VIRAL IMMUNOLOGY

Volume 16, Number 3, 2003

Mary Ann Liebert, Inc.
Pp. 279289

Review

Upper Respiratory Tract Immunity

ADRIAN W. ZUERCHER

INTRODUCTION

M OST VIRAL INFECTIONS occur via mucosal surfaces like the respiratory, gastrointestinal, or genital ep-
ithelium. The mucosal immune system is an important component of the bodys defense against such
infections and consequently induction of mucosal, in addition to systemic immunity, might improve vac-
cine efficacy. Several orally administered vaccines, for example, against poliovirus and gastrointestinal bac-
terial infections, have been developed and are widely used. In contrast, to date most vaccines against res-
piratory pathogens are applied parenterally and thus do not induce significant mucosal immunity. For the
development of effective mucosal vaccines a more profound understanding of the immune mechanisms op-
erative at mucosal surfaces and of the interplay between different mucosal compartments is needed. More-
over, factors like the dose, form of application, and type of mucosal adjuvants are critical to the induction
of effective mucosal immunity. This brief review will focus mainly on the nasal route and will summarize
some recent findings concerning the function of the mucosal immune system of the upper respiratory tract.
Furthermore, routes of cross-immunization between distinct mucosal compartments and how they might be
relevant to vaccine development will be addressed. Finally, I will outline critical factors for the rational de-
sign of nasal vaccines and in this context highlight some recent preclinical and clinical developments in the
field.

MUCOSAL IMMUNITY OF THE UPPER RESPIRATORY TRACT

Nasal-associated lymphoid tissue (NALT) is a mucosal inductive site in the upper respiratory tract of ro-
dents like mice and rats. It is a small, paired, secondary lymphoid organ, containing no more than 105106
lymphocytes (in mice), and is located at the floor of the nasal cavity (48,73) (Fig. 1). NALT is lined by a
ciliated epithelium which, towards the luminal side of the nasal cavity, contains cells that can be stained
with Ulex europeaeus agglutinin I (UEA) (20). This indicates the presence of M-cells and follicle-associ-
ated epithelium (FAE), which enables it to efficiently absorb particulate antigens (44,76,82,88). The organo-
genesis of NALT appears to occur independently of the IL-7R, lymphotoxin signaling cascade. But in the
absence of these cytokines and CD41 CD32 cellsthe providers of themNALT is lymphopenic and
lacks organization (32,40). This indicates a distinct mechanism of organogenesis compared to Peyers patches
(PP) or peripheral lymph nodes (LN). However, under normal conditions of development, NALT is simi-
lar to PP in organization and lymphocytic composition, that is, it displays typical segregation into B- and

Research Immunology, Berna Biotech Ltd., Bern, Switzerland.

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FIG. 1. Model of mucosal immune response in the upper respiratory tract of the mouse.

T-cell areas and consists mainly of naive B-cells (5075%, depending of the strain) (88) and naive (CD45RBhigh)
CD41 T-cells (1530%) (85). But whereas PP in naive, conventionally reared mice are in a state of con-
stant (low level) activation, which is reflected by chronic germinal center reactions, NALT in nave mice
appears to be more quiescent and germinal centers develop only after direct stimulation with antigen, such
as viral infection. In this respect NALT resembles more PP of germ-free mice (83). Thus, even though both
tissues are primary sites of antigen contact, the lower antigenic burden encountered by NALT compared to
PP might explain these differences.
Similar to PP, IgA is the major isotype of antibody produced by NALT (41,86). Specific IgA producing
B-cells are induced in NALT after intra-nasal infection with viral pathogens like influenza virus (2,77,85)
or reovirus (88,89). Production of IgA antibodies is preceded by the induction of germinal centers and the
expansion of IgA1 B-cells in NALT (88). Moreover, it has been shown functionally (88) as well as on the
molecular level (72) that significant proportions of IgG1 B-cells are generated in NALT. Additionally,
NALT is a site of induction of specific T-cells. We have shown that upon intra-nasal infection with re-
ovirus, virus-specific CD81 CTL are generated in NALT, characterized by a higher precursor frequency
compared to draining cervical LN (88). Others have shown induction and persistence of memory CD41
T-cells in NALT after influenza virus infection (85).
After the initial induction in NALT, immune responses are amplified in the draining LN (Fig. 1). Even
though clear anatomical evidence is lacking, functional observations indicate that the submandibular or pos-
terior cervical LN serve this purpose in the upper respiratory tract, analogous to the mesenteric LN in the
gut-associated lymphoid tissue (GALT). The more pronounced expansion of lymphocytes in these LN, com-
pared to NALT, after viral infection (11-fold vs. twofold), the more protracted response and the delayed
onset of responses support this hypothesis (88). Even more strongly than NALT, the draining LN show high
production of specific IgG antibodies. This represents an important mechanism by which mucosal infection
via the nasal route leads to systemic (serum IgG) in addition to mucosal (IgA) immunity. This is a feature
particularly important for the development of nasal vaccines, since an important prerequisite for them is to
induce systemic immunity comparable to a parenterally applied vaccine.
From the draining LN, specific effector cells, by passing through the systemic circulation, home to mu-
cosal effector sites in the upper respiratory tract, such as the lacrimal glands (16), the palatine salivary
glands (88,89) and non-lymphoid tissue of the nasal passage, or as sometimes called, the diffuse-NALT
(Fig. 1). These non-lymphoid sites, particularly the nasal passage tissue, appear to be the major sites of per-
sistence of memory B-cells (50) and T-cells (84) in the upper respiratory tract. This is in agreement with

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the theory of preferred persistence of effector memory cells (70) in non-lymphoid tissue (51). However, the
mechanisms of homing to these sites of the upper respiratory tract are largely unknown to date and are a
particularly interesting field awaiting exploration.
Humans do not have a lymphoid tissue anatomically and structurally identical to NALT. But the lym-
phoid organs of the so-called Waldeyer?s pharyngeal ring, most importantly the nasopharyngeal tonsil (ade-
noids) for the nasopharynx and palatine tonsils for the oropharynx, fulfill the same function. Similarly to
NALT, the crypt epithelium of palatine tonsils and adenoids contains M-cells and FAE, assuring efficient
uptake of antigen. Furthermore, germinal centers are induced after antigen contact followed by production
of IgA and IgG antibodies. The immunobiology of the human tonsils and adenoids has been excellently
and extensively reviewed by others (9).

IMMUNOLOGICAL CROSS-TALK BETWEEN DISTANT MUCOSAL

COMPARTMENTS: THE INTEGRATED (COMMON) MUCOSAL IMMUNE SYSTEM

Rabbit PP cells, when transferred to homologous, lethally irradiated recipients, predominantly relocate to the
intestinal lamina propria (LP), where a large proportion of them give rise to IgA secreting plasma cells (25).
Using a similar approach, Rudzik et al. demonstrated that there are many similarities of B cells from PP and
bronchus-associated lymphoid tissue (BALT) and, importantly, that transfer of either PP or BALT cells resulted
in repopulation of both, the intestinal and the bronchial LP (6567). The conclusion of these findings led to the
concept of a common mucosal immune system (57), proposing that cells primed in one mucosal inductive site
may home to many different if not all mucosal effector sites. Elucidation of the mechanisms responsible for the
immunological cross-talk within this common or integrated mucosal immune network could be most relevant
to the development of mucosally administered vaccines. A substantial body of evidence indicates the existence
of various routes of cross-immunization, despite a distinct compartmentalization within the integrated mucosal
immune system (14,15). Best described is the fact that intestinal priming can lead to protection of the lower
respiratory tract. It has been well established in animal models (1,17,63,64), and human studies (19,31), that
oral immunization can prevent infection of the lung. However, several facts suggest that this protection is not
achieved by specific trafficking or homing of lymphocytes to the lower respiratory tract but rather through
mostly unspecific mechanisms. IgG antibodies can reach the lower lung by non-specific transudation from the
systemic circulation (71) or, as recently proposed, maybe also by transported via FcReceptorN (75). In line with
this, it is known that IgA antibodies play a minor role in protection of the lower respiratory tract, but that IgG
antibodies are of central importance (53,59). Furthermore, using a reovirus infection model, we have recently
shown, that CTL induced in GALT by local intestinal infection effectively relocate to the lungs but not the up-
per respiratory tract. In the lower respiratory tract they played a critical role in viral clearance and protection
from challenge, in immunocompetent as well as in SCID mice after transfer of gut-primed lymphocytes or sub-
sets thereof (89). Considering these facts, it can be reasonably explained how protection of the lower respira-
tory tract can even be achieved by parenteral/systemic immunization (81). However, it was proposed by Bi-
enenstock et al. (1012) that mucosal immune responses might be directly induced in the lower respiratory tract
in BALT. But this active role of BALT seems to be restricted to some rodents like rabbits or rats. Mice do not
even have detectable BALT, whereas in humans it only develops as an organized secondary lymphoid struc-
ture after chronic stimulation, like in the case of chronic hypersensitivity pneumonitis. But under normal con-
ditions it plays a minor role only (58).
Furthermore, we have shown that BALT in rats is structurally and functionally clearly different from PP
and NALT. It lacks characteristic organization of secondary lymphoid tissue with distinct B- and T-cell ar-
eas. Most notably, no indications of induction of local immune responses, such as development of germi-
nal centers or appearance of isotype switched cells, were identified in BALT after lower respiratory tract
infection by intra-tracheal instillation of reovirus (90). Nevertheless, due to the presence of M-cell con-
taining, absorptive epithelium overlaying BALT, it might be that these dynamically developing lymphoid
aggregates serve a function in antigen uptake and transport to (systemic) inductive sites.
In contrast to the well-established gut-to-lower respiratory tract-axis it is not clear to what extent priming of
the gut can afford protection from upper respiratory tract challenge. After local duodenal priming with reovirus

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no significant antibody responses in the upper respiratory tract were observed. But after local nasal challenge
of intestinally primed mice B-cell responses with the characteristics of memory responses were detected in
NALT, submandibular LN and palatine salivary glands (89). These included rapid onset and peak of specific
IgA and IgG antibody production as well as increased magnitude of responses compared to primary responses
upon intranasal infection without previous intestinal priming. We concluded that trafficking of memory B-cells
from the GALT to the upper respiratory tract was responsible for protection, thus indicating a gut-to-upper res-
piratory tract homing pathway. Interestingly, the ability to home to the upper respiratory tract was restricted to
memory B-cells, whereas no trafficking of (effector) memory CTL was observed.
Coffin et al. have shown that intra-nasal but not oral immunization of mice with inactivated rotavirus led
to specific B-cell responses in NALT and bronchial LN as well as in intestinal lymphoid tissue. Impor-
tantly, intra-nasal priming afforded protection from subsequent intestinal challenge with live virus (21).
Likewise, it was shown in a mouse model that intranasal immunization of pregnant mothers with virus-like
particles conferred protection from intestinal rotavirus infection and diarrhea in the offspring (23). These
data thus suggest an upper respiratory tract to gut axis of immune trafficking and it was proposed that ex-
pression of aE integrin might play a role in homing of nasally induced effector B-cells to the intestinal lam-
ina propria (26). Another particularly interesting route of cross-immunization within the integrated mucosal
immune system is induction of vaginal immunity by intranasal immunization (68,69,87). It was shown in
mice that herpes simplex specific immunity in the genital tract could be established by nasal immunization
with a recombinant adenoviral vector (3335). Similarly, nasal vaccination with human papilloma virus 16
virus-like particle resulted in specific immunity in the genital tract (3). Interestingly, lymphoid compart-
ments in the lower respiratory tract (trachea, lung, tracheobronchial LN) appeared to be the sites of induc-
tion of specific responses whereas NALT played a minor role only (4).
In summary, the above examples document various routes of cross-protection between distant mucosal
compartments. However, the underlying mechanisms remain largely elusive. Particularly little is known
about the cellular components responsible for cross-protection and whether their relocation to distant mu-
cosal compartments is guided specifically by homing receptors or alternatively occurs randomly. Some at-
tempts have been undertaken to elucidate such mechanisms. As outlined above, our studies indicated that
after intestinal priming protection of the lower respiratory tract is achieved by effector memory CTL, whereas
specific memory B-cells relocate to the upper respiratory tract and are responsible for rapid secondary re-
sponses and protection (89). This represents an example showing distinct cellular components induced in
the GALT relocating to different distant mucosal compartments. Another approach has been the study of
expression of integrins in the upper respiratory tract and their potential role in homing. Csencsits et al. have
demonstrated that the pattern of integrins expressed by NALT high endothelial venules differs significantly
from PP (29). Notably, it is clear that the interaction of a4b7 integrins with MadCAM is a major mecha-
nism of nave lymphocyte homing to PP as well as homing of effector cells to the intestinal lamina propria
(8). In contrast, a4b7MadCAM interactions played a minor role for homing to NALT, but binding to
NALT HEV mainly involved L-selectin and peripheral node adressin (PNAd). These findings were con-
firmed by studies in L-selectin2/2 mice, which displayed a marked reduction of lymphocytes homing to
the upper respiratory tract resulting in decreased local immune responses (27,28). Also involvement of the
CCR9-TECK/CCL25 interaction for homing to the intestinal lamina propria is well established (13,46).
Whether similar interactions are operative in guiding mucosally induced lymphocytes to non-intestinal mu-
cosal compartments remains to be investigated. Recently a chemokine ligand termed MEC/CCL28 that is
specifically expressed on mucosal epithelial tissue (60) and shares homology with TECK/CCL25 as well
as with CTECK/CCL27 was identified. All of these appear to be involved in homing to mucosal sites (47).
This may indicate a network of integrin and chemokine receptors and ligands controlling homing to non-
intestinal mucosal sitesa hypothesis that awaits deeper investigation.

DESIGNING NASAL VACCINES

For the rational design of nasal vaccines several factors need to be considered. First, and most impor-
tantly, it has to be decided whether nasal application of a particular vaccine is useful and superior to an al-

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ternative route. Generally, local mucosal immunity will be best at the site of initial antigen contact and
therefore local immunity should preferably be induced directly at the site where natural challenge most
likely will occur. This has been underlined by several comparative studies, for example, by Asanuma et al.
who compared intranasal, intravenous and subcutaneous administration of inactivated influenza virus vac-
cine and showed that local primary and memory IgA responses in NALT were best after single or repeated
intra-nasal vaccination and superior to administration by any of the other routes or combinations of differ-
ent routes (2).
Similarly, Thompson et al. have used upper and lower respiratory tract infection with reovirus in mice
to elucidate the optimal route or combinations thereof to induce respiratory mucosal immunity. They found
that the most robust responses were achieved by a combined upper/lower respiratory stimulation with anti-
gen (78). Consequently, the major focus for developing nasal vaccines so far has been against pathogens
that infect the airways via the upper respiratory tract, the most important being influenza, respiratory syn-
cytial or parainfluenza virus (62). On the other hand, several advantages such as the simplicity of applica-
tion, the induction of effective systemic as well as mucosal immunity, and the safety (no need for needles)
are arguments to consider the nasal route even for vaccines used mainly to attempt induction of systemic
immunity (49). And as outlined above, by means of cross-immunization the nasal route might be useful for
the induction of mucosal immunity in compartments distant from the upper respiratory site. Particularly,
the approach of inducing vaginal immunity by intranasal immunization has been well established, includ-
ing studies in primates (69). Thus, it may be speculated that these finding also are applicable to humans.
Therefore, development of intranasal vaccines for the induction of local genital immunity against viruses
like herpes or papilloma virus seems reasonable.
A possible drawback to the broad use of the nasal route for vaccination could be the fact that antigens
applied via mucosal surfaces tend to be tolerogenic. This limitation can be overcome by appropriate for-
mulation of the antigen. To induce anti-viral immunity via the nasal route the use of live attenuated instead
of dead vaccines, such as inactivated whole cell, or subunit vaccines is a strategy to circumvent the prob-
lem, since replicating antigens rarely are tolerogenic. Indeed, in humans cold-adapted influenza virus proved
to afford better protection from natural infection than inactivated influenza (6,80). However, live vaccines
require a more extensive biosafety assessment than inactivated vaccines. Therefore formulating subunits
into liposomes/virosomes (36,37) represents an attractive alternative to live vaccines. Yet another com-
pletely different approach might be the use of genetic vaccines (DNA vaccines); for example, Cox et al.
have recently reported that intramuscular DNA vaccination with NP and HA expressing vectors protected
from subsequent intranasal challenge with wild-type influenza virus (24). Moreover, it was shown recently,
that intra-nasal administration of plasmid DNA included in virosomes led to generation of specific CTL in
mice as well as in human peripheral blood lymphocytes stimulated with autologous, in vitro infected den-
dritic cells (22). However, knowledge on mucosal administration of DNA vaccines is still scarce and it ap-
pears to be more difficult to induced effective immune responses via mucosal routes than by intra-muscu-
lar or intra-dermal (gene gun) administration (5). Thus, the feasibility of DNA vaccines for the induction
of mucosal immunity remains to be convincingly proven.
Another important factor for the development on nasal vaccines is the choice of adjuvant. Adjuvants can
efficiently override the tolerogenic potential of most antigens. However, the safety of some adjuvants that
show promising effects in animal models remain to be established in humans. Heat-labile toxin of E. coli
and the exotoxin of V. cholerae (cholera toxin; CT) as well as chemically or genetically detoxified variants
thereof have been evaluated in numerous animal studies. Despite this wealth of preclinical work, few have
been evaluated in human trials and none are currently used in vaccines licensed for human application. CpG
motives of bacterial DNA (18) are effective adjuvants with a strong propensity to induce Th1-like cytokine
responses by stimulation of antigen-presenting cells via toll-like receptors. In animal models CpG-contain-
ing oligonucleotides also showed convincing effects as mucosal adjuvants (35,5456). Moreover, they have
proven safe for the use in humans (74). Thus, this interesting type of adjuvants might be useful in bring-
ing more nasal vaccines from the preclinical to the clinical level. They might be particularly suited for vac-
cines aiming at inducing strong Th1/cellular responses.
In this respect, it has to be kept in mind that the type of vaccine (live vs. inactivated) as well as the choice
of adjuvant will determine the type of T helper cells mainly activated and consequently the quality of im-

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munity induced. Indeed, it is known that local cytokine patterns in NALT greatly vary depending of the
type of antigen used. The quiescent NALT is characterized by a non-committed Th0 cytokine environment
(42) that can be biased towards either a Th1- or Th2-like cytokine pattern dependent of the antigen. Intra-
nasal infection of BALB/c mice with live influenza virus induced a Th1like cytokine pattern with pro-
duction of IFNg, IL-2, and IL-6, whereas intranasal immunization with inactivated influenza in combina-
tion with CT induced strong IL-4 and IL-6 responses. These distinct cytokine patterns were reflected in the
IgG isotype subclasses that predominated, that is, a major IgG2a response in the former and a strong IgG1
response in the latter case (52).
Despite the large quantity of preclinical data and obvious advantages of intranasal vaccines, currently
none are licensed for the use in humans. Nevertheless, some promising developments have been clinically
tested, some of which have been summarized in an excellent review by Davis (30). Several nasally ad-
ministered influenza virus vaccines have been evaluated in humans. Among these, Tomoda et al. used cold
adapted, live attenuated strains of influenza for immunization via the nasal route (79). They found that se-
cretory nasal IgA as well as IFNg production by specific CD41 T-cells was critical for protection against
challenge during a subsequent epidemic outbreak. Similarly, in a comparative study using a trivalent in-
tranasal influenza virus vaccine the effectiveness of cold-adapted, live attenuated influenza virus vaccine at
preventing the shedding of virus was proven (6). The safety and efficacy of this nasal influenza virus vac-
cine was subsequently confirmed in children (7) as well as HIV-patients (45). However, a possible disad-
vantage of this vaccine is the low level of specific serum IgG1 achieved. In an alternative approach, a vi-
rosome based influenza vaccine, containing HLT as a mucosal adjuvant, proved to be immunogenic in
humans and induced strong specific mucosal and systemic antibody responses (36,37). In another recent
study, Greenbaum et al. reported the effects of intranasal vaccination with an inactivated trivalent influenza
virus vaccine in humans (39). In their study, a single immunization resulted in seroconversion in 4070%
of vaccinees (strain dependent) and an overall shift from non-immune to immune in about 50% of the vol-
unteers. Taken together, these studies demonstrate that intranasal influenza vaccineseither in live atten-
uated or virosomal formulationsare effective for the use in humans.
In contrast to these advanced developments in the influenza virus field, no human trials for nasally ap-
plied RSV vaccines have been performed so far. Nevertheless, a parenterally administered RSV subunit
vaccine consisting of a fragment of RSV G-protein coupled to a carrier protein (BBG2Na) (61) has been
evaluated in a phase III clinical trial in humans. The same vaccine was tested via the nasal route in mice
and induced robust systemic and mucosal immunity that conferred protection from respiratory challenge,
importantly without signs of lung pathology (38). No nasally administered parainfluenza vaccines are cur-
rently evaluated clinically, however, in a green monkey model it was demonstrated that parenteral immu-
nization with Sendai virus prevented subsequent nasal infection with parainfluenza virus (43). No nasal vac-
cines for genital infections such as herpes or papilloma virus have progressed to human trials to date.

ACKNOWLEDGMENTS

I would like to thank John J. Cebra for his continuing support and for his helpful suggestions in writing this
manuscript. Furthermore, I thank Guido Dietrich, Christian Moser, and Jean-Francois Viret for critically read-
ing the manuscript. This work was supported by grant AI-23970 from the National Institutes of Health.

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