Vaccine 23 (2005) 36343641

Immune responses induced by lower airway mucosal immunisation

with a human papillomavirus type 16 virus-like particle vaccine
Denise Nardelli-Haeiger
a,
, Floriana Lurati
b,2
, Daniel Wirthner
a,3
, Francois Spertini
b,2
,
John T. Schiller
c,4
, Douglas R. Lowy
c,4
, Francoise Ponci
a,1
, Pierre De Grandi
a,3
a
Department of Gynecology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
b
Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
c
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
Received 19 July 2004; received in revised form 20 December 2004; accepted 24 February 2005
Available online 14 March 2005
Abstract
Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Previous studies have shown that
intramuscular vaccination of women with an HPV16 virus-like particle (VLP) vaccine induced a strong IgG response and protected against
genital HPV16 infection. However, an alternative route of administration that avoids parenteral injection while inducing mucosal immunity
might facilitate vaccine implementation in some settings, and partially overcome the substantial variation in HPV16 antibodies at the cervix
seen in ovulating women. In this study, women were vaccinated with escalating doses of HPV16L1 VLPs via nasal nebulisation, bronchial
aerosolisation, or a combination of intramuscular and aerosol vaccination. The alternative routes of vaccination were well tolerated and many
of the volunteers who received aerosol vaccinations exhibited serum antibody titers that were comparable to those induced by intramuscular
vaccination. A mucosal immune response was induced by aerosol vaccination as demonstrated by the induction of anti-HPV16 VLP IgA
secreting cells in PBMCand SIgAin secretions. Our data suggest that aerosol administration of HPVVLPs may represent a potential alternative
to parenteral injection.
2005 Elsevier Ltd. All rights reserved.
Keywords: Aerosol vaccination; Virus like particles; Human papillomavirus; Cervical cancer
1. Introduction
Cervical cancer is the second leading cause of cancer
deaths in women world-wide, and virtually all of these tu-
mours are attributable to infection with a sub-set of human
papillomavirues (HPV), of which HPV16 is found most fre-
quently [1,2]. An effective vaccine against these HPVs would
therefore be expected to have a dramatic impact on the inci-
dence of this cancer and its precursor lesions, as well as on the

Corresponding author. Tel.: +41 21 314 40 81; fax: +41 21 314 40 95.
E-mail address: dnardell@hospvd.ch (D. Nardelli-Haeiger).
1
Fax: +41 21 314 40 95.
2
Fax: +41 21 314 07 91.
3
Fax: +41 21 314 34 34.
4
Fax: +1 301 480 53 22.
less common tumours attributable to these viruses (reviewed
in [3]). The leading candidate is a sub-unit prophylactic HPV
virus-like particle (VLP) vaccine (reviewed by [4,5]).
Early phase clinical trials of HPV VLP vaccines have
found that a series of three intramuscular (i.m.) immunisa-
tions are well tolerated and can be highly immunogenic even
without adjuvant [68]. Aproof of principle efcacy trial has
shown that women fully vaccinated with an HPV16 VLP vac-
cine were highly protected against genital mucosal infection
by this viral type [9]. However, the requirement for multiple
injections for a vaccine whose anticipated target population
will be older than those of most vaccines may represent a
substantial hurdle for widespread implementation, particu-
larly in the developing world, which accounts for more than
three-quarters of the world-wide cases of cervical cancer [2].
In addition, it has recently been found that although i.m.
0264-410X/$ see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2005.02.019
D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641 3635
VLP vaccination induces readily detectable IgGat the cervix,
the level of antibody decreases several fold during ovula-
tion, which might impair the protective effects of the vaccine
[10].
Mucosal vaccination might represent a potential approach
to overcome these difculties. While both systemic and mu-
cosal immunisation can induce serum-derived Igs in the fe-
male genital tract, the mucosal route has the theoretical ad-
vantage of being given without injection and of inducing
locally produced specic secretory IgA (SIgA) in mucosal
secretions [1015]. Evidence in animals and in people sug-
gests that menstrual cycle-dependent changes of SIgA in the
female genital tract differ from those of serum-derived Igs,
which implies that the presence of specic SIgA might at
least partially compensate for the decrease in serum-derived
Igs at the cervix in the ovulatory phase of the menstrual cycle
([14,16] and references therein, [17]).
Among the different mucosal routes of immunisation,
nasal vaccination has been reported frequently to be an ef-
fective method for inducing specic immunity in the genital
tract [13,1825]. However, we have found in female mice that
HPV16 VLP mucosal immunisation via the lower respiratory
tract can induce high antibody levels in the genital tract and
that this route is more efcient than the nasal route [13,26],
but it is not known whether any immunogen delivered via the
lower respiratory tract can induce antibodies in the human fe-
male genital tract. We have therefore undertaken a pilot study
to compare the safety and immunogenicity of HPV16 VLPs
administered by nasal spray or aerosol to female volunteers
with the response obtained by systemic immunisation.
2. Material and methods
2.1. Volunteers
The clinical protocol was approved by the local ethical
committee. Healthy adult female volunteers, 1845 years old,
with no history of positive Papanicolaou (Pap) smear were
recruited. Written informed consent was obtained for each
participant after the study had been explained in detail. Ex-
clusion criteria included: atopic diseases (food, perennial or
seasonal allergies, rhinitis, asthma, or atopic dermatitis), ab-
normal Pap smear, HIVor HPV16 seropositivity, any chronic
medication, or pregnancy. The volunteers were recruited over
a 17-month period and successively assigned (ve per group)
to either nasal spray or aerosol vaccinations while the vaccine
dose was escalated (2, 50 and 250 g HPV16 VLPs). A nal
group was assigned to receive an i.m. priming dose (50 g
VLP) followed by a mucosal boosting dose (50 g VLP by
aerosol, see below). Following the screening visit, 33 vol-
unteers were included in this study, while 11 were excluded
because of HPV16 VLP IgG titers >50 (n =4) or abnormal
Pap smear (n =7). The responses were compared with a par-
allel group of nine volunteers who were immunised i.m. at
week 0 and week 4 with two doses of 50 g [10]. The eli-
gibility criteria were similar, and the same bulk vaccine lot
was used for the i.m. immunisations and the 50 and 250 g
nasal and aerosol vaccinations. The shorter interval before
boosting after mucosal delivery (2 weeks) was based on our
previous ndings in the murine model [13].
2.2. Vaccination
Clinical lots of recombinant HPV16 L1 VLPs vaccine
were puried from insect cells and suspended in saline with-
out adjuvant, as described previously [6,10]. Nasal adminis-
tration was via a Devilbiss

nebulizer sprayed alternatively

into each nostril. Aerosol administration to the lower airway
was performed with a Systam

nebulizer (aerosolisation by
sonication) and delivered via a mouth piece. All volunteers
received two immunisations. The rst group of volunteers
(n =9) was randomly assigned to receive 2 g VLP doses
of vaccine with the nasal spray (n =4) or the aerosol (n =5)
at week 0 and week 2. Serum and saliva were collected at
weeks 0, 2, 6 and 8, while cervical secretions were collected
at weeks 0 and 8. PBMCwere taken at week 0 and 10 days af-
ter the second immunisation. As there were only mild symp-
toms from vaccination, a second group of volunteers (n =7)
was randomly assigned to receive 50 g VLP doses with the
nasal spray (n =4) or the aerosol (n =3), and sampling was
performed as described. Because an adverse reaction was
observed in one of the volunteers after the second aerosol
immunisation (see results), the subsequent group (n =7) in-
cluded three volunteers who received the 50 g VLP dose
with the aerosol, and four who received 250 g dose with
the nasal spray. In the absence of additional adverse reac-
tions to the aerosol administration, a new group (n =5) was
immunised with 250 g VLP doses with the aerosol. Finally,
an additional group (n =4) received one i.m. priming dose
with 50 g VLP at week 0 and was boosted at week 4 with
an aerosol dose of 50 g VLP. Serum and saliva in this last
group were collected at weeks 0, 4 and 8, cervical secretions
were taken at weeks 0 and 8, and PBMC were collected at
week 0 and 10 days after the aerosol vaccination.
2.3. Sampling
Cervical samples were collected with Weck-cell sponges
duringa gynaecological examination, as previouslydescribed
[10]. Saliva was collected with Weck-cell sponges in a spear
format. Two sponges were placed under the tongue and left
in place for 23 min. Saliva was extracted and stored with
protease inhibitors at 70

C.
2.4. Adverse reactions
Each volunteer had a physical examination prior to vac-
cination and also at weeks 0 and 2, and 10 days after the
second vaccination. For nasal spray vaccination, the exami-
nation included an anterior rhinoscopy to assess the integrity
of the nasal mucosa and the quality of the secretions. For
3636 D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641
Fig. 1. Comparison of the HPV16 VLP specic titers induced by the different vaccination protocols. Specic IgG and IgA titers in serum are shown in A and
B, respectively. Specic titers in cervical secretions normalised to their content in total Igs (U/g) are shown in C (IgG), D (IgA) and in E (SIgA). The different
vaccination routes and/or VLP doses are indicated below the x-axis. Fishers test exact p-values are indicated.
D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641 3637
aerosol vaccination, the examinations included a pharyngeal
examination, a pulmonary auscultation and a measure of peak
airow. The volunteers were also given symptom diaries to
complete. Those receiving nasal spray vaccination were in-
structed to record nasal itch, runny nose, stuffy nose, sneez-
ing, and dry nose, while those receiving aerosol vaccination
recorded throat itch, sore throat, dyspnea, and cough. Each
symptomwas categorised as weak, mild, or strong, and as oc-
curring immediately, 1 h, or 45 h after vaccination, as well
as during each of the three following days. In addition, all
volunteers were instructed to record any other symptoms and
their daily temperature.
2.5. ELISA
The levels of total and HPV16L1 VLP specic IgA and
IgGwere determinedbyELISA, as previouslydescribed[10].
The total or HPV16 VLP specic SIgA was developed by
adding anti-human secretory component (DAKO #A0187)
as secondary antibody. Each sample was tested in duplicate,
with puried human serum IgA (Cappel #55906), IgG (Cap-
pel #55908) or SIgA (Cappel #55905) included in the as-
says as a reference standard. For HPV16 VLP specic Igs,
two-fold serial dilutions were tested, starting at a dilution of
1:50 for sera, 1:5 for cervical secretions, and 1:20 for saliva.
There was less than 15% variation between duplicate sam-
ples. The titers were arbitrarily determined as the reciprocal
end-point dilution that yielded an OD>0.100 (twothree-
fold the mean preimmune OD+3 S.D.). Volunteers were con-
sidered to be HPV16 seropositive at recruitment if their serum
yielded an OD>0.100 at a 1:50 dilution. OD corresponding
to the mean preimmune OD+3 S.D. was used to determine
the titers for SIgA in cervical secretion (OD>0.150) and for
IgA (OD>0.200) and SIgA (OD>0.300) in saliva. In addi-
tion, for comparative purposes, the specic titers in secretions
were normalised to their content in total Ig and expressed as
U/g of total Igs, to compensate for the variation in Ig content
between samples (see Fig. 1).
2.6. Enzyme-linked immunospot assay (ELISPOT)
Trafcking antibody-secreting cells (ASC) that secreted
anti-HPV16 VLP IgA were measured by the ELISPOT
method [27]. PBMC separated by a Ficoll gradient (Ficoll-
paque, Pharmacia, Uppsala, Sweden) were added to VLP-
coated Maxisorb plates (NUNC, 4 10
5
cells/well), and
specic IgA secreted by individual lymphocytes were de-
tected with a biotinylated goat anti-human IgA (Chemi-
con AP #110B), and visualised by alkaline phosphatase-
conjugated streptavidin (DAKO) with 5-bromo-4-chloro-
3-indolyphosphate as substrate. Detection of 20 or more
spots/4 10
6
cells after vaccination was dened as a posi-
tive response, as it corresponded to the mean number of spots
counted in the PBMC of the vaccinee before vaccination +3
S.D.
2.7. Statistics
Toallowstatistical comparisonwithlarger groups between
vaccination protocols, all doses in the nasal group were con-
sidered together, while for the aerosol protocol the 50 and
250 g doses were considered together. Comparison with the
i.m. group were made with a Fishers exact test for serocon-
version (HPV16 VLP-specic IgG and IgA, see Fig. 1A and
B) andfor detectable responses incervical secretions (HPV16
VLP specic cervical IgG and IgA, see Fig. 1C and D) using
GraphPad Prism.
3. Results
3.1. Safety prole of the vaccine
This was a dose escalation study of nasal (nasal groups)
and aerosol (aerosol groups) immunisation of HPV16 VLPs
(two doses of 2, 50, or 250 g) as well as the combination of
a 50 g priming i.m. injection followed by a 50 g aerosol
booster (systemic/aerosol group). The responses were able
to be compared against two doses of 50 g i.m. vaccination
(systemic group) from a parallel study [10]. The nasal and
aerosol vaccinations were generally well tolerated, with no
serious adverse events. Among the 12 volunteers who re-
ceived VLPs as a nasal spray, the only local symptom noted
was some mild minor local discomfort, and one volunteer
reported a mild possibly related systemic adverse event (fa-
tigue). Among the 15 volunteers in the aerosol groups and
the 4 volunteers in the systemic/aerosol group, the only local
symptom was mild pharyngeal discomfort. One volunteer, in
the 50 g aerosol group, reported a systemic side effect of
moderate intensity that was probably related to the vaccine:
5 h after her second dose she experienced dyspnea, chills,
fever (39.8

C), myalgias, and arthralgias that were relieved

by self-administration of aspirin. The patient rst informed
us about these symptoms 36 h after they began; by that time
she had no symptoms, the physical examination was normal,
and blood tests suggested mild systemic inammation (ery-
throcyte sedimentation rate 28 mm/h {normal: <20 mm/h}, C
reactive protein 46 mg/l {normal: <10 mg/l}). Interestingly,
this volunteer exhibited a very high anti-VLP response in her
serum and cervical secretions (see Tables 1 and 2). On the
day following her rst vaccination, another volunteer, in the
2 g aerosol group, reported mild fatigue, which persisted
for 48 h and was considered possibly vaccine-related.
3.2. Immunogenicity of the vaccine
We measured ELISA titers of HPV16 VLP-specic an-
tibodies in serum and at the cervix; for both of these uids
a good correlation has been shown between this assay and
HPV16 neutralisation [6,28,29]. In contrast to reports with
other antigens nasal vaccination was poorly immunogenic
for most volunteers, including the 250 g nasal group (see
3638 D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641
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y
.
Fig. 1). Only three volunteers (3/11) exhibited specic serum
IgG titers (200800) after administration of two nasal doses
(two after 2 g VLP doses and one after 50 g VLP doses,
data not shown). Only the volunteer with the highest serum
titer (800 for IgG and 100 for IgA) had HPV16 IgG, IgA and
SIgA in her cervical secretion.
Aerosol vaccination was more immunogenic (see
Tables 1and2). AlthoughnoVLP-specic immune responses
were detected in the group that received the 2 g dose, se-
roconversion was seen for 4/6 volunteers who received the
50 g dose and 5/5 who received the 250 g dose. For all
these seroconverters, the predominant specic Ig in the serum
was IgG, as was also true for the systemic/aerosol group
and the systemic group, with serum IgA titers being 220
times lower. In the 50 and 250 g aerosol groups and the
systemic/aerosol group, an increase in the number of VLP-
specic trafcking ASC of the IgA isotype was found in the
PBMC of most vaccinees (11/14). Most of the vaccinees
who seroconverted had detectable VLP-specic IgG titers
in their cervical secretions (9/12). The seroconverters who
did not have detectable cervical IgG came from those with
lower serum IgG titers (100400). By contrast, VLP-specic
IgA in cervical secretions was detected in only six samples,
and did not appear to correlate closely with IgA seroconver-
sion and/or detection of IgASCin PBMC. Salivary antibodies
were also determined; VLP-specic IgAwas only detected in
the saliva of ve vaccinees, and did not correlate with serum
and cervical responses.
Rate of seroconversion after aerosol vaccination (50 and
250 g doses) was signicantly higher than after nasal vac-
cination (all doses, p =0.03 and p =0.06 for IgG and IgA,
respectively, see Fig. 1A and B), but similar to the systemic
group (p =0.47 and p =0.65 for IgG and IgA, respectively).
Since the antibody content in cervical secretions may vary
during the menstrual cycle or under different oral contracep-
tive regimens, the VLP-specic Igs titers were normalised to
their content in total Igs (expressed in arbitrary U/g total
Igs see Fig. 1CE). This normalisation allowed direct com-
parison of cervical titers for different vaccination protocols,
including the systemic group. All vaccinees in the i.m. group
(9/9) developed signicant levels of normalised VLP-specic
IgG in their cervical secretions, and similar titers were in-
duced in 7/14 volunteers who received 50 or 250 g VLP
aerosol doses (including the systemic/aerosol group). How-
ever, rate of detectable cervical IgGresponses seemed higher
(p =0.09) in the systemic group as compared to the aerosol
group (50 and 250 g doses, 6/10). The detection rate was
lower in nasal vaccinees (2/11, p =0.08 and p =0.0003 as
compared to aerosol and systemic groups, respectively). Nor-
malised VLP-specic IgA titers in cervical secretions were
detected in 6/9 i.m. vaccinated volunteers, which is similar
to the rate of detection in the aerosol group (5/10), p =0.65),
but higher than after nasal vaccination (p =0.06 and p =0.02
whencomparedtoaerosol andi.m. vaccination, respectively).
Interestingly the highest normalised VLP-specic IgA titers
were observed in two volunteers of the 50 g aerosol group,
D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641 3639
Table 2
Anti-HPV16 VLP titers
a
in secretions after aerosol and i.m. vaccination
50 g VLP doses 250 g VLP doses I.m. priming +aerosol boosting Two i.m. 50 g VLP doses
Cervical secretion Saliva Cervical secretions Cervical secretions Saliva Cervical secretions
Week 8 Week 2 Week 6 Week 8 Week 8 Week 8 Week 8
IgG IgA SIgA IgA SIgA IgA IgA IgG IgA IgG IgA IgA sIgA IgG IgA
640 20 21
b
30 80
2430
c
610 305 14 180 920 115
180 90 45 100 40 150 50 210 55 136 136 1216 76
84 17
30 44 44 23 832 104
800 50
320
36 18
72 9
a
Only detectable titers are indicated, but the individual subjects follow the same order as those in Table 1 i.e. each lane within a dose group represent the
data from the same volunteer as in Table 1.
b
This volunteer has been only immunized once.
c
This volunteer reported a sytemic side effect of moderate intensity.
and they consisted mainly in SIgA, while no SIgA were de-
tected in the i.m. vaccinated volunteers.
4. Discussion
Papillomavirus VLPs when given systemically have been
well tolerated and remarkably immunogenic even without
adjuvant. I.m. immunisation with a series of 50 g doses of
HPV16 VLPs without adjuvant induced potent serum im-
mune responses that were similar in magnitude to those in-
duced by the same dose of vaccine adjuvanted with alum
or the proprietary adjuvant MF-59 [6]. The current report
indicates that many normal volunteers immunised via an
aerosol route with HPV16 VLPs without adjuvant can also
mount a strong serum response. All ve volunteers in the
250 g aerosol group seroconverted, and the magnitude of
their serum IgG and IgA responses was similar to that seen
with the 50 g i.m. group. The 50 g aerosol immunisation
was somewhat less effective, although 4/6 volunteers in this
group seroconverted, and three of the seroconverters devel-
oped serumtiters comparable to those in the 50 g i.m. group.
Aerosol vaccination was well tolerated, with mild local or
systemic symptoms, except for one volunteer who, 5 h af-
ter her second aerosol dose, experienced a systemic reaction
consisting of dyspnea, chills, fever, myalgias, and arthral-
gias that were relieved by self-administered aspirin. Testing
in additional volunteers will be needed to determine whether
this type of episode represents a coincidental event, a very
unusual side effect of aerosol administration, or a more com-
mon side effect. In contrast to the positive results obtained
with the high dose aerosol groups and to reports with other
antigens [25,30], nasal HPV16 VLP vaccination was poorly
immunogenic for most volunteers, even those in the 250 g
nasal group.
The results indicate there may be substantial differences
in the immune response to the same immunogen when de-
livered by these two mucosal routes. The superiority of
aerosol administration over nasal administration noted here
is similar to what we have observed with HPV16 VLPs
in mice [13], where VLPs were mainly presented to the
immune system by dendritic cells in the tracheobronchial
lymph nodes and not by the nasal associated lymphoid tis-
sues [26]. To our knowledge this feature has not been de-
scribed for other antigens and may be peculiar to HPV
VLPs.
The positive aerosol results also demonstrate that a sub-
unit VLP vaccine given without adjuvant can induce a strong
immune response via this route. Adding a mucosal ad-
juvant to the vaccine might enhance its immunogenicity
[31].
Our data raise the possibility that administration of the
VLP vaccine via the aerosol route may represent an alterna-
tive to systemic immunisation, if more extensive studies con-
rm that vaccination via this route is safe, immunogenic, and
protective against genital HPV infection. The need for mul-
tiple immunisations via needle injection with the i.m. route
may represent a barrier to widespread vaccine implementa-
tion in the developing world [32], where an HPV vaccine
would be expected to have the greatest public health impact,
since cervical cancer is the most common cancer of women
in many developing countries. The experience with hepati-
tis B virus vaccine, which is given by a series of i.m. in-
jections, suggests that widespread implementation may take
many years. Although the hepatitis B vaccine is at least 90%
effective, has been licensed since the 1980s, and national pro-
grams have been recommended by the World Health Organ-
isation and other advisory groups, as of 2001 more than 80
countries around the world lacked such programs [33]. The
barrier to widespread implementation of an HPV vaccine in
3640 D. Nardelli-Haeiger et al. / Vaccine 23 (2005) 36343641
the developing world may be even greater, as the presumed
target population receiving the vaccine would be older than
the infants and young children who are the focus of current
national vaccine programs [34]. The ability to administer an
HPV vaccine via the aerosol route, which has less need for
medical personnel or a medical setting, could facilitate vac-
cine implementation.
Compared with systemic immunisation, mucosal immu-
nisation also has the theoretical advantage of being able to
induce trafcking ASC, leading to the local production at
distant mucosal sites of SIgA, which specically protect mu-
cosal surfaces [35] (reviewed in [36]). Indeed, most (9/10)
of the aerosol vaccinees in the 50 and 250 g groups devel-
oped VLP-specic trafcking ASCof the IgAisotype in their
PBMC, IgA was detected at the cervix in a subset of these
vaccinees, and the two vaccinees with the highest serum IgA
titers were from the aerosol vaccinees. Most of the vacci-
nees with trafcking ASC did not have detectable SIgA at
either mucosal site, however, this low proportion may be a
reection of the relative insensitivity of the SIgA ELISA (ca.
500 ng/ml for total SIgA compared with 50 ng/ml for total
IgA). It is unclear what may account for cervical IgGand IgA
levels in the 250 g aerosol group being lower than those in
the 50 g aerosol group. For some volunteers, this difference
might be attributable to sampling during a portion of the men-
strual cycle associated with low levels of cervical antibodies
[10].
Saliva was also analysed as it is easy to sample, and
specic IgA has been reported previously after various mu-
cosal vaccination routes [3739]. However, only six volun-
teers developed VLP specic antibodies in the saliva. Only
three of them had concomitant VLP IgA in cervical secre-
tions, while the salivary VLP response was isolated in the
three others volunteers. In contrast to our nding in mice
[13], the VLP specic salivary response thus appeared to
be poor predictor of an anti-VLP IgA response in cervical
secretions.
Different routes of immunisation, including vaginal, rec-
tal, oral and nasal have been explored to induce specic anti-
bodies in female genital secretions with variable success de-
pendingonthe type of antigenused[25,3843], but withnasal
vaccination appearing the most practical and efcient method
[25,44,45]. Althoughaerosol immunisationhas beensuccess-
fully used for live attenuated measles vaccination, with an
efcacy similar to the subcutaneous route [46], aerosol ad-
ministration is not commonly used for other vaccines. To our
knowledge, this pilot study is the rst attempt to use the lower
respiratory tract to generate specic antibodies in female gen-
ital secretions. The strong immune response elicited by the
aerosol administration of the HPV subunit vaccine suggests
that further exploration of this route is warranted. Aerosol
delivery may be further optimised by various modications,
such as changing the size of aerosolised particles and/or the
access to lung or alveoli, and might be rendered more practi-
cal by the use of powdered formulations as shown for measles
vaccine [47].
Acknowledgements
This work was supported by the Fonds de Service of the
Dept. of Gynecology and by grants from the Swiss Cancer
League #KFS-00941-09-1999 and #OCS-01179-09-2001 to
DNH and the Swiss National Fund #631-057969.99 to DNH
and #3100A0-100561 to FS.
We thank the nurses of the Dpt. of Gynecology and Div.
of Immunology for their help in the management of the vol-
unteers.
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