U.S. patent application number 11/684062 was filed with the patent office on 2007-07-05 for edible vaccine.
This patent application is currently assigned to ASAHI GLASS CO., LTD.. Invention is credited to Yuko Hama, Toshiyuki SASAGAWA, Hideki Tohda.
Application Number | 20070154491 11/684062 |
Document ID | / |
Family ID | 36036496 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154491 |
Kind Code |
A1 |
SASAGAWA; Toshiyuki ; et
al. |
July 5, 2007 |
EDIBLE VACCINE
Abstract
The object of the present invention is to provide an edible
vaccine which is effective for human papilloma virus (HPV) type 16
and available in large amounts inexpensively. An edible human
papilloma virus vaccine obtained by culturing a transformant of an
avirulent fission yeast host, wherein the transformant carries a
gene encoding an antigenic protein of human papilloma virus
introduced therein and accumulates the expressed antigenic protein
in it.
Inventors: |
SASAGAWA; Toshiyuki;
(Kanazawa-shi, JP) ; Tohda; Hideki; (Yokohama-shi,
JP) ; Hama; Yuko; (Yokohama-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
ASAHI GLASS CO., LTD.
Tokyo
JP
NATIONAL UNIVERSITY CORP. KANAZAWA UNIVERSITY
Kanazawa-shi
JP
|
Family ID: |
36036496 |
Appl. No.: |
11/684062 |
Filed: |
March 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP05/16638 |
Jun 6, 2005 |
|
|
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11684062 |
Mar 9, 2007 |
|
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Current U.S.
Class: |
424/204.1 ;
424/195.16; 977/802 |
Current CPC
Class: |
A61K 2039/542 20130101;
A61K 2039/543 20130101; A61P 35/00 20180101; A61K 39/12 20130101;
A61K 2039/523 20130101; A61P 31/12 20180101; A61K 36/06 20130101;
A61K 2039/55544 20130101; A61K 2039/5258 20130101; A61K 9/19
20130101; A61P 31/00 20180101; C12N 2710/20034 20130101 |
Class at
Publication: |
424/204.1 ;
424/195.16; 977/802 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 36/06 20060101 A61K036/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2004 |
JP |
2004-263580 |
Claims
1. An edible human papilloma virus vaccine obtained by culturing a
transformant of an avirulent fission yeast host, wherein the
transformant carries a gene encoding an antigenic protein of human
papilloma virus introduced therein and accumulates the expressed
antigenic protein in it.
2. The vaccine according to claim 1, wherein the avirulent fission
yeast host is Schizosaccharomyces pombe.
3. The vaccine according to claim 1, wherein the human papilloma
virus is human papilloma virus type 16 (HPV16).
4. The vaccine according to claim 1, wherein the gene encoding an
antigenic protein is a gene encoding a capsid protein of the human
papilloma virus.
5. The vaccine according to claim 4, wherein the capsid protein of
the human papilloma virus is the L1 protein.
6. The vaccine according to claim 4, wherein the capsid protein of
the human papilloma virus is the L1 protein of human papilloma
protein type 16 (HPV16-L1).
7. The vaccine according to claim 4, wherein the protein
accumulated in the transformant is assembled into virus-liked
particles.
8. The vaccine according to claim 1, wherein the vaccine is a
freeze-dried preparation of the transformant.
Description
TECHNICAL FIELD
[0001] The present invention is an edible vaccine effective against
human papilloma virus (sometimes hereinafter referred to simply as
"HPV").
BACKGROUND ART
[0002] HPV is a small del DNA virus having an icosahedral structure
and no envelope. The genome of the virus contains open reading
frames (ORFs) called E1-E7 and L1 and L2: "E" means early, and "L"
means late. L1 and L2 encode capsid proteins of the virus. The
early (E) genes are associated with functions such as virus
replication and cell transformation. The L1 protein is the major
capsid protein having a molecular weight of from 55 to 60 kDa when
measured by polyacrylamide gel electrophoresis. The L2 protein is
the minor capsid protein which also has an estimated molecular
weight of from 55 to 60 kDa and an apparent molecular weight of
from 75 to 100 kDa.
[0003] Although the mortality from cervical cancer has recently
decreased in developed countries, it is the fifth leading cause of
malignancy deaths and the second most common malignancy in women
worldwide. Certain sexually transmitted types of HPV are the most
important risk factor for cervical cancer. Recent reports show that
from 30 to 50% of young women who recently had their first sexual
intercourse have HPV infection in the cervix. Surprisingly, most
cervical HPV infections are caused by high-risk types of HPV which
can induce cancer. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 67 and 68, possibly some other types as well, are
considered to be high-risk types.
[0004] This alarmingly high prevalence of HPV infection among young
women suggests that educational and social health programs aimed at
preventing of HPV infection may not be sufficiently effective in
combating cervical cancer. Especially, prevention of infection with
high-risk types of HPV is a priority for women in developing
countries and young women who have not had uterine cervical cancer
screening. The current cytologic screening (uterine cervical cancer
screening) and post-onset cancer treatment are not cost-effective
choices. Nationwide use of prophylactic vaccines against high-risk
types of HPV can decrease the incidence of cervical cancer. It is
estimated that even single use of a HPV16 vaccine decreases
cervical cancer by half.
[0005] In pursuit of development of HPV vaccines, it was reported
that high-level production of the HPB11 L1 protein led to the
assembly of virus-like particles (hereinafter sometimes referred to
simply as "VLPs") in an insect cell system (non-patent document 1).
Successful synthesis of HPV16 VLPs in this insect cell system was
also reported (non-patent document 2).
[0006] Later, we succeeded in production of HPV6- and HPV16-derived
VLPs in the fission yeast Schizosaccharomyces pombe (hereinafter
sometimes referred to simply as "S. pombe") (non-patent document
3). Although the yield of VLPs from the fission yeast is less than
that from the insect cell system, the expression system using the
fission yeast confers advantages in large-scale production of
virus-like particles (hereinafter sometimes referred to simply as
"VLP") and safety of use in humans.
[0007] Koustsky et al. were the first to report that parenteral
vaccination (by injection) with HPV16 VLPs conferred 100%
protection against HPV16 infection in women (non-patent document
4).
[0008] Unfortunately, the injectable HPV16-VLP vaccine is expensive
because it requires advanced techniques and special facilities for
its production and storage. In addition, repetitive injection
vaccinations required for efficacy has limited feasibility and are
impractical in developing countries with limited numbers of trained
members in their clinical staffs. Furthermore, it has been reported
that injection vaccination with VLPs is a poor inducer of secretory
IgA, which plays a major role in mucosal immunity (non-patent
document 5). Immunization of the mucosa-associated lymphoid tissue
(MALT), which is an immune tissue located in the respiratory and
the digestive tract, can protect against viruses such as HPV that
cause infections in the uterine and the vaginal mucosal epithelia.
Balmelli et al. succeeded in inducing mucosal antibodies that
neutralize HPV16 in the vagina by intranasal administration of
HPV16-VLPs (non-patent document 6).
[0009] However, intranasal vaccination is also problematic like
injection vaccination, because it requires preparation of
relatively large amounts of purified HPV-VLPs. Stimulation of the
gut-associated lymphoid tissue (GALT) with edible human papilloma
virus vaccines (hereinafter sometimes referred to simply as "edible
HPV vaccines") was attempted to induce strong mucosal immunity in
the vagina. Two groups have produced edible HPV vaccines from
tobacco and potato plants that express the HPV11 (non-patent
document 7) and HPV16 (non-patent document 8) L1 genes.
[0010] Purification of HPV virus-like particles (HPV-VLPs) was
disclosed in patent document 1. Patent documents 2-4 and patent
document 5 disclose HPV vaccine preparations from expression
systems in baculovirus and in insect cells, respectively. A nucleic
acid vaccine for immunotherapy of HPV was also disclosed (patent
document 6).
[0011] A therapeutic microorganism delivery system using
non-vaccine active ingredient was also reported (patent document
7). [0012] Patent Document 1: JP-A-2003-520188 [0013] Patent
Document 2: JP-A-2001-519161 [0014] Patent Document 3:
JP-A-2002-516291 [0015] Patent Document 4: JP-A-2002-510976 [0016]
Patent Document 5: JP-A-2004-269 [0017] Patent Document 6:
JP-A-2004-121263 [0018] Patent Document 7: JP-A-10-506791 [0019]
Non-patent Document 1: Rose R C, et al. J Virol. 1993; 67: 1936-44.
[0020] Non-patent Document 2: Kirnbauer R, et al. J Virol. 1993;
67: 6929-36.9 [0021] Non-patent Document 3: Sasagawa T, et al.
Virology. 1995; 206:126-35. [0022] Non-patent Document 4: Koutsky L
A, et al. N Engl J Med. 2002; 347: 1645-51. [0023] Non-patent
Document 5: Hagensee M E, et al. Virology 1995; 206: 174-82. [0024]
Non-patent Document 6: Balmelli C, et al. J Virol 1998; 72: 8220-9.
[0025] Non-patent Document 7: Warzecha H, et al. J Virol. 2003; 77:
8702-11. [0026] Non-patent Document 8: Biemelt S, et al. J Virol.
2003; 77: 9211-20.
DISCLOSURE OF THE INVENTION
PROBLEMS THAT THE INVENTION IS TO SOLVE
[0027] The object of the present invention is to provide an edible
HPV vaccine which is available in large amounts inexpensively.
MEANS FOR SOLVING THE PROBLEMS
[0028] The present inventors pursued their research for solutions
to the above-mentioned problems by examining usefulness of a
recombinant fission yeast expressing HPV proteins as a vaccine and
have accomplished the present invention.
[0029] The present invention provides the following:
[0030] 1. An edible human papilloma virus vaccine obtained by
culturing a transformant of an avirulent fission yeast host,
wherein the transformant carries a gene encoding an antigenic
protein of human papilloma virus introduced therein and accumulates
the expressed antigenic protein in it.
2. The vaccine according to 1, wherein the avirulent fission yeast
host is Schizosaccharomyces pombe.
3. The vaccine according to claim 1 or 2, wherein the human
papilloma virus is human papilloma virus type 16 (HPV16).
4. The vaccine according to 1, 2 or 3, wherein the gene encoding an
antigenic protein is a gene encoding a capsid protein of the human
papilloma virus.
5. The vaccine according to 4, wherein the capsid protein of the
human papilloma virus is the L1 protein.
6. The vaccine according to 4 or 5, wherein the capsid protein of
the human papilloma virus is the L1 protein of human papilloma
protein type 16 (HPV16-L1).
7. The vaccine according to 4, 5 or 6, wherein the protein
accumulated in the transformant is assembled into virus-liked
particles.
8. The vaccine according to any one of 1 to 7, wherein the vaccine
is a freeze-dried preparation of the transformant.
EFFECTS OF THE INVENTION
[0031] Immunization with the edible HPV vaccine, especially the
edible HPV16 vaccine, of the present invention and intranasal
immunization with HPV-VLPs were observed to induce IgG in serum and
the vagina and IgA in the vagina. All the induced antibodies
strongly reacted with the HPV-VLP antigen. This indicates that the
edible HVP16 vaccine is effective as a prophylactic vaccine against
HPV16.
BRIEF EXPLANATION OF THE DRAWINGS
[0032] FIG. 1A Digestion of a freeze-dried yeast in the stomach
(Test Example 1)
[0033] FIG. 1B Digestion of a freeze-dried yeast in the abdominal
cavity (Test Example 1)
[0034] FIG. 1C Digestion of a freeze-dried yeast in the intestine
(Test Example 1)
[0035] FIG. 2 Induction of antibodies by immunization with HPV16
vaccines (Test Example 2)
[0036] FIG. 3 Induction of antibodies by immunization with HPV16
vaccines followed by intranasal boosting with HPV16-VLPs Test
Example 3)
[0037] FIG. 4 Change in antibody responses to immunization HPV16
vaccines followed by intranasal boosting with HPV16-VLPs (Test
Example 5)
[0038] FIG. 5 Changes in antibody responses to immunization with
HPV16 vaccines followed by intranasal boosting with HPV16-VLPs
(Test Example 5)
EXPLANATION OF REFERENCE SIGNS
[0039] .quadrature. Reaction with denatured HPV16-L1 antigen (FIGS.
4 and 5)
[0040] .box-solid. Reaction with HPV16-VLP antigen (FIGS. 4 and
5)
BEST MODE FOR CARRYING OUT THE INVENTION
[0041] The present invention provides a cultured transformant which
carries a gene encoding an antigenic protein of HPV and expresses
and accumulates the protein, as an edible HPV vaccine.
[0042] In the present invention, the host into which a gene
encoding an antigenic protein of HPV is introduced to obtain the
transformant is an avirulent fission yeast, specifically the
fission yeast S. pombe.
[0043] In the present invention, "the antigenic protein of HPV"
specifically means a capsid protein of HPV, preferably L1. In the
present invention, the HPV is preferably a high-risk type of HPV
with the danger of cervical cancer, specifically HPV16.
[0044] In the present invention, any inducible expression vector
for S. pombe into which a foreign gene is inserted, such as
multicloning vectors disclosed in JP-A-7-163373 and JP-A-11-192094,
may be used to introduce a gene encoding an antigenic protein of
HPV into the host S. pombe without any restrictions. For example, a
vector having a HPV16-L1 gene insert under the control of a
thiamine-repressible promoter may be used to synthesize virus-like
fragments recombinantly (non-patent document 3). The presence of
the 55-kDa L1 protein in the virus-like fragments can be confirmed
by Western blotting or the like.
[0045] In the present invention, "the transformant which
accumulates an antigenic protein of HPV in it" is a transformant of
an avirulent fission yeast host which accumulates the antigenic
protein of HPV, and there are any particular restrictions.
Specifically speaking, the transformant (hereinafter referred to
"recombinant S. pombe") is cultured at 23 to 37.degree. C. for 6 to
192 hours in a known culture medium, preferably YPD medium under
appropriate conditions to accumulate the expressed antigenic
protein of HPV in it. After culturing, the culture medium
containing the recombinant S. pombe is centrifuged under
appropriate conditions, for example, at 0 to 50.degree. C. for 1 to
60 minutes at an acceleration of 500- 3000 g, to collect the
precipitate before oral administration.
[0046] The centrifugally collected recombinant S. pombe is called a
yeast pellet and may be used as the "transformant which accumulates
an antigenic protein of HPV in it" of the present invention. The
yeast pellet may be processed appropriately, if necessary, for
example, by freeze-drying, and the "transformant which accumulates
an antigenic protein of HPV in it" of the present invention covers
such a processed pellet. Freeze-drying may be carried out under
ordinary conditions, for example, at a maximum shelf temperature of
-20.degree. C. overnight, though there are no particular
restrictions as long as S. pombe is freeze-dried. The edible HPV
vaccine of the present invention covers a transformant having
accumulated an antigenic protein of HPV thus obtained.
[0047] Oral immunization has some advantages over prophylactic
immunization through other routes. For example, edible vaccines are
readily administered and acceptable to vaccine recipients. In
addition, edible vaccines may contain active ingredients at lower
concentrations than injection vaccines and therefore, can be
produced at low production costs.
[0048] The edible HPV vaccine of the present invention may be
formulated into preparations with at least one medicinally
acceptable additional ingredient such as a carrier, a diluent, an
adjuvant and/or a buffer. Further, the edible HPV vaccine of the
present invention may be used as a mixture with food or the like.
Known adjuvants such as an E. coli-derived mucosal adjuvant LT
(R192) may be used.
[0049] The edible HPV vaccine of the present invention is
administered at a dose of yeast (on a wet basis) selected within
the range of from 10 to 500 mg/kg, preferably from 20 to 200 mg/kg,
or at a dose of the HPV16-L1 protein selected within the range of
from 0.05 to 5 mg/kg, preferably from 0.1 to 2 mg/kg. The edible
HPV vaccine may be administered only once or repeatedly.
[0050] It is suggested that the edible HPV vaccine of the present
invention is useful as a prophylactic vaccine against HPV16. The
edible HPV vaccine of the present invention may be co-administered
with conventional injection HPV vaccines and/or intranasal HPV
vaccines in ordinary vaccination regimens for them, for example, as
"a concomitant vaccine" to be used in combination with known
vaccines. It may also be used as a "booster vaccine" to keep
induced antibody titers.
EXAMPLES
[0051] Now, the present invention will be described by referring to
Examples and Comparative Examples. However, the Examples are mere
embodiments of the present invention which help reproduction of the
present invention and by no means limits or restricts the present
invention.
Example 1
Construction of Recombinant S. pombe Strain Expressing the HPV16-L1
Protein
[0052] A recombinant S. pombe strain was obtained in accordance
with non-patent document 3. In order to increase L1 gene
expression, the HPV16-L1 gene (B27;
[0053] wild-type HPV16) was inserted into a new vector, pTL2M
(JP-A-7-163373). The recombinant S. pombe strain was cultured in 2
L of YPD medium to allow the expression of the HPV16-L1 protein. It
was confirmed by Western blotting that the recombinant S. pombe
(pTL2-HPV16-L1) expressed high levels of the 55-kDa L1 protein. It
was confirmed by electron microscopy that the expressed protein
assembled into virus-like particles.
[0054] The total amount of the proteins expression by the yeast was
about 10% of the wet weight of the yeast and about 50% of the
freeze-dried yeast, and the L1 protein accounted for from 5 to 10%
of the expressed proteins.
Example 2
Purification of Inactivated Freeze-Dried Yeast for Vaccine
Preparation
[0055] A recombinant S. pombe strain was cultured in 2 L of YPD
medium and collected by centrifugation at 4.degree. C. for 10
minutes at 2000 g. The yeast pellet was washed with phosphate
buffered saline (PBS) and resuspended in PBS at 150 mg/mL on a wet
basis. The S. pombe suspension was freeze-dried overnight at a
maximum shelf temperature of -20.degree. C. The freeze-dried
recombinant S. pombe was sealed in air-tight plastic tubes and
stored at 4.degree. C. until use. The resulting edible vaccine is
referred to as "HPV16-L1 yeast" for the sake of simplicity.
[0056] The freeze-dried HPV16-L1 yeast was resuspended in more than
ten volumes of 70% ethanol in water and incubated at 4.degree. C.
for 30 minutes, in order to inactivate the yeast, then separated by
filtration and dried, before use as an edible vaccine. Separate
aliquots of the freeze-dried S. pombe strain was treated with
ethanol similarly before oral administration. Further, "fresh-live
yeast cells" were treated with ethanol similarly before oral
administration.
Example 3
Vaccines for Study on Yeast Digestion in the Mouse Digestive
Tract
[0057] A recombinant S. pombe strain expressing a red fluorescent
protein (RFP) was obtained in the same manner as in Example 1. The
recombinant S. pombe strain was cultured as described above to
allow it to express the two proteins. After culturing, the yeast
strain was centrifuged at 40.degree. C. for 10 minutes at 2000 g,
and the yeast pellet was collected.
Example 4
Adjuvant
[0058] The Escherichia coli heat-labile toxin LT (R192G), provided
in accordance with Curr Top Microbiol Immunol 1999; 236: 216-36,
was used as a mucosal adjuvant. The powdery adjuvant was suspended
in PBS and stored at -30.degree. C. until use.
Comparative Example 1
Purification of HPV16-VLPs for Intranasal Immunization
[0059] The HPV16-VLP protein was purified from the recombinant S.
pombe , pTL2-HPV16-L1, obtained in Example 1 was purified by cesium
chloride gradient ultracentrifugation (Giga-Hama Y, et.
Biotechnology (N Y), 1994; 12: 400-4).
[0060] The cells were centrifuged at 4.degree. C. at 2000 g for 5
minutes, resuspended in 50 mM potassium phosphate buffer [50 mM
KH.sub.2PO.sub.4 (pH 6.5) containing 20 mM
ethylenediaminetetraacetic acid (EDTA) and cooled on ice. The
suspension was centrifuged again, and the yeast pellet was
resuspended in 10 ml KKC buffer [20 mM KPO.sub.4 (pH 6.5), 800 mM
KCl, 0.1 mM CaCl.sub.2, 1.5 mM MgCl.sub.2 containing 5 mg/ml
Novozyme] and incubated at 32.degree. C. for 30 minutes to digest
the yeast cell walls, and sonicated at for 1 minute at 60 W. The
cell extract was recovered by centrifugation at 4.degree. C. for 10
minutes at 7000 g. The pellet was resuspended in 10 mL VLP buffer
(10 mM HEPES, 10 mM KCl (PH 7.0)) containing 0.5 % surfactant
(NP-40) and sonicated gently as previously mentioned.
[0061] The supernatant was collected and layered on 40% sucrose VLP
buffer and centrifuged at 4.degree. C. for 2 hours at 27000 g in a
Beckman SW28 rotor (Beckman Coulter). The pellet was resuspended in
VLP buffer and centrifuged at 4 .degree. C. for 20 hours at 27000 g
in CsCl-equilibrated VLP buffer in the SW rotor. The appropriate
fractions were diluted to a density of 1.29 g/ml and centrifuged at
4.degree. C. for 2.5 hours at 27000 g.
[0062] The pellet was resuspended in VLP buffer and stored at
-30.degree. C.
[0063] The assembly of the HPV16-VLP protein was confirmed by
enzyme-linked immunosorbent assay (ELISA) using two anti-HPV16
monoclonal antibodies, Camvir-5 and Camvir-6 (provided from
Margaret Stanley, Cambridge University), which recognize
conformation-dependent epitopes.
[0064] The HPV16-VLP protein was used for intranasal immunization.
The intranasal vaccine is called "HPV16-VLP" for the sake of
simplicity.
Test Example 1
Yeast Digestion in Mouse Digestive Tract
[0065] Four 9-week-old BALB/c mice were fed 20 mg (on a wet basis)
of the fresh-live or freeze-dried yeast expressing a fluorescent
protein (RFP) obtained in Example 3, 6 times at hourly intervals
after 12 hours of starvation. After the final feed, the mice were
dissected and examined for yeast digestion. The digestive tracts of
the mice were cut, and touch smears were obtained on glass slides.
Fluorescent yeast cells were observed on each slide under Axiovert
S-100 Microscope (Carl Zeiss, Germany) and photographed with Fuji
3CCD camera (Fuji Film).
[0066] The fresh-live yeast cells were not digested because they
were observed at most parts of the digestive tract and excreted in
stools.
[0067] In contrast, the freeze-dried yeast cells were not disrupted
in the stomach (FIG. 1A) or the jejunum (FIG. 1B), but cells
decreased from the ileum to the large 5 intestine and were very few
in the rectum (FIG. 1C). This suggests that freeze-dried yeast
cells are digested in the intestine where the gut-associated
lymphoid tissue is located.
Test Example 2
Antibody Production induced by Edible Vaccine "HPV16-L1 Yeast"
[0068] Female 9-week-old BALB/c mice were fed the freeze-dried
yeast suspended in PBS after 12 hours of starvation. The mice were
divided into 6 groups as shown in Table 1 and vaccinated at 4-week
intervals and examined for antibody production. Group 1 is a
negative control, and Group 2 is a positive control. The doses are
expressed on a wet basis. TABLE-US-00001 TABLE 1 Ad- Vaccination
Mouse ID Vaccine Dose juvant route Group 1 Nos. 1-3 Wild-type 50 mg
-- Oral yeast Group 2 Nos. 4-6 Purified 5 .mu.g 10 .mu.g Intranasal
HPV16-L1 Group 3 Nos. 7-9 HPV16-L1 50 mg -- Oral yeast Group 4 Nos.
10-12 HPV16-L1 50 mg 10 .mu.g Oral yeast Group 5 Nos. 13-18
HPV16-L1 150 mg -- Oral yeast Group 6 Nos. 19-24 HPV16-L1 150 mg 10
.mu.g Oral yeast
[0069] After the vaccinations, serum samples and vaginal samples
were collected from the mice. The serum samples were obtained from
blood collected from the mouse tail, and the vaginal samples were
obtained by washing the vaginae with 100 .mu.l of PBS using a
micropipette. To avoid the influence of the mouse estrous cycle on
antibody production, the vaginal samples were collected twice with
a 5-day interval, and the two samples were mixed and used for
analysis. The samples were collected a few days before the first
oral immunization and four weeks after each immunization. All the
samples were divided into aliquots and stored at -30.degree. C.
until use to avoid repeated thawing.
[0070] After three immunizations, the levels of HPV16-specific
antibodies (IgG and IgA) were evaluated by ELISA (FIG. 2).
1) ELISA
[0071] HPV16-VLP protein purified from insect cells in accordance
with Rose (J Virol. 1933; 67: 1936-44., J Gen Virol 1994; 75:
2445-9.) was used as the HPV16-VLP antigen for ELISA.
[0072] For antigen coating, 100 ng and 300 ng of HPV16-VLP antigen
was incubated in PBS on ELISA plates (NUNC Immunoplate Maxisorp;
Nalgene Nunc International) at 4.degree. C. overnight.
[0073] The coated plates were washed with PBST (PBS, 0.1% Tween-20)
once and incubated with blocking buffer (3% albumin, 0.5% FCS in
PEST) at room temperature (RT; 20-24.degree. C.) for 1 hour. All
the subsequent washes were carried out with PEST.
[0074] In the antibody reaction, 1 .mu.l of serum samples or 20
.mu.l of vaginal samples were mixed with reaction buffer (1.5%
bovine albumin, 0.25% FCS in PEST), added to the ELISA plates and
incubated at room temperature for 3 hours.
[0075] After three washes, a biotinylated anti-mouse IgA or IgG
antibody (diluted with the reaction buffer at a ratio of 1:1500 for
IgA and 1:1000 for IgG) was added to the plates and incubated at
room temperature for 1 hour.
[0076] After three washes, 100 .mu.l of strepavidin-horseradish
conjugate (DAKO, Germany) diluted with PEST at a ratio of 1:5000
was added to the plates and incubated for 30 minutes.
[0077] After three washes, 100 .mu.l of 50 mM citrate buffer (pH
5.0; 0.0075% hydrogen peroxide) containing one tablet of AET
[2,2'-azino-bis(3-ethylbenzeothiazoline-6-sulfonic acid) (Sigma)
was added to the plates. After 1 hour of color development at room
temperature, the optical density (OD) values were measured at dual
wavelengths (405/540 nm) with an automated plate reader (Iems
Reader MS; Labsystems). The final OD values were calculated by
subtraction of the OD values at 540 nm from those at 405 nm.
[0078] 2) Evaluation The cut-off points for IgA and IgG titers in
serum were set at the average OD values plus 2.5 times the standard
deviations of 10 serum and vaginal samples from non-immunized mice.
ELISA was carried out in duplicate or more for each sample, and the
mean OD values were used for evaluation. Two sets of positive and
negative control samples were included in each experiment to
compensate for inter-assay variation.
[0079] All the positive control mice showed high OD values for
serum IgG, vaginal IgG and vaginal IgA, although no serum IgA
responses were detected.
[0080] Finally, two of the 18 mice (11%) (No. 7 in Group 3 and No.
14 in Group 5) showed transient weak serum IgG responses after the
second immunization (FIG. 2A). In contrast, no vaginal IgG or IgA
responses were observed in any orally vaccinated mice (FIGS. 2B and
21C).
Test Example 3
Boosting of Immune Responses by Intranasal Administration of
Purified HPV16-VLPs
[0081] A suboptimal dose (1 .mu.g) of the HPV16-VLP was
intranasally administered as a booster to all the mice, including
the positive controls, negative controls and those orally immunized
with the HPV16-L1 yeast, 12 weeks after the final immunization.
[0082] The results are shown in Table 2 and FIG. 3. TABLE-US-00002
TABLE 2 Titers of serum IgG and vaginal IgA induced in mice
Antibody titer Mouse Vaccination Antigen and dose (dilution) ID
route HPV16 Serum Vaginal No. 1st-3rd/4th Yeast VLP Adjuvant IgG
IgG No. 1 Oral/Intranasal Wt.sup.a 50 mg 1 .mu.g 0 0 No. 2
Oral/Intranasal Wt 50 mg 1 .mu.g 0 0 No. 4 Intranasal/ 0 5 .mu.g +
1 .mu.g 10 .mu.g 6400 1600 Intranasal No. 5 Intranasal/ 0 5 .mu.g +
1 .mu.g 10 .mu.g 25600 3200 Intranasal No. 6 Intranasal/ 0 5 .mu.g
+ 1 .mu.g 10 .mu.g NA NA Intranasal No. 7 Oral/Intranasal
HPV16.sup.b 50 mg 1 .mu.g -- NA.sup.c 0 No. 8 Oral/Intranasal HPV16
50 mg 1 .mu.g -- 3200 NA No. 9 Oral/Intranasal HPV16 50 mg 1 .mu.g
-- 1600 NA No. 10 Oral/Intranasal HPV16 50 mg 1 .mu.g 10 .mu.g NA 0
No. 11 Oral/Intranasal HPV16 50 mg 1 .mu.g 10 .mu.g 1600 400 No. 12
Oral/Intranasal HPV16 50 mg 1 .mu.g 10 .mu.g 1600 800 No. 13
Oral/Intranasal HPV16 150 mg 1 .mu.g -- 1600 NA No. 15
Oral/Intranasal HPV16 150 mg 1 .mu.g -- 1600 800 No. 17
Oral/Intranasal HPV16 150 mg 1 .mu.g -- 3200 NA No. 19
Oral/Intranasal HPV16 150 mg 1 .mu.g 10 .mu.g 1600 100 No. 21
Oral/Intranasal HPV16 150 mg 1 .mu.g 10 .mu.g NA 100 No. 22
Oral/Intranasal HPV16 150 mg 1 .mu.g 10 .mu.g 3200 800
.sup.awild-type yeast, .sup.bHPV16-L1 yeast, .sup.cnot detected
(Not applicable)
[0083] All the three positive controls (100%) and 9 orally
vaccinated mice showed positive reactions for serum IgG four weeks
later (FIG. 3A). In terms of vaginal IgG, the three positive
controls (100%) and 6 orally vaccinated mice (33%) were positive,
while none of the negative controls were positive (FIG. 3B). With
respect to vaginal IgA, the three positive controls (100%) and 7
orally vaccinated mice (39%) were positive, and none of the
negative controls were positive (FIG. 3C).
[0084] No antibodies were induced in the negative controls even by
intranasal boosting with a suboptimal amount of HPV16-VLPs after
oral yeast vaccination, whereas mice orally vaccinated with the
HPV16-L1 yeast showed positive immune responses, which suggests
that HPV16-specific immune responses can be primed by the HPV16-L1
yeast.
Test Example 4
Effects of Oral Doses of HPV16-L1 Yeast and Adjuvant on Immune
Responses
[0085] The antibody-positive rates in the HPV16-L1 yeast-vaccinated
mice did not differ at a low HPV16-L1 yeast dose (50 mg) and a high
HPV16-L1 yeast dose (100 mg).
[0086] The mucosal adjuvant LT (R192G) was administered with the
HPV16-L1 yeast to some mice to enhance the antibody response. The
adjuvant did not cause serious side effects in mice. The adjuvant
did not any difference in serum IgG responses among groups orally
vaccinated with the HPV16-L1 yeast, while the positive rates were
two-fold higher for vaginal IgG and 2.5-fold higher for vaginal IgA
in the adjuvant groups than in the non-adjuvant groups, though
there were no statistically significant differences. The OD values
for vaginal IgA were slightly higher in the adjuvant group of
HPV16-L1 yeast-vaccinated mice than in the non-adjuvant group of
HPV16-L1 yeast-vaccinated mice (P=0.085; Mann-Whitney test), which
suggest a slight effect of the adjuvant.
Example 5
Characterization of the Antibodies Elicited by Oral and Intranasal
Vaccination
[0087] The reactivity of antibodies was assayed by ELISA using
HPV16-VLP and denatured HPV16-L1 protein coatings.
[0088] HPV16-VLPs were boiled in bicarbonate buffer (100 g for IgG
and 300 ng for IgA) for 10 minutes and used as the denatured
HPV16-L1 protein antigen in the ELISA. The reactivities to the VLP
antigen or the denatured HPV16-L1 antigen expressed in OD were
compared. The sera and the vaginal washings were serially diluted,
and the antibody titers were measured.
[0089] Previous studies demonstrated that antibodies that recognize
conformation-dependent epitopes on HPV-VLPs neutralize the virus.
Here, it was investigated whether the induced antibodies react more
strongly to the HPV16-VLP antigen than to the denatured HPV16-L1
antigen.
[0090] The antibodies induced in two intranasally
HPV16-VLP-vaccinated mice (No. 4 and No. 6) by the first intranasal
immunization reacted more strongly with the HPV16-VLP antigen.
However, the serum IgG antibodies induced at the second
immunization or later reacted more strongly with the HPV16-VLP
antigen (FIG. 4A). This indicates seroconversion from a
non-specific type of reaction to a HPV16-specific type of reaction
after the second immunization (FIG. 4A).
[0091] The antibodies induced in orally HPV16-L1 yeast-vaccinated
mice after intranasal boosting (No. 12 and No. 12 and No. 22)
reacted more strongly with the HPV16-VLP antigen (FIG. 4B).
[0092] In contrast, two mice orally vaccinated with the HPV16-L1
yeast were transiently positive but eventually negative after
boosting. The antibodies in one (No. 7) of the two reacted more
strongly with the denatured HPV16-L1 antigen, while the antibodies
in the other (No.14) reacted equally with both types of HPV16-VLP
antigen throughout the time course of the experiment (FIG. 4B).
[0093] The results of the assays of vaginal samples from mice
orally vaccinated with the HPV16-L1 yeast showed that IgG
antibodies were elicited in 6 mice (FIG. 5A). The antibodies
elicited in all these mice reacted more strongly with the HPV16-VLP
antigen.
[0094] The edible HPV16-L1 yeast vaccine elicits HPV16-specific
vaginal IgA and IgG antibodies, as well as serum IgG. Although oral
administration of the freeze-dried HPV16-L1 yeast alone did not
induce anti-HPV16 antibodies, but when it was followed by
intranasal boosting with a suboptimal amount of HPV16-VLPs, serum
IgG, vaginal IgG and vaginal IgA were elicited in 50%, 33% and 39%
of the mice, respectively. In the negative control mice, no
anti-HPV16 antibodies were elicited even after the same intranasal
boosting. Intranasally administered HPV16-VLPs increased antibody
production probably by enhancing the activity of primed memory B
cells that recognize HPV16. All of the induced antibodies reacted
more strongly with HPV16-VLP antigen than with the denatured
HPV16-L1 antigen, which suggests that these antibodies recognize
conformation-dependent HPV16-L1 epitopes and are neutralizing.
Industrial Applicability
[0095] As described above, the edible HPV16-L1 yeast vaccine of the
present invention induces neutralizing antibodies and is functional
as a vaccine. Therefore, a vaccine is provided which is less
painful for patients than intranasal vaccines and injectable
vaccines to be used singly. The edible vaccine needs no
purification and therefore, is available in large amounts
inexpensively. The vaccine may be used in various ways, primarily
to induce anti-HPV antibodies in unimmunized people, and also as a
booster to keep induced antibody titers.
[0096] The entire disclosure of Japanese Patent Application No.
2004-263580 filed on Sep. 10, 2004 including specification, claims,
drawings and summary is incorporated herein by reference in its
entirety.
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