nasal passages as a paired non encapsulated accumulation of lymphoid cells consisting of a T and B cell area, and covered by a specialized epithelium with rare goblet cells and M cells. The basic structure was comparable to MALT and therefore Spit et al. [38] called this structure nasal lymphoid tissue (NALT). Thus, there are two bell-shaped aggregations of lymphoid tissue best seen on frontal sections (Fig. 3). Kuper et al. [39] used immunohistochemical and enzyme histochemical techniques to characterize T and B cells and also macrophage subsets, and hypothesized that they may play an important role in immune reactions to inhaled antigens. The
Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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Fig. 1. Lateral wall and roof of the nasal cavity in humans, documenting the olfactory nerves (Putz and Pabst [78] with permission of the publisher).
postnatal development of NALT was described by Hameleers et al. [40].
4.2. Microbial influence on NALT Germfree (GF) and conventional (CV) rats were studied and the effect of an infection with Mycoplasma pulmonis shown [16] There were significant differences in the size of NALT between GF and CV rats and in showing larger NALT in CV rats. In both groups the size of NALT dramatically increased after M. pulmonis infection.
4.3. Mouse The development and structure of NALT in the mouse was studied by van der Ven and Sminia [41]. It was very similar to that described in the rat. The exposure to formaldehyde for about one month did not result in hyperplasia as in the rat [42]. This observation is of major relevance since formaldehyde has often been associated with nasal carcinomas. The high annual production of formaldehyde and occupational exposure (see Kuper et al. for details [42]) underline the interest in animal models to extrapolate the limits of exposure doses for humans.
Fig. 2. Paramedian section of the nose and pharynx in adults, showing the localization of the pharyngeal tonsil (Putz and Pabst [78] with permission of the publisher).
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Fig. 3. Frontal section of the head of a young adult mouse, showing the bell shaped NALT at the bottom of the nasal ducts. Decalcified by Cali clear Rapid, H & E stain.
Restraining the mouse for 3 h for four days resulted in a decrease of lymphocyte numbers and subsets, but for 8 days IgA was higher than in control animals [43], indicating effects of corticosteroids and norepinephrine on the structure and cellular composition of NALT in mice. This phenomenon has not been studied for other lymphoid organs except the thymus which is extremely stresssensitive. The presence of M cells was documented by Park et al. [44]. In a very recent paper this was also documented and dendrites of DC
extending into the nasal cavity were also demonstrated are showing the morphological prerequisites for antigen uptake by NALT [45].
4.4. Adhesion molecules in mouse NALT The development of HEV (the entry site for lymphocytes) is mediated by the chemokine receptor CXCR 5 and also the
Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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R. Pabst / Vaccine xxx (2015) xxx–xxx Table 1 Lymphocyte subpopulations in NALT and other organs in the mouse (%), based on published data [48].
CD4+ CD8+ B (B220+ ) IgM+ , IgA− IgM+ , IgA+
NALT
PP
Spleen
Blood
32 9 55 49 4
23 2 70 56 6
32 12 45 31 3
75 3 12 7 2
lymphotoxin ß receptor (LTßR) in adult mice. However, NALT can be rescued by other signalling routes [45]. On frozen sections of mouse NALT the presence of different adhesion molecules was tested: all HEV expressed the peripheral lymph node adhesion molecule (PNAd) and this was either associated with the expression of the mucosal vascular addressin MAdCAM-1 (double positive 63%) or PNAd (positive only 37%) [46]. This pattern was completely different from that in Peyer’s patches or peripheral lymph nodes [46]. The postnatal development of NALT was studied by immunohistology in the rat and also in the mouse without major species differences [40]. In further experiments a unique homing pattern of naïve lymphocytes was documented in NALT [47]. It was also documented that PNAd in lymphocyte homing to NALT and allergic responses play a critical role [47]. Thus, the expression pattern of adhesion molecules in this MALT organ is unique [46,47].
4.5. Lymphocyte subpopulations in NALT In normal adult female BALB/c mice a total 1 × 106 cells were obtained per site of the palate. The subset composition was different from that in the blood, spleen or Peyer’s patches [48], as shown in Table 1. Interestingly the rate of CD4/CD8 lymphocytes in NALT differed from that in Peyer’s patches and was similar to that in the spleen. Unfortunately the breeding facilities and the environmental situation of the animal unit were not described in that paper. The composition of lymphocytes in NALT of adult female mice was about 50% B and 50% T cells. There were about 2–3 times more CD4+ than CD8+ lymphocytes [49]. Furthermore, the NALT lymphocytes produced IFN gamma after culture for 24 h and secreted IgA and IgG virus specific antibodies [50]. The same group compared the IgA antibody forming cell response in NALT after vaccination by intranasal, intravenous and/or subcutaneous routes [50]. The combination of intranasal priming and intranasal boosting resulted an increased in anamnestic IgA antibody formation [42].
4.6. Immune reactions in NALT IgA class switch is an important aspect of mucosal immunology and takes place only in organized MALT and not at the effector sites such as the lamina propria [6,50]. In adult mice IgA + B220+ cells were increased in NALT after nasal stimulation with cholera toxin but not after antigen exposure alone [50]. Fernandez et al. [51] documented the induction of germinal centres in NALT and specific mucosal IgA and serum IgG production after intranasal vaccination with a recombinant SEB vaccine on nasal secretion. The efficacy of intranasal vaccination was dependent on NALT as after surgical removal the mice were not protected from toxic shock. In a more recent paper the same group studied NALT by ex vivo culturing NALT and as an alternative to gene knockout mice surgical removal of NALT was used, which is a difficult technique in the mouse [54].
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4.7. NALT in the chinchilla The structure of the nose with its turbinates’ differs greatly between species (for details see [55]). In adult chinchillas (600–800 g body weight) in coronal sections through the dorsal part of the nose lymphoid tissue with well developed follicles was found as paired structures on the palate in each nasal passage [55]. On sagittal sections lymphoid tissue similar to the pharyngeal tonsil was found, which is not present in the mouse or rat. The same group recently published very innovative but also surprising data [56]. They documented a preventive and therapeutic concept to protect against otitis media due to non-typeable Haemophilus influenza in the chinchilla model. This is of enormous clinical relevance. The animals were injected with the bacteria via the skin of the pinna. To track the migration of DC from the pinna the vital dye CFSE was used. The surprising finding was the presence of these DC in the NALT, as it had never been shown in any species that there are lymphatics from the skin of the ear to NALT, but lymphatics would drain to cervical lymph nodes only as far as I know. 4.8. NALT in rabbits The nose of rabbits has not been studied in much detail. In three adult rabbits NALT was found at the bottom of the ventral nasal meatus similar to the site in rodents. Furthermore, isolated lymphoid follicles on the nasal conchae and lateral walls of the nasal cavity were described [57]. 4.9. NALT in farm animals No organized lymphoid tissue similar to that of rodents has been documented (for review [58]). However, isolated lymphoid follicles have been described in sheep and horses, made visible by acetic acid treatment, and M cells have also been documented [59]. In a recent publication the different tonsils were examined in farm animals (ox, sheep, goat, pig, horse, dog, and cat), and NALT was not found in any of them [59]. Thus these species cannot be used as models for vaccination via NALT. 4.10. NALT in humans Human nasal tissue blocks from 150 children, who had died in the first two years of life [60] (cause of death: sudden infant death syndrome (SIDS), traumatic or other causes of death) were studied by routine histological techniques. In contrast to rodents NALT was not found at a definite site but disseminated. NALT was found in a mean of 38% of the children with no difference in respect to the cause of death or age. It was found in the upper nasal cavity 30.1%, on the middle concha 26.4%, inferior nasal concha 13.5%, superior nasal concha 10.4% etc. (Fig. 4). There are no data to my knowledge on the frequency of NALT in adolescents and adults. This is probably due to the problems in obtaining this tissue at autopsy and the need for decalcification. For the situation in humans there is also the previously mentioned problem of definition. Fujimura et al. [61] described M cells which are well defined for antigen uptake in particular in the epithelium covering Peyer’s patches. These M cells, however, were documented for the pharyngeal tonsil only and not for lymphoid tissue in the nose. 4.11. Absence of NALT had little effect in some infection models A classical approach to test the relevance of an organ is to repeat the experiments after surgical removal of the organ. Sabirov and Metzger [62] succeeded in surgically removing NALT in eight day old mice. They later vaccinated the animals intranasally with a
Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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Fig. 4. Sites of organized lymphoid tissue in the nose of children in percentage of cases. Data modified after Debertin et al. [52].
pneumococcal vaccine and interleukin 12 as an adjuvant. The animals lacking NALT developed nasal and serum antibodies and were protected against pneumococcal colonization of the middle ear and nasopharynx. This excellent technique has recently been applied also by Fernandez et al. [53]. Surgical removal of cervical lymph nodes on the other hand resulted in an increased immune response [63]. Thus, the influenza infection might not be induced in the nose but further down in the respiratory tract [63]. Also in a model of challenge with Mycobacterium tuberculosis after an adenoviral vaccine NALT contributed little in protection after aerosol challenge [64]. Resistance to upper respiratory tract infections by influenza was not provided by NALT or cervical lymph nodes [63]. 5. Antigen uptake by the nasal mucosa 5.1. Mouse Kim et al. [65] have documented M cells in the nasal passages of mice. These cells were capable of picking up bacterial antigens, and were also present and active in mice genetically lacking NALT. Already in 1991 the different types of DC in the conducting airways of rats were characterized [66]. 5.2. Antigen-presenting cells in the human nasal mucosa It is a problem to obtain normal human material for basic research. Nasal biopsies from patients undergoing nasal surgery with no known allergies were studied in 14 patients [67]. Not only DC but also macrophages were identified by typical surface markers. Macrophages outnumbered immature DC. These two types of antigen-presenting cells formed a dense network. In conclusion, the high density of DC and macrophages in the mucosa of the nose forms the structural basis for an effective antigen uptake, a prerequisite for successful nasal vaccination protocols as documented by Cisney by combining surgical removal of NALT with vaccination protocols [54]. 5.3. Adjuvants in intranasal vaccination Fujihashi and Kiyono [29] discussed the relevance of specific adjuvants in mucosal vaccination and in particular for the nasal mucosal immune system. Only a few examples will be given. Neutralizing antibodies and T helper cell response to anthrax were
induced by adding choleratoxin to the antigen [68]. In the experiments on vaccination against P. aeruginosa infection the intranasal route was only as effective as the intramuscular route when cholera toxin was used as an adjuvant [69]. Bacillus firmus had an adjuvant effect via the expression of cytokines [70]. An expansion of DC in the nose was induced by the use of P6 with outer membrane protein and alpha-galactosylceramide in nontypeable Haemophilus influenza [71]. Vajdy and Singh [27] summarized the data on intranasal vaccination protocols against HIV. The intranasal route of influenza vaccination has been discussed by Hagenaars et al. [72] and the comparison to other routes in healthy adults by Keitel et al. [20]. Berstad et al. [73] documented the adjuvant effect of inactivated meningococci and pertussis bacteria in intranasal influenza vaccination. The toxic agent acetic acid in a low dose and small volume (12.5 l of 5% acetic acid per nostril) was used to immunize mice with S. suis effectively [14]. The adenovirus serotype 5 vector expressing Flt 3 ligand activated DC and these started a Notch-ligand expression and the induction of antigen-specific Th1 and Th2 cytokine responses [74]. The epithelium of the respiratory tract is not only a physical barrier for microbial and other antigens but influence via different mediators innate and adaptive immune reactions via dendritic cells as well as T and B cells as reviewed by Schleimer et al. [75]. However, the epithelium of the nose has not been studied in this respect. 6. Conclusions Intranasal vaccination has many advantages in contrast to injection with needles using the subcutaneous or intramuscular route. This is in particular obvious for vaccinating children not forgetting the genetic regulation of immune responses in children [76] or specific problems such as influenza vaccination in the elderly [77]. However, there are still many aspects which have to be studied in more detail which antigen is taken up by organized lymphoid tissue of the nose (NALT) and/or by the specialized cells in the epithelium like dendritic cells, which adjuvants are needed for the first and/or booster vaccination? In any case the enormous age a species differences have to be considered, when data obtained e.g., in the mouse are extrapolated to the situation in man. Acknowledgements My apologies to all authors whose publications have not been quotes but are mainly mentioned in the review articles. The technical assistance in preparing Fig. 3 by Cornelia Höpfel, the preparation of Fig. 4 by Marita Peter, the secretarial help by Sabine Buhmann and Silke Wallbaum and the correction of the English by Sheila Fryk are gratefully acknowledged. References [1] Holmgren J, Lycke N. Principles of mucosal vaccine strategies. In: Smith PD, Mac Donald TT, Blumberg RS, editors. Principles of mucosal immunology. London: Garland Science; 2013. p. 413–28. [2] Fukuyama Y, Tokuhara D, Kataoka K, Gilbert RS, McGhee JR, Yuki Y, et al. Novel vaccine development strategies for inducing mucosal immunity. Expert Rev Vaccines 2012;11:367–79. [3] Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12:592–605. [4] Kunkel EJ, Bucher EC. Plasma-cell homing. Nat Immunol 2003;3:822–9. [5] Brandtzaeg P, Pabst R. Let’s go mucosal: communication on slippery ground. Trends Immunol 2004;25:570–7. [6] Brandtzaeg P, Kiyono H, Pabst R, Russel MW. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol 2008;1:31–7. [7] Czerzinsky C, Holmgren J. Mucosal delivery routes for optimal immunization: targeting immunity to the right tissues. Curr Top Microbiol Immun 2012;354:1–18. [8] Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 2006;6:148–58.
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Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences Reinhard Pabst ∗ Institute of Immunomorphology Centre of Anatomy Medical School, Hannover, Germany
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 25 June 2015 Accepted 8 July 2015 Available online xxx Keywords: Intranasal vaccination Nose-associated lymphoid tissue (NALT) Nasal mucosa Species differences
a b s t r a c t The advantage of mucosal vaccination in viral and bacterial infections in different age groups is of enormous clinical relevance. The advantages and potential hazards of intranasal vaccination have always to be considered. The intranasal route for vaccination is very successful for some antigens. Specific adjuvants are necessary. In the nose of rodents there is a structured lymphoid tissue (nose-associated lymphoid tissue (NALT)). This abbreviation should not be used for nasopharynx-associated lymphoid tissue, as this includes parts of the tonsils. In children lymphoid tissue is more dispersed in the nose and not concentrated at the bottom of the dorsal nose ducts as in rodents. There are no data on organized lymphoid tissue in the nose of adults. In NALT of rodents there is a unique structure of adhesion molecule expression; the postnatal development and the different composition of T and B lymphocytes in comparison with Peyer’s patches document the uniqueness of this lymphoid organ. There is also a mucosa in the nose with antigen-presenting dendritic cells. Thus, it is often unclear whether intranasal vaccination is initiated via NALT or the diffuse nasal mucosa. There are still many open questions e. g., which adjuvant is necessary for a specific virus, bacterium or other allergen, how many doses are critical for an effective nasal vaccination. Species differences are of major importance when extrapolating results from rodents to humans. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction More than 60 years ago the oral polio vaccine was introduced. However, this needle free route has not resulted in many commercially available mucosa vaccines as summarized by Holmgren and Lycke and Fukuyama et al. [2]. There are still several unsolved aspects like safe adjuvants and live vectors [3]. There is an obvious lack of knowledge why mucosal immunization of an organ does not a result in secretory antibody production in all organs covered by a mucosa [4]. Therefore the often used term common mucosa-associated-lymphoid tissue [2] (cMALT) should be replaced by integrated MALT [5,6]. An excellent example for this
Abbreviations: BrdU, Bromodesoxy Uridine; CV, conventional; DC, dendritic cell; GF, germ free; HEV, high endothelial venule; MAdCAM-I, mucosal adhesion molecule; MALT, mucosa-associated lymphoid tissue; M cell, microfold cell; NALT, nose-associated lymphoid tissue; PNAd, peripheral node adhesion; SEB, staphylococcal enterotoxin B; Th1, T helper 1 cell; Th2, T helper 2 cell. ∗ Correspondence to: Immunomorphology, Medical School Hannover, CarlNeuberg-Str. 1, 30625 Hannover, Germany. Tel.: +49 511 532 6742; fax: +49 511 532 8256. E-mail address: [email protected]
compartmentalization is the substantial IgA and IgG antibody response in the vagina after nasal immunization [7]. Organized mucosal-associated lymphoid tissue/MALT has to meet certain criteria: follicular-like accumulation of lymphoid cells with typical B and T lymphocyte compartments (follicles and the interfollicular area with high endothelial venules (HEV), the epithelium to the lumen of the organs is mostly bulging into the lumen, infiltrated by lymphoid cells and covered by specialized epithelial cells—Microfold cell (M cells). These are effective in the uptake of particulate antigen, and can be used as an entry site for different antigens as reviewed by Neutra and Kozlowski [8]. MALT is the typical inductive site for mucosal immunity. The best known and characterized part of MALT are the Peyer’s patches in the small intestine. As antigen uptake is via the epithelium there is no need for afferent lymphatics, but all MALT organs have efferent lymphatics. NALT drains into the cervical lymph nodes. MALT should not be mixed up with the dispersed immune cells in the epithelium and lamina propria of the mucosal organs [6]. NALT stands for nose-associated lymphoid tissue and not for nasal as this would be linguistically wrong. About 20 years ago Kuper [9] reviewed the structure of NALT and Kraal focussed on NALT in rodents [10]. More recently Kiyono [11] summarized the nasopharyngeal immune system and compared it to the oral immune system.
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Waldeyer’s ring of tonsils is well described for many species. It consists of the paired palatine tonsils, a single pharyngeal tonsil at the roof of the pharynx, the lingual tonsil and the paired tonsils at the back of the pharynx. In humans there is a typical sequence of hypertrophy, e.g., in small children at kindergarten age the pharyngeal tonsil is often hypertrophied and can obstruct the breathing through the nose. This is often an indication for an adenoidectomy. At primary school age the palatine tonsils are frequently hypertrophied after repetitive tonsillitis. It is debateable whether these tonsils are an indication for a tonsillectomy. Rodents do not have tonsils and it has often been argued that the NALT takes over the role of the tonsils in other species. The nose can be used for drug delivery such as protein hormones. The problems and possibilities for the route to the brain via the olfactory nerve have been cortically reviewed by Illum [12]. The pharmaceutical aspects of delivering vaccines and the role of particulate systems are extensively reviewed by Sharma et al. [13]. The nasal route is also effective in a mouse model for vaccination against Streptococcus suis, a bacterium of great relevance in pigs and also in humans [14]. Mucosal vaccinations against bacterial respiratory infections have been summarized by Baumann [15]. Commensal bacteria were of relevance in rat nasal vaccination against Mycoplasma pulmonis [16]. The current literature will be reviewed to answer these clinically relevant questions. Vaccination is very effective in preventing different infections. The parenteral route is often only effective against some viruses and bacteria, and needles or other expensive delivery devices are necessary. In a recently published review the relevance of upper and lower respiratory vaccination in contrast to the parenteral or oral route was outlined and species differences described [15–17]. The mucosa-lined organs function more or less independent of the systemic immune system or the skin. Thus, mucosal or topical immunization strategies are of outstanding relevance as reviewed in 2010 [18] and also in 2013 [1]. The airways and the digestive tract are exposed to different microbial agents and it has to be considered that there are not only a number of common structural and functional aspects, but also major differences between these two groups of mucosa-lined systems (for review see Kunisawa [19]). The intranasal route for vaccination against influenza has already been described in 2001 [20] and summarized recently [21]. Inactivate bacteria like menigococcus and pertussis have been reported as adjutants in inactivated influenza virus vaccination [21]. Pseudomonas aeruginosa is a further bacterium of great clinical relevance and different routes and techniques to enhance the immunogenicity have been tested in humans [21–26]. Furthermore, the intranasal deliveries of vaccines against HIV have been reported [27]. So far it is not known, why an intranasal vaccination preferentially stimulates immunoreactions in the female genital tract but not in the gut (for review see [1,2]). Upper respiratory tract infections are very common, in particular in young children. Pneumococci play an important role in respiratory tract infections. The impact of vaccines on the epidemiology and risk factors of Streptococcus pneumoniae have been critically discussed by Lynch and Zhanel [28]. Therefore, several groups have tested the intranasal route for vaccination. Effective immune responses in the nose depend on specific adjuvants ranging from interleukins to toxins. Some very basic aspects such as nasal clearance will also be of important in nasal vaccination, because bioadhesion and absorption enhancement are not only relevant in nasal drop application [30]. The critical questions, however, are: will the protective immune reaction be initiated in an organized lymphoid tissue such as NALT or by the diffusely distributed immune cells in the nasal mucosa? Are there species differences which are critical when extrapolating animal data to the situation in
humans? What is known about age effects of NALT in the different species?
2. Structure of the nose In humans the back bone of the nose consists of bone and cartilage. The base consists of the palate, the border to the oral cavity. There are three ducts: the superior, middle and lower meatus, the right and left part being separated from each other by the septum. It is of great clinical relevance that at the roof of the superior meatus the olfactory epithelium is found, as these are the endings of the first cranial nerve which directly connects the nose to the brain. This is a rather small area and about 10% of the nasal surface, in man but of enormous clinical relevance. There are major species differences such as in dogs this olfactory area is about 80% of the total nasal cavity [32]. Thus, viruses can adhere to these nerves and use the nerves as travel routes to the brain (Fig. 1). This was the reason to withdraw an anti-influenza vaccine from further testing. Mutsch et al. [31] documented an increased risk of Bell’s palsy after intranasal influenza vaccination in comparison to the parenteral route. The problems of nasal drug delivery have been discussed [32]. The olfactory nerve is quite unique because the olfactory surgery neurons undergo programmed cell death during development and also in the adult [33]. A further unique aspect is that there are olfactory ensheathing cells, which are able to impact the apoptotic neurons [33,34]. The olfactory ensheathing cells are therefore be suggested to be part of the innate immune reactions [35]. This is part of a strategy to prevent or minimize entry and spreading of neurotropic viruses [36]. It is very likely that the entry of a virus into the brain during intranasal immigration happens via this route. Therefore, it is recommended to test potential life virus vaccines in vitro, because the olfactory ensheathing cells can be cultured [37].
3. Confusion in terminology of NALT The term NALT had been introduced to describe organized lymphoid tissue in the nose of rodents [9,29]. However, other authors have used the same abbreviation [2,11] for nasopharynx-associated lymphoid tissue by including the pharyngeal tonsils, often also called “adenoids” (Fig. 2). Mucosa-associated lymphoid tissue “MALT” is found in different parts of the body [e.g., [1,5,6]]. There is the inductive site (organized tissue) and the effector site (by differently distributed immune cells). However, as there are dendritic cells (DC) in the mucosal layers which very effectively take up antigens and transport these to draining lymph nodes, this might also be called inductive site for immune reactions.
4. NALT in different species 4.1. Rat Organized lymphoid tissue was documented at the bottom of both nasal passages as a paired non encapsulated accumulation of lymphoid cells consisting of a T and B cell area, and covered by a specialized epithelium with rare goblet cells and M cells. The basic structure was comparable to MALT and therefore Spit et al. [38] called this structure nasal lymphoid tissue (NALT). Thus, there are two bell-shaped aggregations of lymphoid tissue best seen on frontal sections (Fig. 3). Kuper et al. [39] used immunohistochemical and enzyme histochemical techniques to characterize T and B cells and also macrophage subsets, and hypothesized that they may play an important role in immune reactions to inhaled antigens. The
Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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Fig. 1. Lateral wall and roof of the nasal cavity in humans, documenting the olfactory nerves (Putz and Pabst [78] with permission of the publisher).
postnatal development of NALT was described by Hameleers et al. [40].
4.2. Microbial influence on NALT Germfree (GF) and conventional (CV) rats were studied and the effect of an infection with Mycoplasma pulmonis shown [16] There were significant differences in the size of NALT between GF and CV rats and in showing larger NALT in CV rats. In both groups the size of NALT dramatically increased after M. pulmonis infection.
4.3. Mouse The development and structure of NALT in the mouse was studied by van der Ven and Sminia [41]. It was very similar to that described in the rat. The exposure to formaldehyde for about one month did not result in hyperplasia as in the rat [42]. This observation is of major relevance since formaldehyde has often been associated with nasal carcinomas. The high annual production of formaldehyde and occupational exposure (see Kuper et al. for details [42]) underline the interest in animal models to extrapolate the limits of exposure doses for humans.
Fig. 2. Paramedian section of the nose and pharynx in adults, showing the localization of the pharyngeal tonsil (Putz and Pabst [78] with permission of the publisher).
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Fig. 3. Frontal section of the head of a young adult mouse, showing the bell shaped NALT at the bottom of the nasal ducts. Decalcified by Cali clear Rapid, H & E stain.
Restraining the mouse for 3 h for four days resulted in a decrease of lymphocyte numbers and subsets, but for 8 days IgA was higher than in control animals [43], indicating effects of corticosteroids and norepinephrine on the structure and cellular composition of NALT in mice. This phenomenon has not been studied for other lymphoid organs except the thymus which is extremely stresssensitive. The presence of M cells was documented by Park et al. [44]. In a very recent paper this was also documented and dendrites of DC
extending into the nasal cavity were also demonstrated are showing the morphological prerequisites for antigen uptake by NALT [45].
4.4. Adhesion molecules in mouse NALT The development of HEV (the entry site for lymphocytes) is mediated by the chemokine receptor CXCR 5 and also the
Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022
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CD4+ CD8+ B (B220+ ) IgM+ , IgA− IgM+ , IgA+
NALT
PP
Spleen
Blood
32 9 55 49 4
23 2 70 56 6
32 12 45 31 3
75 3 12 7 2
lymphotoxin ß receptor (LTßR) in adult mice. However, NALT can be rescued by other signalling routes [45]. On frozen sections of mouse NALT the presence of different adhesion molecules was tested: all HEV expressed the peripheral lymph node adhesion molecule (PNAd) and this was either associated with the expression of the mucosal vascular addressin MAdCAM-1 (double positive 63%) or PNAd (positive only 37%) [46]. This pattern was completely different from that in Peyer’s patches or peripheral lymph nodes [46]. The postnatal development of NALT was studied by immunohistology in the rat and also in the mouse without major species differences [40]. In further experiments a unique homing pattern of naïve lymphocytes was documented in NALT [47]. It was also documented that PNAd in lymphocyte homing to NALT and allergic responses play a critical role [47]. Thus, the expression pattern of adhesion molecules in this MALT organ is unique [46,47].
4.5. Lymphocyte subpopulations in NALT In normal adult female BALB/c mice a total 1 × 106 cells were obtained per site of the palate. The subset composition was different from that in the blood, spleen or Peyer’s patches [48], as shown in Table 1. Interestingly the rate of CD4/CD8 lymphocytes in NALT differed from that in Peyer’s patches and was similar to that in the spleen. Unfortunately the breeding facilities and the environmental situation of the animal unit were not described in that paper. The composition of lymphocytes in NALT of adult female mice was about 50% B and 50% T cells. There were about 2–3 times more CD4+ than CD8+ lymphocytes [49]. Furthermore, the NALT lymphocytes produced IFN gamma after culture for 24 h and secreted IgA and IgG virus specific antibodies [50]. The same group compared the IgA antibody forming cell response in NALT after vaccination by intranasal, intravenous and/or subcutaneous routes [50]. The combination of intranasal priming and intranasal boosting resulted an increased in anamnestic IgA antibody formation [42].
4.6. Immune reactions in NALT IgA class switch is an important aspect of mucosal immunology and takes place only in organized MALT and not at the effector sites such as the lamina propria [6,50]. In adult mice IgA + B220+ cells were increased in NALT after nasal stimulation with cholera toxin but not after antigen exposure alone [50]. Fernandez et al. [51] documented the induction of germinal centres in NALT and specific mucosal IgA and serum IgG production after intranasal vaccination with a recombinant SEB vaccine on nasal secretion. The efficacy of intranasal vaccination was dependent on NALT as after surgical removal the mice were not protected from toxic shock. In a more recent paper the same group studied NALT by ex vivo culturing NALT and as an alternative to gene knockout mice surgical removal of NALT was used, which is a difficult technique in the mouse [54].
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4.7. NALT in the chinchilla The structure of the nose with its turbinates’ differs greatly between species (for details see [55]). In adult chinchillas (600–800 g body weight) in coronal sections through the dorsal part of the nose lymphoid tissue with well developed follicles was found as paired structures on the palate in each nasal passage [55]. On sagittal sections lymphoid tissue similar to the pharyngeal tonsil was found, which is not present in the mouse or rat. The same group recently published very innovative but also surprising data [56]. They documented a preventive and therapeutic concept to protect against otitis media due to non-typeable Haemophilus influenza in the chinchilla model. This is of enormous clinical relevance. The animals were injected with the bacteria via the skin of the pinna. To track the migration of DC from the pinna the vital dye CFSE was used. The surprising finding was the presence of these DC in the NALT, as it had never been shown in any species that there are lymphatics from the skin of the ear to NALT, but lymphatics would drain to cervical lymph nodes only as far as I know. 4.8. NALT in rabbits The nose of rabbits has not been studied in much detail. In three adult rabbits NALT was found at the bottom of the ventral nasal meatus similar to the site in rodents. Furthermore, isolated lymphoid follicles on the nasal conchae and lateral walls of the nasal cavity were described [57]. 4.9. NALT in farm animals No organized lymphoid tissue similar to that of rodents has been documented (for review [58]). However, isolated lymphoid follicles have been described in sheep and horses, made visible by acetic acid treatment, and M cells have also been documented [59]. In a recent publication the different tonsils were examined in farm animals (ox, sheep, goat, pig, horse, dog, and cat), and NALT was not found in any of them [59]. Thus these species cannot be used as models for vaccination via NALT. 4.10. NALT in humans Human nasal tissue blocks from 150 children, who had died in the first two years of life [60] (cause of death: sudden infant death syndrome (SIDS), traumatic or other causes of death) were studied by routine histological techniques. In contrast to rodents NALT was not found at a definite site but disseminated. NALT was found in a mean of 38% of the children with no difference in respect to the cause of death or age. It was found in the upper nasal cavity 30.1%, on the middle concha 26.4%, inferior nasal concha 13.5%, superior nasal concha 10.4% etc. (Fig. 4). There are no data to my knowledge on the frequency of NALT in adolescents and adults. This is probably due to the problems in obtaining this tissue at autopsy and the need for decalcification. For the situation in humans there is also the previously mentioned problem of definition. Fujimura et al. [61] described M cells which are well defined for antigen uptake in particular in the epithelium covering Peyer’s patches. These M cells, however, were documented for the pharyngeal tonsil only and not for lymphoid tissue in the nose. 4.11. Absence of NALT had little effect in some infection models A classical approach to test the relevance of an organ is to repeat the experiments after surgical removal of the organ. Sabirov and Metzger [62] succeeded in surgically removing NALT in eight day old mice. They later vaccinated the animals intranasally with a
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Fig. 4. Sites of organized lymphoid tissue in the nose of children in percentage of cases. Data modified after Debertin et al. [52].
pneumococcal vaccine and interleukin 12 as an adjuvant. The animals lacking NALT developed nasal and serum antibodies and were protected against pneumococcal colonization of the middle ear and nasopharynx. This excellent technique has recently been applied also by Fernandez et al. [53]. Surgical removal of cervical lymph nodes on the other hand resulted in an increased immune response [63]. Thus, the influenza infection might not be induced in the nose but further down in the respiratory tract [63]. Also in a model of challenge with Mycobacterium tuberculosis after an adenoviral vaccine NALT contributed little in protection after aerosol challenge [64]. Resistance to upper respiratory tract infections by influenza was not provided by NALT or cervical lymph nodes [63]. 5. Antigen uptake by the nasal mucosa 5.1. Mouse Kim et al. [65] have documented M cells in the nasal passages of mice. These cells were capable of picking up bacterial antigens, and were also present and active in mice genetically lacking NALT. Already in 1991 the different types of DC in the conducting airways of rats were characterized [66]. 5.2. Antigen-presenting cells in the human nasal mucosa It is a problem to obtain normal human material for basic research. Nasal biopsies from patients undergoing nasal surgery with no known allergies were studied in 14 patients [67]. Not only DC but also macrophages were identified by typical surface markers. Macrophages outnumbered immature DC. These two types of antigen-presenting cells formed a dense network. In conclusion, the high density of DC and macrophages in the mucosa of the nose forms the structural basis for an effective antigen uptake, a prerequisite for successful nasal vaccination protocols as documented by Cisney by combining surgical removal of NALT with vaccination protocols [54]. 5.3. Adjuvants in intranasal vaccination Fujihashi and Kiyono [29] discussed the relevance of specific adjuvants in mucosal vaccination and in particular for the nasal mucosal immune system. Only a few examples will be given. Neutralizing antibodies and T helper cell response to anthrax were
induced by adding choleratoxin to the antigen [68]. In the experiments on vaccination against P. aeruginosa infection the intranasal route was only as effective as the intramuscular route when cholera toxin was used as an adjuvant [69]. Bacillus firmus had an adjuvant effect via the expression of cytokines [70]. An expansion of DC in the nose was induced by the use of P6 with outer membrane protein and alpha-galactosylceramide in nontypeable Haemophilus influenza [71]. Vajdy and Singh [27] summarized the data on intranasal vaccination protocols against HIV. The intranasal route of influenza vaccination has been discussed by Hagenaars et al. [72] and the comparison to other routes in healthy adults by Keitel et al. [20]. Berstad et al. [73] documented the adjuvant effect of inactivated meningococci and pertussis bacteria in intranasal influenza vaccination. The toxic agent acetic acid in a low dose and small volume (12.5 l of 5% acetic acid per nostril) was used to immunize mice with S. suis effectively [14]. The adenovirus serotype 5 vector expressing Flt 3 ligand activated DC and these started a Notch-ligand expression and the induction of antigen-specific Th1 and Th2 cytokine responses [74]. The epithelium of the respiratory tract is not only a physical barrier for microbial and other antigens but influence via different mediators innate and adaptive immune reactions via dendritic cells as well as T and B cells as reviewed by Schleimer et al. [75]. However, the epithelium of the nose has not been studied in this respect. 6. Conclusions Intranasal vaccination has many advantages in contrast to injection with needles using the subcutaneous or intramuscular route. This is in particular obvious for vaccinating children not forgetting the genetic regulation of immune responses in children [76] or specific problems such as influenza vaccination in the elderly [77]. However, there are still many aspects which have to be studied in more detail which antigen is taken up by organized lymphoid tissue of the nose (NALT) and/or by the specialized cells in the epithelium like dendritic cells, which adjuvants are needed for the first and/or booster vaccination? In any case the enormous age a species differences have to be considered, when data obtained e.g., in the mouse are extrapolated to the situation in man. Acknowledgements My apologies to all authors whose publications have not been quotes but are mainly mentioned in the review articles. The technical assistance in preparing Fig. 3 by Cornelia Höpfel, the preparation of Fig. 4 by Marita Peter, the secretarial help by Sabine Buhmann and Silke Wallbaum and the correction of the English by Sheila Fryk are gratefully acknowledged. References [1] Holmgren J, Lycke N. Principles of mucosal vaccine strategies. In: Smith PD, Mac Donald TT, Blumberg RS, editors. Principles of mucosal immunology. London: Garland Science; 2013. p. 413–28. [2] Fukuyama Y, Tokuhara D, Kataoka K, Gilbert RS, McGhee JR, Yuki Y, et al. Novel vaccine development strategies for inducing mucosal immunity. Expert Rev Vaccines 2012;11:367–79. [3] Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12:592–605. [4] Kunkel EJ, Bucher EC. Plasma-cell homing. Nat Immunol 2003;3:822–9. [5] Brandtzaeg P, Pabst R. Let’s go mucosal: communication on slippery ground. Trends Immunol 2004;25:570–7. [6] Brandtzaeg P, Kiyono H, Pabst R, Russel MW. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol 2008;1:31–7. [7] Czerzinsky C, Holmgren J. Mucosal delivery routes for optimal immunization: targeting immunity to the right tissues. Curr Top Microbiol Immun 2012;354:1–18. [8] Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 2006;6:148–58.
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Please cite this article in press as: Pabst R. Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.022