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Journal of Virology, October 1998, p. 8220-8229, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Nasal Immunization of Mice with Human
Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing
Antibodies in Mucosal Secretions
Carole
Balmelli,1
Richard
Roden,2
Alexandra
Potts,1
John
Schiller,2
Pierre
De
Grandi,1 and
Denise
Nardelli-Haefliger1,*
Department of Gynecology, Centre Hospitalier
Universitaire Vaudois, CH-1011 Lausanne,
Switzerland,1 and
Laboratory of Cellular
Oncology, National Cancer Institute, Bethesda, Maryland
20892-40402
Received 22 April 1998/Accepted 24 June 1998
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ABSTRACT |
To specifically induce a mucosal antibody response to purified
human papillomavirus type 16 (HPV16) virus-like particles (VLP), we
immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP
immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital
secretions were measured by enzyme-linked immunosorbent assay (ELISA).
Systemic immunizations alone induced HPV16 VLP-specific IgG in serum
and, to a lesser extent, in genital secretions but no secretory IgA.
Oral immunization, even in the presence of CT, was inefficient.
However, three nasal immunizations with 5 µg of VLP given at weekly
intervals to anesthetized mice induced high (>104) and
long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all
samples, including IgA and IgG in saliva and genital secretions. CT
enhanced the VLP-specific antibody response 10-fold in serum and to a
lesser extent in saliva and genital secretions. Nasal immunization of
conscious mice compared to anesthetized mice was inefficient and
correlated with the absence of uptake of a marker into the lung.
However, a 1-µg VLP systemic priming followed by two 5-µg VLP
intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG
and IgA in secretions, although the titers were lower than in
anesthetized mice given three intranasal immunizations. Antibodies in
serum, saliva, and genital secretions of immunized mice were strongly
neutralizing in vitro (50% neutralization with ELISA titers of 65 to
125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols
that induced significant titers of neutralizing IgG and secretory IgA
in mucosal secretions in mice may be relevant to genital HPV VLP-based
human vaccine trials.
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INTRODUCTION |
The "high-risk" human
papillomavirus (HPV) types, most commonly type 16 (HPV16), are
etiologically linked to over 90% of cervical cancers (7).
Cervical cancer is the second leading cause of cancer deaths in women
worldwide, encouraging the development of a prophylactic vaccine to
prevent genital infection by these viruses. Vaccine development has
been hindered by the difficulty of virus propagation in culture and the
lack of animal models for the genital mucosatropic HPV type
(34). However, expression of the papillomavirus major capsid
protein L1 in mammalian, insect, yeast, or bacterial cells has been
shown to generate virus-like particles (VLPs) (22, 24, 28, 30, 43,
51, 53). Parenteral injection of these VLPs elicits high titers
of neutralizing antibodies in serum and protection from experimental
challenge with infectious virus in animal papillomavirus models
(10, 28, 29, 51, 53). Protection from experimental infection
with cottontail rabbit papillomavirus and canine oral papillomavirus by
passive transfer of immunoglobulin G (IgG) from immunized to naive
animals has been demonstrated for rabbits (10) and dogs
(56), respectively, indicating that cell-mediated effector
immune responses are not required for protection.
Neutralizing antibodies must be present at the genital mucosal site of
infection to completely prevent cervical HPV infection. Antibodies both
pass from plasma into genital secretions and are synthesized by local
plasma cells (48, 58, 64). The plasma cell precursors that
migrate to the genital tract are derived primarily from mucosal
lymphoid tissues and mostly secrete IgA (9, 39). Stimulation
of these cells requires that antigens have access to mucosa-associated
lymphoid tissue (MALT). In several experimental systems, nasal
instillation was the most effective route of immunization to generate
specific antibodies in genital secretions in mice (15, 16, 20, 25,
27, 43, 47, 55) and in monkeys (52).
Systemic immunization of mice with purified HPV VLPs did not induce
detectable genital mucosal antibodies (21), while low-titer VLP-specific IgG, but not IgA, was detected in cervicovaginal washes of
parenterally immunized monkeys (33). The experiments in
monkeys, however, showed that the transudated IgG alone partially and
transiently neutralized HPV11 in vitro, suggesting that a local,
sustained production of secretory IgA (sIgA) and/or specific IgG may be
required for long-lasting sterilizing immunity. In this study, we have
compared different protocols of immunization of mice, including nasal
and oral mucosal routes, by using HPV16 VLPs purified from insect cells
for their ability to induce HPV16 VLP-specific antibodies in serum and
mucosal secretions of mice. Furthermore, the in vitro neutralizing
activity of salivary and genital secretions containing specific IgA
and/or IgG, and sera were compared by using an HPV16 pseudovirion
neutralization assay (50).
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MATERIALS AND METHODS |
Purification of HPV16 VLPs, immunization, and sampling of
mice.
Baculovirus-derived HPV16 VLPs were purified as
described previously (28) and diluted to a final inoculum
volume of 20 µl with either phosphate-buffered saline (PBS) for
subcutaneous immunizations or PBS-0.5 M NaCl either alone or mixed
with 5 µg of cholera toxin (CT; Sigma) for nasal immunizations.
Six-week-old female BALB/c mice were used in all the experiments. For
oral immunization, the mice were fed 30 µl of 10% sodium bicarbonate
to neutralize stomach acidity 5 min prior to administration of the
20-µl inoculum. For nasal immunization, the mice were either
conscious or anesthetized by intraperitoneal injection with 100 µl
each of both 0.2% xylazine hydrochloride (Rompun; Bayer) in PBS and 10 µg of ketamine hydrochloride per ml (Ketavet; Parke-Davis) per
10 g of body weight. The inoculum (20 µl) was introduced
dropwise into one nostril. Subcutaneous immunization was performed at
the base of the tail. Blood, saliva, and genital samples were taken as
described previously (25). All samples were stored at
70°C. For neutralization experiments (see below), the saliva and
genital washes were sterilized by irradiation (3,000 rads).
ELISA.
The amounts of total IgA and IgG as well as
anti-HPV16 VLPs antibodies were determined by enzyme-linked
immunosorbent assay (ELISA) with biotinylated goat anti-mouse IgA
(Kirkegaard & Perry Laboratories) or IgG (Amersham), respectively, as
described previously (25, 43). End-point dilution of all
samples was carried out. The specific IgA or IgG titers were expressed
as the reciprocal of the highest dilutions that yielded an optical
density at 492 nm four times that of preimmune samples. These
reciprocal dilutions were normalized to the amount of total IgA or IgG
in saliva and vaginal washes (25).
In vitro HPV16 neutralization assay.
Infectious pseudotyped
virions consisting of the HPV16 capsid, comprising L1 and L2,
surrounding the bovine papillomavirus type 1 genome, designated HPV16
{BPV1}, were generated as previously described (50).
Infectious pseudotype HPV16 in cell extracts was quantitated by the
induction of transformed foci in monolayers of mouse C127 cells.
Neutralizing activity was measured after preincubation of the cell
extracts with samples from mice at the indicated dilutions (final
volume, 1 ml) for 1 h on ice. Preimmune samples and samples from
mice immunized with an unrelated antigen (a recombinant
Salmonella typhimurium strain expressing the nucleocapside of hepatitis B virus: HBc antigen [25, 43]) were used
as negative controls. Mouse monoclonal antibody H16.V5 raised against
HPV16 VLP (a gift of N. Christensen, Milton S. Hershey Medical Center, Hershey, Pa.) was used as a positive control.
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RESULTS |
Intranasal immunization with purified HPV16 VLPs induces systemic
and mucosal antibody responses.
Groups of four anesthetized mice
were immunized intranasally three times at weekly intervals with 5 µg
of purified VLPs, either with or without 5 µg of CT, in a final
volume of 20 µl. Serum, saliva, and genital washes were taken 3, 4, 5, 7, 10, and 15 weeks after the first immunization. Both methods
induced sustained high HPV16 VLP-specific antibody titers in serum and
in genital and oral secretions (Fig. 1).
The addition of CT resulted in a 10-fold increase in the specific IgG
titers in the serum, while the effect on specific antibody titers in
both saliva and vaginal washes was generally smaller. Overall, the
titers of antibody were quite stable during the 15-week study. In
contrast, oral immunization of mice with the same amount of VLPs did
not induce VLP-specific antibodies in either serum or secretions
whereas the addition of CT induced very low titers of VLP-specific IgG
in sera and IgA in secretions (mean titers of 50 and 10, respectively;
data not shown). Because the significant toxicity of CT precludes its use in humans and because of the highly immunogenic nature of VLPs, CT
was not used in the subsequent experiments.
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FIG. 1.
Anti-HPV16 VLP systemic and mucosal antibody responses
after intranasal immunization with purified HPV16 VLP in the presence
or absence of the CT adjuvant. Two groups of four 6-week-old BALB/c
anesthetized mice were immunized three times weekly with 5 µg of
HPV16 VLP alone or mixed with 5 µg of CT by the intranasal route.
Data are expressed as the geometric means (log10) of the
reciprocal dilutions of specific IgG in serum and specific IgA per
microgram of total IgA or specific IgG per microgram of total IgG in
secretions. Error bars indicate the standard errors of the means.
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Effective intranasal immunization requires anesthesia.
We had
previously observed that the titers of antibodies generated by
intranasal immunization with recombinant Salmonella varied
between conscious and anesthetized mice (25), so we tested intranasal immunization with purified VLPs in conscious mice. The
schedule of immunization and sampling was similar to that in the first
experiment (Fig. 1). In contrast to mice immunized under anesthesia,
mice immunized while conscious generated only very low titers of
specific antibody titers in serum and in saliva (mean titers of 50 and
10, respectively) and no detectable antibody in vaginal washes (data
not shown). To examine the role of anesthesia in the induction of
anti-VLP antibodies after nasal immunization, we compared the fate of a
live Salmonella typhimurium inoculum administered
intranasally to conscious and anesthetized mice. The number of bacteria
recovered in various organs was assayed to trace the inoculum (Fig.
2). The fate of the inoculum 15 min after
immunization was quite different in conscious and anesthetized mice: a
high percentage of inoculum (30%) was recovered from the lungs of mice
immunized under anesthesia, but the mice immunized while conscious had
a very small amount of inoculum in the lungs (0.1%). Instead, much of
the intranasal inoculum (45%) in conscious mice was rapidly swallowed
and found in the intestine. Similar portions of inoculum remained in
the nasal cavity 15 min postimmunization in both protocols. These data
suggest that the strong antibody response induced by nasal immunization
with purified HPV16 VLPs of mice under anesthesia results from
deposition of the antigen in the lungs.
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FIG. 2.
Fate of an inoculum 15 min after intranasal immunization
of anesthetized or conscious mice. Two groups of three 6-week-old
BALB/c mice were immunized intranasally with 108 CFU of
S. typhimurium PhoPc in 20 µl of PBS (25,
43) while they were under anesthesia or conscious. At 15 min
later, the nasal tissue, the trachea and lungs ("lung"), the
esophage, pharynx, and stomach ("stomach"), and the intestine and
rectum ("intestine") were recovered and homogenized in a Dounce
homogenizer in 2 to 4 ml of 15% sucrose in PBS (43). Data
are expressed as the geometric mean (log10) of recovered
CFU per organ. Error bars indicate the standard errors of the means.
The percentages of the recovered inoculum in each organ is also
indicated.
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The efficacy of nasal immunization is dose and regimen
dependent.
The HPV16 VLP-specific antibody responses were compared
in mice immunized intranasally under anesthesia with a dose of either 5 or 1 µg of purified HPV16 VLPs (Fig. 3)
and a booster immunization with the same dose 8 weeks later. Samples of
serum, saliva, and vaginal washes were taken 3, 4, 6, and 8 weeks after
the first immunization and 2, 5, and 11 weeks after the booster dose. A single 1-µg VLP dose induced only barely detectable titers of specific IgG in serum and specific IgA in saliva, and the booster immunization had no effect, whereas a single 5-µg VLP dose induced low specific IgG titers in serum and genital secretions and a 10-fold
increase in all specific antibody titers after the booster immunization. Although the specific IgG titers in serum after two
5-µg VLP immunizations were similar to those measured with the three
weekly 5-µg doses (Fig. 1), the titers of specific antibodies in
secretions were lower and less stable over time after only two more
widely spaced immunizations.
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FIG. 3.
Anti-HPV16 VLP systemic and mucosal antibody responses
after single or double nasal immunizations. Two groups of four
6-week-old BALB/c anesthetized mice were immunized at week 0 and at
week 8 with 1 or 5 µg of HPV16 VLP by the intranasal route. Data are
expressed as the geometric means (log10) of the reciprocal
dilutions of specific IgG in serum and specific IgA per microgram of
total IgA or specific IgG per microgram of total IgG in secretions.
Error bars indicate the standard errors of the means.
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Systemic priming influences the mucosal response induced by nasal
immunization.
Although systemic immunization alone fails to induce
sIgA in mucosal secretions, it has been previously shown to enhance the outcome of associated mucosal immunizations (18, 32, 38, 57). Moreover, systemic immunization is usually more effective at
inducing specific systemic antibodies than are immunizations by mucosal
routes, and the serum-specific IgG concentration can influence the
amount of specific IgG transudating into genital secretions. We have
therefore evaluated different combinations of systemic, i.e.,
subcutaneous, and intranasal immunizations. The effects of systemic
boosting are depicted in Fig. 4. Mice immunized previously with three weekly intranasal 5-µg doses (Fig. 1
and conscious mice) were given booster doses of 1 µg of HPV16 VLP
subcutaneously and samples of serum and secretions were taken 2, 6, and
12 weeks later (weeks 17, 21, and 27, respectively, in Fig. 4). A sharp
but transient increase in specific IgG titers was observed in serum and
secretions of all animals, including the nonresponder mice that had
been immunized intranasally while conscious, thus indicating that those
mice had not been tolerized. The systemic boost had no effect on the
IgA titers in secretions and, at best, transiently boosted mucosal IgG
titers (Fig. 4).
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FIG. 4.
Anti-HPV16 VLP systemic and mucosal antibody responses
following systemic boosting. Three groups of mice previously immunized
(Fig. 1 and conscious mice) by the intranasal route, three times
weekly, with 5 µg of HPV16 VLP mixed with CT or alone under
anesthesia or conscious were given subcutaneous (s.c.) booster doses at
week 15 with 1 µg of HPV16 VLP. Data are expressed as the geometric
means (log10) of the reciprocal dilutions of specific IgG
in serum and specific IgA per microgram of total IgA or specific IgG
per microgram of total IgG in secretions. Error bars have been deleted
to simplify reading.
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To analyze the effect of systemic priming followed by nasal boosting,
16 mice were subcutaneously immunized with 1 µg of HPV16
VLPs,
divided into four groups of four mice, and subsequently
immunized at
weeks 2 and 10 by different methods. The first and
second groups were
given two intranasal booster doses under anesthesia
with 1 and 5 µg
of VLPs respectively, while the third group received
5-µg doses
intranasally without anesthesia and the fourth group
received 1-µg
doses subcutaneously. Samples of serum, saliva,
and vaginal washes were
taken 2 weeks after the systemic priming;
2, 4, 6, and 8 weeks after
the second immunization; and 2, 4,
and 8 weeks after the third
immunization (Fig.
5). After a single
subcutaneous immunization with a 1-µg VLP dose, the specific IgG
titers measured in the serum were about 10-fold higher than those
measured with a single 5-µg VLP dose administered nasally under
anesthesia (compare Fig.
3 and
5), indicating that systemic
immunization
is indeed more efficient than intranasal immunization at
inducing
IgG responses. After the second immunization, a similar rise
in
the specific IgG titer in serum was observed in all groups of
mice
independently of the route of immunization. The low specific
IgG titers
measured in saliva became undetectable at week 10.
Variable but higher
specific IgG titers were measured in vaginal
washes, and this was seen
more consistently in mice given 5-µg
booster doses either
subcutaneously or intranasally while anesthetized
(Fig.
5). As
expected, no specific IgA was detected in mice given
subcutaneous
booster doses whereas significant VLP-specific IgA
titers were measured
in saliva and vaginal washes in mice given
intranasal booster doses.
This is in contrast to single or double
intranasal immunizations, by
either dose regimen or mode, which
induced no or barely detectable
specific IgA (Fig.
3). The data
indicate that systemic priming both
overcomes the nonresponsiveness
to nasal immunization in conscious mice
and enhances overall the
efficacy of intranasal immunization. Although
the second boost
induced some increase in the specific antibody titers
in serum
and secretions, this effect was transient, except for IgG in
saliva,
and thus was of little benefit.
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FIG. 5.
Anti-HPV16 VLP systemic and mucosal antibody responses
in mice systemically primed. Four groups of four 6-week-old BALB/c mice
were primed with 1 µg of HPV16 VLP subcutaneously and then given
booster doses at weeks 2 and 10 either intranasally with 1 or 5 µg of
HPV16 VLP under anesthesia or conscious or subcutaneously (s.c.) with 1 µg of HPV16 VLP. Data are expressed as the geometric means
(log10) of the reciprocal dilutions of specific IgG in
serum and specific IgA per microgram of total IgA or specific IgG per
microgram of total IgG in secretions. Error bars indicate the standard
errors of the means.
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To further compare a three-dose protocol of immunization either with or
without systemic priming, we immunized three groups
of four mice with
three weekly VLP doses. The first group received
the 5-µg VLP doses
intranasally under anesthesia, while the second
and third groups were
subcutaneously primed with 1 µg of VLP and
then given two booster
doses with 5 µg of VLPs intranasally under
anesthesia or while
conscious, respectively (Fig.
6). From
this
direct comparison, it appears that three intranasal 5-µg VLP
doses
were more effective at inducing specific antibodies in secretion
than were the other methods tested in this study.
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FIG. 6.
Direct comparison of anti-HPV16 VLP systemic and mucosal
antibody responses in mice systemically primed or only intranasally
immunized. Three groups of four 6-week-old BALB/c mice were either
primed subcutaneously with 1 µg of HPV16 VLP and then given
intranasal booster doses of 5 µg at weeks 1 and 2 under anesthesia or
conscious or only intranasally immunized three times weekly with 5 µg
of HPV16 VLP under anesthesia. Data are expressed as the geometric
means (log10) of the reciprocal dilutions of specific IgG
in serum and specific IgA per microgram of total IgA or IgG per
microgram of total IgG in secretions. Error bars indicate the standard
errors of the means.
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Mucosal secretions containing HPV16 VLP specific IgA and/or IgG
neutralize HPV16 pseudotyped viruses.
To assess the neutralization
potential of mucosal secretions containing anti-HPV16 VLP antibodies,
saliva and genital secretions were tested in the HPV16 pseudovirion in
vitro neutralization assay (50) at two different dilutions
(Table 1). Serum and secretions obtained
by a triple intranasal immunization under anesthesia in the presence
and absence of CT (Table 1; Fig. 7), as
well as by a triple spaced subcutaneous immunization (Fig. 7), were
tested. The absolute titers of VLP-specific IgG and/or IgA in the
samples that were used for neutralization experiments (Table 1; Fig. 7)
were plotted against their efficiency of neutralization (Fig. 7). In
samples containing both specific IgA and IgG, the titers of the two
specific isotypes were added, and the total titers are indicated (Fig.
7). The neutralizing activity of the anti-HPV16 VLP monoclonal antibody
H16.V5 (isotype IgG2b), was also determined. The data presented were
derived from three independent experiments involving an HPV16
pseudovirion input of 22, 44, and 54 focus-forming units. Best-fitting
sigmoidal curves were drawn (GraphPad, Prism [Fig. 7]), and the
VLP-specific antibody titers at which 50% HPV16 pseudovirion
neutralization occurs in vitro were calculated for each group, i.e., 64 for secretions containing both specific IgA and IgG, 98 for secretions
containing specific IgG alone, 125 for sera (specific IgG alone), and
214 for H16.V5. The values for 50% neutralization by serum and
secretions containing specific IgG alone were not statistically
different by Tukey's multiple-comparison test (P > 0.05). The neutralization efficiency of the VLP-specific IgG from serum
and secretions was very similar for individual animals that had been
immunized parenterally (data not shown). Examination of the titers of
IgA and IgG in the samples in Table 1 suggests that the VLP-specific
IgA elicited in secretions of immunized mice is neutralizing, since
many samples contain too low an IgG titer to account for the
neutralization observed, as judged from the IgG titer determination
curve for serum or secretions containing only IgG (Fig. 7).
Furthermore, the titer for 50% neutralization by secretions containing
both IgA and IgG, 64, was significantly (P < 0.05)
lower than for serum (titer of specific IgG, 125) and was lower than
for secretions containing specific IgG alone (titer, 98), although not
significantly so.
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TABLE 1.
Analysis of saliva and vaginal washes obtained from mice
immunized three times intranasally while anesthetized by using 5 µg
of HPV16 VLPs, either with or without 5 µg of CT
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FIG. 7.
Neutralization efficacy of anti-HPV16 IgA and/or IgG in
secretion and serum. The final titers of anti-HPV16 VLP IgA and/or IgG
in the samples that were used for neutralization experiments were
plotted against the neutralization efficacy elicited. Best-fitting
sigmoidal curves were drawn with the different groups (GraphPad,
Prism). In the secretions that contained both specific IgA and IgG
(Table 1), the titers of the two specific isotypes were added and the
summed value is indicated.
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DISCUSSION |
Infection by genital HPV is believed to occur when minor trauma
expose the basal cells of the genital squamous epithelium to virus.
Sterilizing immunity therefore would be mediated by neutralizing
antibodies located in the vaginocervical secretion or close to the
basal cells at the time of infection. Furthermore, neutralizing
antibody in mucosal secretions could limit transmission of breakthrough
infection. A prophylactic vaccine against genital HPVs should therefore
be capable of inducing HPV neutralizing antibodies in genital
secretions throughout the menstrual cycle and over long periods. It
might also be advantageous to produce neutralizing antibodies in other
mucosal sites of HPV infection, i.e., oral and anal sites, in which
HPV-associated cancers are known to occur. Systemic immunizations
induce only transudated antibodies, mainly IgG, from the serum in
genital secretions (8, 45), while mucosal immunizations can
induce both transdudated IgG and locally produced, mainly sIgA,
antibodies (9, 39). However, the induction of a strong
and/or protective immune response in the genital tract is highly
variable and depends on the nature of the immunogen, the addition of
mucosal adjuvants, the route of immunization, and the host species
(63).
In this study, we have measured the antibody response induced in serum
as well as in oral and genital secretions of mice by intranasal
immunization with purified HPV16 VLPs. Our data demonstrate that low
doses of the VLP antigen, probably due to its particulate and
repetitive, closely packed antigenic characteristics (2), are highly immunogenic when delivered by the intranasal route even in
the absence of the mucosal adjuvant CT. The same doses induced only
barely detectable specific antibodies via the oral route, probably due
to antigen degradation in the stomach and intestine. However, the doses
used were 50- to 1,000-fold lower than the amounts of soluble antigen
previously used successfully to induce antibody responses via the oral
route (26, 36). It is therefore possible that high doses of
VLPs will be capable of generating a substantial response after oral
delivery, as has been shown with rotavirus VLPs (46). The
nasal route also appeared to be the most efficient route of
immunization for other antigens (1, 14, 16, 17, 20, 25, 32, 37,
43, 46, 47, 52, 55, 61). Immunization by a triple 5-µg
intranasal dose of VLP was more efficient (Fig. 1) than systemic
immunization by a triple, spaced, 1-µg VLP dose (Fig. 5), since it
induced similar titers of specific IgG in secretions but also induced specific sIgA. However, our data suggest that the VLP antigen should
contact the lungs to induce an effective antibody response in mice. If
safe, immunization via the lungs in humans could be achieved with
aerosols.
The mechanisms by which intranasal immunization induces an immune
response are not fully understood. Nasal-associated lymphoid tissue
(NALT) appears to play a key role in rodents (65), but the
contributions of the bronchus-associated lymphoid tissue (BALT [54]) and/or intraepithelial dendritic cells are
unclear. In our experimental system, BALT appears to be crucial for
generating an effective antibody response and NALT is less effective.
Intranasal immunization of conscious mice, i.e., via NALT, does not
induce immune tolerance or suppression (60), since strong
specific antibody responses could be induced by a subsequent systemic
boost (Fig. 4).
Our data obtained with mice suggest that the nonresponse after
intranasal immunization in the absence of anesthesia may be overcome by
systemic priming followed by an intranasal boost. Systemic immunization
with microencapsulated antigens has previously been shown to prime for
the induction of disseminated mucosal IgA responses by subsequent
mucosal boosting by either the oral or intratracheal route in mice
(18) and in monkeys (37). It is, however, unclear
how the systemic and mucosal immune systems interact. Antigen delivery
to the MALT leads to a generalized secretory immune response. After
antigen processing and presentation by dendritic cells, committed B
lymphocytes proliferate in the MALT and then migrate via the lymph and
eventually reach all secretory tissues. This results in the ultimate
appearance of antigen-specific sIgA in all of the mucosal secretions
(6, 9), including the female genital secretions, as
predicted by the concept of a common mucosal system (39,
40). The homing of mucosally primed immunoblasts to mucosal
tissue is mediated by specific homing receptors (
4
7 [4,
23]) that recognize their counterparts, mucosal addressins
(MAdCAM-1 [4, 11]), in the high endothelial venules of
the gut. The mucosal addressins responsible for homing to nonintestinal
mucosal sites such as the salivary gland or the genital mucosa are not
known. In contrast, immunoblasts primed in peripheral lymph nodes (PLN)
bear L-selectin homing receptor and home to PLN (3).
Interestingly, intranasally primed human immunoblasts have been shown
to coexpress mucosal and systemic homing receptors (49),
suggesting that these lymphocytes might home to both mucosal sites and
PLN, where interactions with systemically primed lymphocytes could
occur.
sIgA antibodies are believed to be the primary effectors of mucosal
immunity, and their superiority over IgG in immune defense mechanisms
has often been suggested (5, 13). Resistance to mucosal
infection has been strongly correlated both with the presence of
specific IgA in mucosal secretions (41, 44) and with the number of IgA-secreting cells at the site of infection (66). Although systemic passive transfer of immune serum IgG protected animals from experimental papillomavirus infection, even at oral sites,
the ability of a systemic IgG response and the importance of sIgA in
protecting from natural genital transmission remain unknown. Induction
of both antibody classes may be important to achieve continuous
protection, since it has been shown that both IgA and IgG levels in
genital secretions vary greatly and inversely during the estrous cycle
(19, 31, 59, 62). Low levels of transudated IgG and partial
in vitro neutralization by anti-VLP IgG alone in genital secretions of
monkeys immunized parenterally with HPV11 VLPs have been demonstrated
(33).
Our data provide an opportunity to compare the efficacy of in vitro
neutralization of pseudotype HPV16 by antibodies in saliva and genital
secretions with those in serum. The neutralizing activities of the
polyclonal IgG antibody samples were not statistically different
(P > 0.05 by Tukey's multiple-comparison test),
regardless of whether the antibody was secreted or serum derived (50%
neutralization at titers of 98 and 125, respectively). Since the
secreted IgG of mice immunized parenterally is thought to be primarily
the result of transudation from the serum, its properties would be expected to match those of serum IgG.
Several reports indicate that the neutralizing efficacy of IgA
antibodies may be greater than that of IgG (5, 35, 42), and
50% neutralization was obtained by secretions containing both IgG and
IgA with a titer of 64, yet titers of 98 (not significantly different)
and 125 (P < 0.05 by Tukey's multiple-comparison
test) were required by secretions containing only IgG and serum,
respectively (Fig. 7). However, the ratios of IgA and IgG varied
between secretions (Table 1), and there is potential for subtly
different ELISA sensitivities for each antibody isotype. Study of HPV16
pseudovirion neutralization by purified antibodies of each isotype is
required to determine differential efficacy of neutralization by IgG
and IgA.
Determination of the titers of VLP-specific mouse antibodies
demonstrated similar activity to the anti-HPV11 virion sera derived from rabbits and African green monkeys tested (12, 33) by using the athymic mouse xenograft assay for HPV11 neutralization. This
suggests that the two in vitro neutralization assays have similar
sensitivities and that monkeys, rabbits, and mice produce VLP-specific
antibodies of quite similar neutralizing efficacy regardless of whether
the antigen was purified from warts, insect cells, or yeast. Further,
the VLP ELISA data correlated well with the in vitro neutralization
data in these studies, implying that the VLP ELISA is a good surrogate
assay for in vitro neutralization.
Although caution must be used in extrapolating our observations to
humans, due to substantial differences in the anatomy of the nasal
passages and the associated lymphoid tissues, our findings raise the
possibility that nasal immunization with purified HPV VLPs may be an
effective and well-tolerated method for inducing both HPV-neutralizing
IgA and IgG in the genital secretions of women.
| |
ACKNOWLEDGMENTS |
We thank Jean-Pierre Kraehenbuhl and Douglas Lowy for critical
reading of the manuscript.
This work was supported by Fonds de Service of the Dept. of Gynecology
and Swiss National Funds 31-52892.97 to D.N.-H. and by the intramural
program of the National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de Gynécologie, c/o Institut de Microbiologie,
Bugnon 44, 1011 Lausanne, Switzerland. Phone: 021/314 40 81. Fax:
021/314 40 95. E-mail: DNARDELL{at}ulrec1.unil.ch.
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Abstract
Introduction
