U.S. patent application number 09/770405 was filed with the patent office on 2001-10-11 for attenuated microorganism strains and their uses.
This patent application is currently assigned to BTG International Limited. Invention is credited to Haefliger, Denise N., Kraehenbuhl, Jean-Pierre.
Application Number | 20010029043 09/770405 |
Document ID | / |
Family ID | 10801186 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010029043 |
Kind Code |
A1 |
Haefliger, Denise N. ; et
al. |
October 11, 2001 |
Attenuated microorganism strains and their uses
Abstract
This application relates to the use of attenuated prokaryotic
miccrooganism strains (such as Salmonella) expressing nucleic acid
encoding HPV proteins as vaccines against HPV infection and the
associated increased risk of cancer. In particular, the work shows
that it is possible to assemble VLPs in a prokaryotic organism and
that nasal immunization of mice with the strains HPV-specific
conformationally dependent and neutralizing antibodies in serum and
genital secretions. The experiments described herein show that it
is also possible to assemble chimeric VLPs of a HPV including a
fusion partner and that tumour protection can be induced.
Inventors: |
Haefliger, Denise N.;
(Lausanne, CH) ; Kraehenbuhl, Jean-Pierre; (Rivaz,
CH) |
Correspondence
Address: |
Nixon & Vanderhye P.C.
8th Floor
1100 N. Glebe Rd.
Arlington
VA
22201-4714
US
|
Assignee: |
BTG International Limited
|
Family ID: |
10801186 |
Appl. No.: |
09/770405 |
Filed: |
January 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09770405 |
Jan 29, 2001 |
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09413807 |
Oct 7, 1999 |
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6251406 |
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09413807 |
Oct 7, 1999 |
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09288861 |
Apr 9, 1999 |
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Current U.S.
Class: |
435/252.3 ;
424/258.1; 435/5 |
Current CPC
Class: |
C12N 2710/20022
20130101; A61P 31/20 20180101; C12N 15/74 20130101; A61K 2039/51
20130101; A61K 39/00 20130101; Y02A 50/30 20180101; A61P 31/12
20180101; C07K 14/005 20130101; Y10S 977/804 20130101; C12N
2710/20023 20130101; Y10S 977/904 20130101; Y10S 977/917 20130101;
C07K 2319/00 20130101; A61P 35/00 20180101; Y02A 50/476 20180101;
Y02A 50/484 20180101 |
Class at
Publication: |
435/252.3 ;
424/258.1; 435/5 |
International
Class: |
C12Q 001/70; C12N
001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 1996 |
GB |
9621091.9 |
Oct 7, 1997 |
US |
PCTGB9702740 |
Claims
1. An attenuated strain of a prokaryotic microorganism transformed
with nucleic acid encoding papillomavirus virus major capsid
protein wherein the protein assembles in the microorganism to form
virus like particles (VLPs).
2. The attenuated microorganism strain of claim 1 which is an
attenuated strain of Salmonella.
3. The attenuated microorganism strain of claim 2 wherein the
Salmonella strain is Salmonella typhimurium, Salmonella typhi,
Salmonella dublin, or Salmonella enteretidis.
4. The attentuated microorganism strain of claim 1 which is an
attenuated strain of Escherichia coli, Shigella, Yersinia,
Lactobacillus, Mycobacteria or Listeria.
5. The attenuated microorganism strain of claim 1 wherein the
nucleic acid encodes a human papillomavirus virus major capsid
protein.
6. The attenuated microorganism strain of claim 1 wherein the HPV
strain is HPV16, 18, 31, 45 or 56.
7. The attenuated microorganism strain of claim 1 wherein the
papillomavirus virus major capsid protein is L1 protein.
8. The attenuated microorganism strain of claim 1 wherein the
papillomavirus virus major capsid protein is expressed as a chimera
with a fusion partner.
9. The attenuated microorganism strain of claim 8, wherein the
papillomavirus major capsid protein is coexpressed with L2 protein,
the L2 protein being fused to the fusion partner.
10. The attenuated microorganism strain of claim 8 wherein the
fusion partner is E6, E7 or E2 HPV protein, an immunogenic protein
from a non-HPV pathogen or a tumour specific antigen.
11. The attenuated microorganism strain of claim 1 wherein the
microorganism is transformed with nucleic acid encoding two or more
papillomavirus virus major capsid proteins.
12. A composition comprising one or more of the attenuated
microorganisms of claim 1, in combination with a physiologically
acceptable carrier.
13. A vaccine comprising one or more of the attenuated
microorganisms of claims 1, in combination with a physiologically
acceptable carrier.
14. The vaccine of claim 13 formulated for mucosal
immunization.
15. The vaccine of claim 14 wherein the mucosla immunization is via
oral, rectal, nasal, or genital routes.
16. The vaccine of claim 13 wherein the vaccine provides protection
against papillomavirus infection or cancer of the anogenital
tract.
17. A method for producing assembled papillomavirus virus like
particles comprising culturing an attenuated microorganism strain
of claim 1 and recovering the assembled virus like particles thus
produced.
18. A method of detecting the presence of anti-papillomavirus
antibodies in a sample from a subject, the method comprising
immobilizing the HPV VLPs on a solid support, exposing the support
to the sample and detecting the antibodies binding to the
immobilized HPV VLPs, wherein the HPV VLPs are produced by an
attenuated microorganism strain of claim 1.
19. A method of treating a patient in need of prophylaxis or
therapy of a papillomavirus infection or papillomavirus associated
cancer of the anogenital tract comprising administering to that
patient a prophylactically or therapeutically effective amount of
an attenuated microorganism as claimed in claim 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to attenuated strains of
prokaryotic microorganisms, in particular Salmonella, transformed
with nucleic acid encoding papillomavirus virus proteins, to
compositions comprising these microorganisms, especially for use as
vaccines, and to the medical uses of these strains. In a further
aspect, the present invention provides a method of producing
assembled papillomavirus virus like particles (VLPs).
BACKGROUND OF THE INVENTION
[0002] Human papilloma virus (HPV) 16 is the major type of HPV
which, in association with cofactors, can lead to cervical cancer
(49). Studies on HPV have been hampered by the inability to
propagate the virus in culture, the lack of animal models and the
paucity of virions in clinical lesions. This has led to the
development of alternative approaches of antigen production for
immunological studies. The conformational dependency of
neutralizing epitopes, as observed in experimental animal
papillomavirus systems (8, 22) suggests that properly assembled HPV
particles are critical for the induction and detection of
clinically relevant immune reactivity.
[0003] The HPV capsids are formed by 72 pentameric capsomers of L1
proteins arranged on a T7 icosahedral lattice (15). Recently, a
number of investigators have demonstrated the production of HPV
capsids, i.e. virus like particles (VLP), by utilizing baculovirus,
vaccinia virus or yeast expression systems (15, 22, 45, 48, 61).
The potential of VLPs as subunit vaccines has been demonstrated
using the cottontail rabbit papillomavirus (CRPV) (4), the canine
oral papillomavirus (COPV) (57), and the HPV11 models (45).
[0004] HPV16 infects through the genital mucosa, where benign
proliferative lesions are confined. Protection against infection
with such a pathogen could be provided by specific (anti-VLP)
secretory immunoglobulins A (sIgA) or immunoglobulins G (IgG) in
genital secretions. By analogy with existing animal models. HPV16
VLPs-specific antibodies in cervical secretions might help to
prevent sexually transmitted infection by HPV16 in women. However,
this cannot be formally proven in the absence of an experimental
model for genital PV infection and other scenarios requiring
cell-mediated immunity cannot be excluded.
[0005] Moreover, the mechanism underlying HPV infection is unclear.
HPV may directly infect the basal cells of the stratified cervical
epithelium at the occurrence of breaches. Alternatively, HPV
infection could also occur either directly through Langerhans cells
in intact epithelia or indirectly from an HPV-producing
keratinocyte, and thus neutralizing antibodies will not be
functional as shown for other viruses. This further adds to the
difficulty in providing vaccines effective against HPV
infection.
[0006] Immunosuppressed individuals are more prone to develop
cervical carcinoma as compared to immunocompetent individuals,
suggesting the possibility of using immunotherapy. Therapeutic
vaccines (37) aimed to the treatment of established HPV infection
or HPV associated premalignant and malignant lesions have been
investigated during the last ten years (59). Evidence for
HPV-antigen-directed immunotherapy against cervical cancer comes
from the observations that experimental (13), (34), (83) and
natural (82) PV-associated tumours can be controlled by
immunization with E6 and E7 preparations. These studies suggested
that CTL might be the most effective immunological effector
mechanisms. E6 and E7 preparations consisted in either peptides
(13), bacterially prepared fusion proteins (82), eukaryotic
transfected cells (83) or recombinant vaccinia viruses (34).
[0007] Recently, chimeric VLPs carrying the 17 kD E7 protein as a
fusion with L2 have been shown to induce rejection of syngeneic
tumour cells (84) engineered to express L1 and/or E7 ORF (i.e. C3
cells (13) and TCl cells (85)). This data demonstrates the
possibility of providing prophylactic and therapeutic effects in
the same vaccine preparation. Salmonella that are attenuated, yet
invasive, have been proposed for the delivery of heterologous
antigens to the mucosal and systemic immune systems (10). The
antigen is delivered by the live Salmonella to mucosal inductive
sites, where after priming, antigen-specific B and T cells migrate
from the site of induction and mature into effector cells. The
migrating IgA-expressing B cells home to different mucosal sites,
including the genital tract, where they differentiate into IgA
secreting plasma cells (32). Thus, oral or nasal immunization can
provide protective antibodies in genital secretions. Recently, we
and others have shown that mucosal immunization with recombinant
Salmonella can elicit antibody responses in the genital mucosa of
mice and humans (18, 37, 56).
SUMMARY OF THE INVENTION
[0008] In order to develop a prophylactic vaccine against HPV, we
have expressed the major protein L1 of HPV16 in a PhoP.sup.c (35)
attenuated strain of Salmonella typhimurium. Surprisingly, the
inventors found for the first time that it is possible to assemble
VLPs in a prokaryotic organism and that nasal immunization of mice
with an HPV16-L1/Salmonella recombinant strain induces
HPV16-specific conformationally dependent and neutralizing
antibodies in serum and genital secretions. The experiments
described herein also show that it is possible to assemble chimeric
VLPs of a HPV protein and a fusion partner.
[0009] Accordingly, in a first aspect the present invention
provides an attenuated strain of a prokaryotic microorganism
transformed with nucleic acid encoding papillomavirus virus major
capsid protein wherein the protein assembles in the microorganism
to form virus like particles (VLPs).
[0010] Thus, the present invention provides a way of producing
properly assembled papillomavirus VLPs in an attenuated strain of a
prokaryotic microorganism such as Salmonella so that they can be
used as a vaccine to raise an immune response in a subject.
Preferably, the VLPs are delivered to mucosal sites, having the
advantage of generating the immune response to the papillomavirus
VLPs at the locations where infection actually takes place, as well
as at other mucosal surfaces.
[0011] The term "papillomavirus" used herein covers both human and
animal PVs. However, preferably, the papillomavirus is a human
papillomavirus (HPV). About 70 different types of HPV have been
cloned and characterized (denoted HPV1 to HPV70 . . . ), and all
have an 8 kb double stranded Gnome which encodes different early
products and two late products L1 and L2, and are either
epitheliotropic or mucosatropic. L1 is a major capsid protein and
is relatively well conserved among the different HPV types. For a
review of the HPV types and their nucleic and amino acid sequences,
see Human Papillomaviruses "A Compilation and Analysis of Nucleic
Acid and Amino Acid Sequences", 1994, ed. Myers et al, Theoretical
and Biophysics Group T-10 Los Alamos National Laboratory.
Clinically, the most important HPV types are those that infect the
anogenital tract, and that have high oncogenic risk and a high
prevalence. This group includes HPV16, 18, 31, 45 and 56, with
HPV16 alone accounting for more than 50% of invasive cancer in the
anogenital tract, as well as being the most prevalent single type
of HPV.
[0012] The papillomavirus proteins correspond to wild type major
capsid proteins (e.g. L1 and/or L2) or may be chimeras of ail or
part of a HPV protein and a fusion partner. The fusion partner may
be any immunogenic protein against which specific CTL would be
targeted. This protein may be an HPV protein (e.g. E7, E6 or E2 of
any HPV type), a protein from another pathogen or any tumour
specific antigen. In one embodiment the HPV protein is L1 protein
coexpressed with L2, with the fusion partner expressed so that it
is linked to the L2 protein.
[0013] It has been shown that chimeric VLPs can elicit anti-tumour
immunity against carrier and inserted proteins in HPV16 tumour
models. Thus, chimeric VLPs which induce E7-specific CTLs aimed to
the killing of already HPV infected cells or HPV-associated
premalignant lesions. In this event, induction of CTLs to eliminate
already HPV infected cells appears therefore an appealing
complement to the induction of neutralizing antibodies, and
chimeric VLPs have been shown to induce both functions.
[0014] Thus, in one embodiment of the invention, Salmonella strains
able to induce neutralizing antibodies and CTLs by expressing
chimeric VLPs could be therapeutic at least for early or
premalignant HPV lesions in which the downregulation of MHC I or
other factors observed in more advanced cancers has not yet
occurred.
[0015] Preferably, the prokaryotic microorganism is an attenuated
strain of Salmonella. However, alternatively other prokaryotic
microorganisms such as attenuated strains of Escherichia coli,
Shigella, Yersinia, Lactobacillus, Mycobacteria, Listeria or Vibrio
could be used. Examples of suitable strains of microorganisms
include Salmonella typhimurium, Salmonella typhi, Salmonella
dublin, Salmonella entereuidis, Escherchia coli, Shigella
flexeneri, Shigella sonnei, Vibrio cholera, and Mycobacterium bovis
(BC6).
[0016] Attenuated Salmonella strains are one of the best
characterized mucosal vaccine carriers. Recombinant Salmonella
strains that are attenuated yet invasive have been used as oral
vaccine vectors to carry protective epitopes of several pathogens
into the mucosal associated lymphoid tissue thus inducing mucosal,
systemic and CTL immune responses against both the carrier and the
foreign antigens (58, 65, 67, 69, 75, 77).
[0017] The currently licensed oral vaccine against typhoid fever S.
typhi Ty21a (72) administered as a three-dose regimen of
enteric-coated capsules (10.sup.9 CFU/capsule) provided a 67%
efficacy over a 3 year period. However, because the S. typhi Ty21a
requires high and multiples doses in liquid formulation for higher
efficacy, and its mutations are not yet all characterised (63, 64,
70, 71, 78), new attenuated Salmonella strains have recently been
developed and tested in humans. These include nutritional
auxotrophs in which pathways for biosynthesis of aromatic compounds
have been interrupted (.DELTA.aro mutants). The .DELTA.aroA,
.DELTA.purA mutants of S. typhi have been tested in human
volunteers (32) and were shown to elicit specific cell-mediated
immune responses but weak humoral responses. Other aro mutants
(aroC and aroD) were insufficiently attenuated and caused fever and
bacteremia (79). A double mutant .DELTA.aroC .DELTA.aroD Ty2 (CD
908) was safe and elicited IgG antibodies against LPS in 80% of the
immunized adult volunteers (73, 80). S. typhi mutants were also
generated in which the adenylate cyclase (cya) and the cAMP
receptor (crp) genes were deleted. These gene products are required
for the transcription of many genes and operons that control
transport processes, expression of fimbriae, flagella and some
outer membrane proteins. One mutant .sub..chi.3927 (.DELTA.cya
.DELTA.crp Ty2) was tested and shown to be immunogenic but some
volunteers developed fever and vaccine bacteremia (79). Therefore,
a novel strain, .sub..chi.4073, was constructed by deleting a third
gene (cdt) responsible for colonization of deep tissue (66, 68,
74). This strain was administered to volunteers and proved to be
completely safe at doses up to 5.times.10.sup.8 CFU and generated a
seroconversion in 80% of the volunteers (66).
[0018] Preferred attenuated Salmonella strains include mutants in a
two-component regulatory system, the PhoP/PhoQ genes. These genes
affect expression of a number of other genes and are responsive to
phosphate levels and to environmental conditions expected to be
experienced by Salmonella residing within macrophages. Preferred
example of these mutants are the PhoP.sup.c strains used in the
examples described below. Recently, a PhoP/PhoQ-deleted Salmonella
typhi (ty800) has been shown to be safe and immunogenic in humans
(81).
[0019] A still more preferred example of such a mutant is one in
which a .beta.-aspartate semialdehyde dehydrogenase (asd) vector is
incorporated in order to maintain selective pressure in vivo to
maintain the expression of HPV16 VLPs. This is surprising as it has
previously been reported that such mutant, when administered
nasally at least, induces much lower levels of anti-HPV16 L1 VLPs
than intact PhoP.sup.c strains (see Benyacoub et al, 16.sup.th
International Papilloma Conference, Siena, Sep. 5-12, 1997),
surviving to a lesser extent and with absence of L1 expression.
[0020] More preferred is use of a PhoP.sup.c .DELTA.asd strain that
places .DELTA.asd and the HPV VLP protein or its fusion together in
a plasmid that has a `medium copy` number eg. 15 to 20 rather than
a higher copy number. Eg. having a pBR ori such as plasmid
pYA3342.
[0021] As mentioned above, the attenuated strain of the prokaryotic
microorganism is transformed with a nucleic acid encoding one or
more major papillomavirus capsid proteins. The inventors found for
the first time that, when this nucleic acid is expressed in the
microorganisms, the capsid proteins produced assemble correctly to
form VLPs, making them especially suitable for the vaccination of
subjects against papillomaviruses. Preferably, the major viral
capsid protein is L1, optionally additionally including nucleic
acid encoding L2 protein. As discussed above, the capsid protein
may be linked to a fusion partner such as another antigen.
[0022] In a further aspect, the present invention provides a
composition comprising one or more of above attenuated prokaryotic
microorganisms, optionally in combination with a physiologically
acceptable carrier. Preferably, the composition is a vaccine,
especially a vaccine for mucosal immunization, e.g. for
administration via the oral, rectal, nasal, vaginal or genital
routes. Our earlier studies using recombinant Salmonella expressing
hepatitis B virus antigen (18) showed that vaccination via any of
these routes produces a sIgA response in the mucosal secretions at
other sites. Advantageously, for prophylactic vaccination, the
compositions comprises one or more strains of Salmonella expressing
a plurality of different VLPs, e.g. VLPs from different
papillomavirus types. This has the advantage of improving the
protective effect of the vaccine to a range of challenges by the
different papillomavirus types. For therapeutic vaccination,
subsequent chimeric VLP constructs can comprise fusion products of
various HPV type L1 capsids with the same L2 fusion partner.
[0023] In a further aspect, the present invention provides an
attenuated strain of a prokaryotic microorganism described above
for use as a medicament, especially as a vaccine.
[0024] In a further aspect the present invention provides the use
of an attenuated strain of a prokaryotic microorganism transformed
with nucleic acid encoding papillomavirus virus major capsid
protein, wherein the protein assembles in the microorganism to form
virus like particles, in the preparation of a medicament for the
prophylactic or therapeutic treatment of papillomavirus infection
or anogenital cancer, especially cervical cancer.
[0025] Generally, the microorganisms or VLPs according to the
present invention are provided in an isolated and/or purified form,
i.e. substantially pure. This may include being in a composition
where it represents at least about 90% active ingredient, more
preferably at least about 95%, more preferably at least about 98%.
Such a composition may, however, include inert carrier materials or
other pharmaceutically and physiologicaly acceptable excipients. A
composition according to the present invention may include in
addition to the microorganisms or VLPs as disclosed, one or more
other active ingredients for therapeutic use, such as an antitumour
agent.
[0026] The compositions of the present invention are preferably
given to an individual in a "prophylactically effective amount" or
a "therapeutically effective amount" (as the case may be, although
prophylaxis may be considered therapy), this being sufficient to
show benefit to the individual. The actual amount administered, and
rate and time-course of administration, will depend on the nature
and severity of what is being treated. Prescription of treatment,
eg. decisions on dosage etc, is within the responsibility of
general practioners and other medical doctors.
[0027] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0028] Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may include, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. Such materials
should be nontoxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material will depend on the route of administration.
[0029] Examples of techniques and protocols mentioned above can be
found in Remington's Pharmaceutical Sciences, 16th edition, Osol,
A. (ed), 1980.
[0030] In a further aspect, the present invention provides a method
for producing assembled papillomavirus virus like particles
comprising culturing an attenuated strain of a prokaryotic
microorganism transformed with nucleic acid encoding papillomavirus
virus major capsid protein wherein the protein is expressed and
assembles in the microorganism to form virus like particles.
Preferably, the method additionally comprises the step of
recovering the VLPs from the prokaryotic microorganism.
[0031] In a further aspect, the present invention provides the use
of a papillomavirus VLP as obtainable by transforming an attenuated
prokaryotic microorganism with nucleic acid encoding the VLPs and
expressing the nucleic acid to produce assembled VLPs, in a
diagnostic method. in one embodiment, present invention provides a
method for detecting the presence of anti-papillomavirus antibodies
in a sample from a subject comprising immobilizing the HPV VLPs on
a solid support, exposing the support to the sample and detecting
the presence of the antibodies, e.g. using ELISA.
[0032] Preferred embodiments of the present invention will now be
described by way of example and not limitation with reference to
the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1. HPV16 L1 expression in the PhoP.sup.c/HPV strain.
Salmonella were grown overnight and prepared as indicated in
Material and Methods. A. Commassie blue staining of a 10%SDS PAGE
gel; Lane M : Molecular weight marker; Lane 1: total lysate of the
PhoP.sup.c strain Lane 2: total lysate of the PhoP.sup.c/HPV
strain, a unique 57 kDa protein is indicated with an arrow. B.
Immunoblot using anti-HPV16-L1 mAb of the total and fractionated
PhoP.sup.c/HPV lysate. Lane tot: total lysate, Lane 1 to 25:
different fractions obtained after fractionation of the
PhoP.sup.c/HPV lysate through a 10-40% sucrose gradient, the
heavier fraction of the gradient being in lane 1. The 57 kDa
protein band identified as being L1 is indicated (arrow).
[0034] FIG. 2. HPV16 L1 assemble into VLPs. Electron micrographs of
(A) PhoP.sup.c/HPV16 VLPs and (B) Baculo-derived HPV16 VLPs. In A,
factions 7 to 13 of PhoP.sup.c/HPV lysate (FIG. 1) were pooled. The
samples were negatively stained with phosphotungstic acid. Bar
represents 53 nm.
[0035] FIG. 3. Anti-HPV16VLP and anti-LPS systemic and mucosal
antibody responses after nasal immunization with the PhoP.sup.c/HPV
strain. Three 6-week-old BALB/c female mice were immunized with
5.times.10.sup.7 CFU, sampled at the indicated weeks, sacrificed
and bled at week 27. Data are expressed as the geometric means 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.
[0036] FIG. 4. HPV viral cycle and vaccination strategies. In the
left portion of the figure the HPV productive viral cycle during
keratinocyte differentiation is schematically drawn (early
infection-CIN I). A late stage of infection (CIN III-tumour) in
which the HPV DNA is integrated into the host genome is shown on
the right. Different protective immune mechanisms are shown with
arrows indicating the sites of action. Antibody-dependent cellular
cytotoxicity (ADCC) mechanims which require viral antigens to be
expressed at the surface of cells are not indicated.
[0037] FIG. 5. In vitro neutralization of HPV16 pseudotype virus
infection of mouse C127 cells. (A) no virus added. (B-H) Equal
aliquots of an HPV16(HPV1) pseudotype virus containing extract was
added. The aliquots were preincubated with (B) no antibodies, (C)
BPV1 neutralizing MoAb B1.A1, (D) HPV16(BPV1) neutralizing MoAb
H16.E70, (E) mouse preimmune sera, (F) mouse#4 immune
sera.sub.(week27), (G) mouse#5 immune sera.sub.(week27), (H)
mouse#4 immune sera.sub.(week27).
[0038] FIG. 6 shows the results of tumour growth experiments in
mice immunized with Salmonella HPV producing strains. Nasal
immunisations were performed three times weekly with either 20
.mu.l PBS (A) or 5 .mu.g of purified HPV16 VLPs+5 .mu.g of cholera
toxin (CT)(E) and two times at week0 and week2 with 10 CFU of
PhoPc/HPV16 L1 (b), .sub..chi.4550/pYA34L1 (C) and
.sub..chi.4550/pYA32L1 (D). All mice were challenged with 5 105 C3
cells into the flank two weeks after the last immunisation. The
mean volume of the tumours in each group are shown, while the
number of mice harbouring a tumour/number of mice injected is
indicated at Day 17.
[0039] FIG. 7 shows coexpression of L1 and L2 in PhoP.sup.c/HPV16
L1-L2. Blot A was revealed with an anti-L2 antibody, while blot B
was revealed with anti-L1 antibody (Camvir).
[0040] FIG. 8 shows the expression of L1 in E. coli BL12 pET 3DL1.
Identical amounts of bacteria were loaded (3.times.10.sup.6 CFU)
after 3 hours incubation with IPTG and the blot was revealed with
an anti-L1 antibody.
[0041] FIG. 9 shows the results of tumour growth experiments with
PhoP.sup.c .DELTA.asd mutants having or lacking the ability to
express L1 VLPs. Plots are for PBS (A), PhoP.sup.c .DELTA.asd/nasal
(B) and PhoP.sup.c .DELTA.asd/HPVL1 nasal (C).
[0042] FIG. 10 is a map showing the essential characteristics of
plasmid pYA3342
DETAILED DESCRIPTION
Materials and Methods
[0043] Plasmid construction and bacterial strains used
[0044] Plasmid pFS14nsd HPV16-L1 was constructed by exchanging in
the plasmid pFS14 NSD (54) the hepatitis B nucleocapsid gene
(HBcAg, NcoI-HindIII fragment) for a NcoI-HindIII fragment encoding
the HPV16-L1 open reading frame. The HPV16-L1 NcoI-HindIII fragment
was generated by Polymerase Chain Reaction (PCR) using the
baculovirus expression plasmid pSynwtVIHPV16 114/B-L1+L2 (23) as a
template with a 28 mer containing a NcoI site:
5'-GGGCCATGGCTCTTTGGCTGCCTTAGTGA-3' and a 27 mer containing a
HindIII site 5'-GGGAAGCTTCAATACTTAAGCTTACG-3'. The final construct
containing the Tac promoter places the HPV16-L1 ATG at position +8
relative to the Shine-Dalgarno sequence and introduces a change in
the second amino acid which becomes an alanine instead of the
serine encoded by the original sequence. Sequencing of the entire
L1 open reading frame was carried out (MycrosynthAG) and no further
nucleotide change was observed Plasmid pFS14nsd HPV16-L1 was
amplified in E. coli JM105 and then electroporated as described
previously (50) into bacterial strain CS022. This strain is derived
from the ATCC 14028 strain, into which the pho-24 mutation was
introduced by P22 transduction, resulting in attenuation in both
virulence and survival within macrophages in vitro (PhoP.sup.c,
(35)). The resultant recombinant strain is called PhoP.sup.c/HPV
hereafter.
[0045] Expression of HPV16-L1 in Salmonella and VLPs
purification
[0046] After overnight growth at 37.degree. C. the recombinant
bacteria were lysed by boiling in Laemmli buffer containing 5% SDS.
The lysates were separated on 10% SDS/PAGE gels and expression of
L1 was analyzed by Western blot using HPV16-L1 mAb CAMVIR-1 (33) as
primary antibody, an alkaline-phosphatase conjugated goat
anti-mouse IgG (Sigma) as secondary antibody and BCIP/NBT
(Boehringer) as substrate.
[0047] To prepare VLPs, bacteria were lysed by sonication and the
lysate fractionated on a 10%-40% sucrose gradient in Phosphate
Buffer Saline (PBS) containing 1M NaCl for 1 hour at 40 Krpm using
a TST41.14 rotor. Fractions of the gradient were then analysed for
the presence of the L1 protein by Western blot. The fractions of
high sedimentation containing the L1 protein were pooled, dialyzed
against PBS/O.5M NaCl. VLPs were pelleted for 1 h at 50 Krpm using
a TST65.1 rotor, adsorbed to carbon-coated grids, negatively
stained with phosphotungstic acid and examined with a Philips
electron microscope.
[0048] Purification of HPV16 VLPs expressed in insect cells from a
recombinant baculovirus
[0049] The transfer vector pSynwtVI-HPV16 114/B-L1+L2 (23) was
cotransfected with the linearized genome of baculovirus
(Baculo-Gold, Pharmingen) using the calcium-phosphate method into
SF9 cells. The recombinant baculoviruses were plaque-purified and
propagated by standard methods (39). Baculo-derived HPV16 VLPs were
purified as described previously (23).
[0050] Immunization and sampling of mice
[0051] Six-week-old female BALB/c mice were immunized at day 0 and
at week 14 by the nasal route with 5.times.10.sup.7CFU of inoculum.
Blood, saliva and genital samples were taken as described
previously (18). All samples were stored at 70.degree. C.
[0052] ELISA
[0053] The amount of total IgA, anti-LPS IgA and IgG antibodies in
samples were determined by enzyme-linked immunosorbent assay
(ELISA) as described previously (18). For the anti-HPV16 VLP, ELISA
plates were coated with 10 ng of a preparation of baculo-derived
HPV16 VLPs in PBS (total protein content was determined with a
BioRad Protein assay with BSA as standard). This amount of VLP was
saturating in our ELISA test. Endpoint dilutions of samples were
carried out. The specific IgA or IgG amounts are expressed as
reciprocal of the highest dilution that yielded an OD.sub.492 four
times that of preimmune samples. These reciprocal dilutions were
normalized to the amount of total IgA or IgG in saliva and genital
washes. ELISA plates were also coated with 10 ng of baculo-derived
HPV16 VLPs in 0.2M carbonate buffer pH9.5 to determine the titer of
antibodies recognizing unfolded VLPs (14).
[0054] In vitro HPV16 neutralization assay
[0055] Infectious pseudovirions consisting of HPV capsid made of L1
and L2 surrounding the bovine papillomavirus type 1 (BPV1) genome,
designated HPV16(BPV1), were generated as recently described (43).
Briefly. BPHE-1 hamster cells harbouring autonomously replicating
BPV1 genomes were co-infected with defective recombinant Semliki
forest viruses that expressed L1 and L2 virion capsid genes of
HPV16. Infectious pseudotype HPV16 virus 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 mouse sera diluted 1:50
(1.0 ml final volume) in culture medium. Mouse monoclonal
antibodies H16.E70 and B1A1 were generated against recombinant
baculovirus expressed HPV16 L1 VLPs and BPV16 VLPs respectively,
and used at a 1:100 dilution. HPV16.E70 and B1.A1 served as
positive and negative controls for HPV16 (BPV1) neutralization,
respectively.
Results
[0056] HPV16-L1 is expressed in PhoP.sup.c and VLP assemble
[0057] The open reading frame of the major protein L1 of HPV16 was
cloned in the plasmid pFS14 NSD (53). L1 is constitutively
expressed under the control of the Tac promoter in S. typhimurium.
A unique 57 kDa protein detected in the lysate of PhoP.sup.c/HPV
overnight cultures (FIG. 1A), was identified as HPV16 L1 by Western
immunoblot using an anti-HPV16-L1 monoclonal antibody (CAMVIR,
(33), FIG. 1B). To determine whether the L1 protein expressed by
PhoP.sup.c/HPV assembled into VLP, the bacterial lysate was
fractionated through a 10-40% sucrose gradient and the heavier
fractions containing the L1 protein FIG. 1B) were analyzed by
electron microscopy. Spherical particles typical of PV capsids were
recovered from the bacterial preparation (FIG. 2A) but the
bacterial VLPs appeared more polymorphic in size with diameters
ranging from 40 to 55 nm (FIG. 2A) when compared to 55 nm VLPs
expressed in insect cells (FIG. 2B).
[0058] Nasal immunization with the PhoP.sup.c/HPV strain induces
systemic and mucosal antibody responses
[0059] Since nasal immunization using recombinant Salmonella was
shown to elicit strong vaginal sIgA responses against an expressed
foreign antigen (18), we immunized mice nasally with the
PhoP.sup.c/HPV strain (5.times.10.sup.7 CFU). Samples of blood,
saliva and vaginal washes were taken 0, 2, 4, and 6 weeks after
immunization. The immune responses against both the carrier, i.e.
anti-LPS and the carried antigen, i.e. anti-HPV16 VLP, were
determined Serum HPV16 VLP specific IgG (FIG. 3) were detected
after 2 weeks in one mouse and after 4 weeks in all mice. The
response peaked after 6 weeks at relatively low titers and
persisted at least until week 14. At that time, no HPV16 VLP
specific antibodies were detected in vaginal secretions, while one
mouse had low titers of IgA in the saliva. The systemic and the
mucosal immune responses against LPS were relatively low (FIG. 3),
but similar to those elicited by the PhoP.sup.c/HBc strain (18)
suggesting a normal take of PhoP.sup.c/HPV Salmonella by the mice.
The low anti-LPS response observed after nasal immunization incited
us to perform a booster immunization. Thus, a second nasal
immunization was performed at week 14 and samples were taken 5 and
10 weeks later (week 19 and 24 respectively). The second
immunization induced, 5 weeks later (week 19), a 15 fold increase
of anti-BPV16 VLP IgG in serum, as well as anti-HPV16 VLP IgA in
the vaginal washes (FIG. 3) from the three mice. Anti-HPV16 VLP IgG
were also found in vaginal washes but only in two mice at week 19
and titers were again almost undetectable at week 24 (FIG. 3).
Anti-HPV16 VLP IgA and IgG were also found in the saliva of the
three mice in amounts comparable or slightly higher to those found
in vaginal washes.
[0060] Anti-HPV16 VLP antibodies recognize only folded VLP
[0061] In order to examine whether the immune responses induced by
the PhoP.sup.c/HPV strain generated conformational antibodies
directed against native but not unfolded VLPs, we measured by ELISA
(Table 1) the binding of antibodies, in the samples from the
immunized mice, to baculo-derived VLPs in PBS (native form) or in
carbonate buffer (pH 9.5, unfolded VLP, (14)). The specific IgG or
IgA elicited by the PhoP.sup.c/HPV strain very poorly recognizes
unfolded VLPs suggesting that the majority of L1 were folded into
highly ordered structures when expressed in PhoP.sup.c/HPV (Table
1).
[0062] In vitro neutralizing activity of the immune sera
[0063] In previous studies of baculo-derived VLPs, neutralizing
activity and protection from experimental infection generally
correlated with ELISA reactivity to native VLPs. We therefore
wished to determine if the conformationally dependent anti-VLP
antibodies elicited by the live Salmonella vaccine were also
neutralizing. Although no infectivity assay or source of the virus
currently exists for authentic HPV16, it has recently been
demonstrated that HPV16 capsid proteins can encapsidate
autonomously replicating BPV1 genomes resulting in HPV16(BPV1)
pseudotype virions whose infectivity can be monitored by focal
transformation of cultured mouse fibroblasts (43). We therefore
used the HPV16(BPV1) infectivity assay to examine the neutralizing
activity of the mouse sera generated above. Each of the three
immune sera displayed strong neutralizing activity against
HPV16(BPV1) (FIG. 5), but did not neutralize BPV1 virions (data not
shown). The preimmune sera had no neutralizing activity. The
neutralizing activities of the immune sera appeared to correlate
with the titers in the native VLP ELISA, although the sera were
only tested at a single dilution.
[0064] Tumour protection assay in the HPV16 mouse tumour model
[0065] It has been recently shown that the growth syngeneic tumour
cells (C3) injected into the flank of C57BL/6 mice was inhibited by
a subcutaneous immunization with purified HPV16 cell (84). We have
tested whether nasal immunization with purified VLPs and
recombinant Salmonella/HPV strains was able to induce the same
effect. Specifically, we have tested the following stains:
PhoPc/HPV16 L1 (86) and the .sub..chi.4550 (56) expressing either
high levels (.sub..chi.4550/pYA34L1- ) or low levels
(.sub..chi.4550/pYA32L1) HPV16 L1. Tumour growth in the different
groups of mice is shown in FIG. 6. Our preliminary results
demonstrate that nasal immunization with purified VLPs is effective
and that all the Salmonella/HPV strain tested induced partial
tumour protection. Of interest, is the strain
.sub..chi.4550/pYA34L1 that prevented complete tumour growth in
4/10 mice.
[0066] Coexpression of the L2 protein into PhoPc/HPV16
[0067] The L2 OR17 was cloned downstream of the L1 ORF by PCR into
the plasmid pPSnsdHPV16 L1 (86). The PCR reaction included a 5'
specific oligonucleotide that contained a synthetic Shine-Dalgarno
sequence in order to allow translation of 12 from a polycistronic
L1-L2 RNA. The resultant PhoPC/HPV16 L1+L2 recombinant strain
expressed both L1 and L2 and VLPs assembled in amount similar to
the parent PhoPC/HPV16 L1 strain as assessed by a sandwich ELISA
This suggests that by fusing the E7 ORF to the L2 ORF, in the
PhoP.sup.c/HPV16 L1+L2 strain, a chimeric VLPs would also assemble
and such recombinant Salmonella strain used to induce HPV16
E7-CTLs.
[0068] High level expression of L1 in the inducible E. coli PET
expression system
[0069] The L1 ORF was cloned in the plasmid pET3 (ovagen).
L1-expression driven by a T7 promoter was assessed in the strain
BL21apLysS (expressing T7 polymerase upon IPTG induction). After
IPTG induction, a 10 fold higher level of L1 expression/bacteria
was achieved in comparison to the Salmonella PhoP.sup.c strain (see
FIG. 8). The lysate of this recombinant E. coli formed a band at a
density of VLPs in a CsCl density gradient, suggesting that the
VLPs self-assembled in this bacteria.
[0070] Induction of therapeutic immune response with .DELTA.asd
mutants
[0071] A deletion in the aspartate-.beta.-semialdehyde
dehydrogenase (asd) gene was introduced into the PhoP.sup.c strain
described above by P22Htint bacteriophage transduction. The
original P22Htint lysate propagated on the .sub..chi.3520 S.
typhimurium .DELTA.asd A1 zhf-4::Tn10 (provided by Dr R Curtiss
III). A tetracycline sensitive PhoP.sup.c .DELTA.asd strain was
then selected (see Maloy et al (1981) J. Bacteriol p1110-1112
incorporated herein by reference). A NcoI-HindIII fragment
containing HPV16 L1 (from the plasmid pFS14nsd HPV16-L1-see
Nardellihaefliger et al (1997) Infection & immunity 65 (8)
3328-3336 incorporated herein by reference) was cloned into the
NcoI-Hind-III sites of the plasmid pYA3342- (provided by Dr R
Curtiss III) and the resultant recombinant PhoP.sup.c
.DELTA.asd/HPV16 L1 strain was used in a tumour protection assay as
follows.
[0072] Nasal immunisations were performed at week 0 and week 2 with
10 .mu.l of PBS (A in FIG. 9), with 1.times.10.sup.7 CFU of
recombinant PhoP.sup.c .DELTA.asd (B in FIG. 9) or 1.times.10.sup.7
CFU recombinant PhoP.sup.c .DELTA.asd/HPV16 L1. All mice were
challenged with 5.times.10.sup.5 C3 cells into the flank four weeks
after the last immunisation.
[0073] Results, displayed graphically in FIG. 9, show the
protection against tumour growth conferred by the immunisation with
the PhoP.sup.c .DELTA.asd/HPV16 L1, No protection was conferred by
the parent strain that lacks expression of the L1 antigen
PhoP.sup.c .DELTA.asd.
Discussion
[0074] In this study, we demonstrate that an attenuated Salmonella
strain expressing the major capsid protein of HPV16 is a promising
vaccine candidate against HPV16 infection, as the VLPs that are
assembled by this recombinant bacteria can induce serum as well as
genital VLP-specific conformational antibodies. The results above
also show that the antibodies are able to neutralize HPV16 viruses.
These results could be readily extrapolated by the skilled person
to other types of HPV or other papillomaviruses, or other
prokaryotic microorganisms.
[0075] The life cycle of papillomavirus is intimately associated
with the differentiation of the epithelial cells in skin or the
oral and genital mucosa (5, 19, 40, 62). It is believed that
viruses gain access to the basal epithelial cells through mucosal
abrasions (21). Upon infection of the cervical epithelium for
instance, the viral DNA released in the cytoplasm of the basal
cells migrates into the nucleus where it remains episomic and early
genes are transcribed leading to a low rate of cell proliferation
and the thickening of the basal layer (Cervical intraepithelial
neoplasia type I, CIN I). As the infected epithelial cells migrate
through the suprabasal layer and undergo differentiation, the
episomal viral genome replicates reaching .about.1000 copies per
cell (29). Concomitant to viral DNA amplification, late genes
become expressed and capsids assemble in terminally differentiated
keratinocytes (FIG. 4), thus facilitating a new round of infection.
In high grade lesions (CIN III and carcinoma) the entire epithelium
consists of undifferentiated basal cells in which the viral DNA has
been integrated into cellular DNA. In these cells, the E6/E7 gene
products constitute the major HPV proteins expressed and viruses
are no longer produced.
[0076] Based on our knowledge of HPV pathogenesis, it appears that
two arms of immunity (humoral and cellular) have to be effective to
prevent viral infection, to decrease the local viral load, or to
cure tumors (FIG. 4, see also (59)). A local or systemic humoral
immune response with neutralizing antibodies is likely to block
early infection, while a cellular response may contribute to the
elimination of untransformed or transformed infected cells. An
ideal vaccine should trigger the two types of response, although
the immunological correlate of protection and of cure have not been
identified so far.
[0077] Prophylactic vaccines inducing type-specific neutralizing
conformational (anti-VLP) antibodies have been shown to prevent
CRPV or COPV infections in cottontail rabbit (4) or dog (57),
respectively. In both cases serum neutralizing antibodies where
generated by vaccination with self-assembled PV capsids. By
analogy, neutralizing antibodies to HPV16 capsid in cervical
secretions are expected to prevent infection. Since the precise
mucosal site where early HPV infection takes place is not known, it
is difficult to predict whether sIgA antibodies acting from the
lumenal site or circulating IgG antibodies reaching the basal
layers will be most efficient.
[0078] The elimination of HPV-infected cells or tumor cells
requires a cellular immune response with cytotoxic T lymphocytes
(CTL) recognizing viral antigens presented by MHC class I molecules
on the infected cells. Therapeutic vaccines aimed at eliminating
HPV-induced tumors have been generated using either peptides
corresponding to T cell epitopes from the E6/E7 oncogenes or E6/E7
expressing vaccinia viruses. Both were shown to elicit CTLs and in
some cases tumor regression was observed (3, 6, 7, 12, 13, 34). One
of the major problems, however, is that MHC class I molecules are
down-regulated in the differentiated keratinocytes that produce
viruses or in tumour cells (9).
[0079] Since both humoral and cellular immunity are believed to
control HPV infection and since local and systemic responses are
desirable, an efficient vaccine should reach inductive sites
associated with mucosal surface and/or peripheral lymph nodes. Live
bacterial vaccines are known to cross mucosal surfaces and elicit
humoral or cellular responses (41). Recombinant and attenuated
enteropathogenic bacteria, such as Salmonella, represent ideal
antigen delivery systems, because they efficiently cross all
mucosal surfaces to gain access to both mucosal organized lymphoid
tissue (MALT) or draining lymph nodes. They exploit the two basic
sampling systems mediating uptake of mucosally administered
antigens including M cells in simple epithelia and dendritic cells
both in simple and stratified epithelia (38). We have selected a
Salmonella typhimurium strain attenuated for macrophage survival,
because long lasting antibody responses were elicited by a single
nasal, oral, rectal or vaginal administration of recombinant
bacteria expressing a foreign antigen (18). In that study, the best
genital responses were obtained after nasal immunization. In the
airways, antigen uptake occurs through M cells found in NALT, the
nasal associated lymphoid tissue (25) and BALT, the bronchial
associated lymphoid tissue (55). The primed IgA-expressing
lymphocytes then migrate into cervical and uterine tissues where
they produce polymeric IgA antibodies, which are transported across
the epithelium by the polymeric Ig receptor (26-28).
Intraepithelial dendritic cells in the bronchial epithelium also
play a major role in antigen presentation by taking up the antigens
in the respiratory epithelium and carrying them to distant draining
lymph nodes where priming occurs (17). This probably explains why
nasal immunization is so efficient in triggering both local and
systemic antibody responses.
[0080] Antigens expressed in Salmonella strains can also elicit
cellular responses with specific CTLs (1, 16, 58). Depending on
which viral antigen is expressed, specific CTLs recognizing
infected cells at different stages of differentiation could be
generated (FIG. 4). For instance, E7-specific CTLs were generated
by immunizing mice with recombinant Salmonella expressing HPV16 E7
epitopes (31).
[0081] To trigger neutralizing antibodies using recombinant
Salmonella, it is essential that the antigen retains its native
conformation. For HPV, this requires that the L1 proteins form
VLPs. Papilloma VLPs haste been shown to assemble in eukaryotic
cells (15, 22, 45, 48, 61), but not in prokaryotes. In bacteria
mainly L1-fusion proteins were expressed (2, 20, 24) and when bona
fide L1 proteins were expressed, VLP assembly was not examined
(11). As shown in this paper, HPV16 VLP assemble in Salmonella
probably because the level of expression achieved in our
experiments was high and capsid assembly does not require
glycosylation (60). Capsid production in bacteria has also been
reported for other viruses such as the nucleocapsid of Hepatitis B
virus (52) and the capsid of Polyomavirus (30, 46). Polyomavirus
VP1 major capsid protein, analogous to HPV L1, forms capsomers when
expressed in E. coli which subsequently self-assembled into VLPs in
vitro (46). The fact that only capsomers but no VLPs were recovered
is probably due to the reducing agents present during purification,
which are known to disrupt capsids (47).
[0082] Nasal immunization with the PhoP.sup.c/HPV strain induced
systemic and mucosal antibodies against native but not denatured
HPV16 VLPs. In contrast, recombinant vaccinia expressing HPV1
capsid protein triggered serum antibodies recognizing both folded
and unfolded VLP, probably reflecting different mode of viral
protein expression, and low HPV-specific genital IgA antibody
titers (14), as expected with a non-mucosal route of
immunization.
[0083] Antibody titers against the foreign antigen induced by
PhoP.sup.c/HPV compared to PhoP.sup.c/HBc Salmonella were about 10
times lower (18). This could reflect differences in immunogenicity
between the two viral antigens (51) or, more likely, differences in
plasmid stability. In contrast to the HBc DNA, the plasmid carrying
the HPV16-L1 DNA was unstable in Salmonella in vivo in the absence
of selective pressure, since less than 1% of the Salmonella
recovered from different tissues two weeks after immunization still
harboured the L1-containing plasmid (data not shown). To increase
the stability of the plasmid we are currently recloning the L1 gene
in .beta.-aspartate semialdehyde dehydrogenase (asd)based vectors
which maintain selective pressure in vivo (36, 56).
[0084] The above work also demonstrates the following points:
[0085] (a) that purified VLPs and Salmonella/HPV strains are
capable of providing tumour protection in a HPV16 mouse tumour
model.
[0086] (b) that chimeras of a HPV protein and a fusion partner
assemble in prokaryotes to form VLPs.
[0087] c) that high levels of expression of HPV proteins that
assemble to form VLPs can be obtained in E. coli, demonstrating
that the invention is applicable in prokaryotes other than
Salmonella.
[0088] In conclusion, we have constructed a recombinant Salmonella
strain expressing HPV16-L1 capsid proteins and assembling VLPs that
induce conformational serum IgG and vaginal sIgA antibodies
recognizing VLPs. Neutralizing activities of these antibodies were
tested and shown to display strong neutralizing activity in an
HPV16(BPV1) infectivity assay.
1TABLE 1 Titers of IgG (in serum) or IgA (in vaginal washes)
against native and unfolded HPV16 VLP in mice immunized with
PhoP.sup.c/HPV anti- Samples anti-HPV16 VLP HPV16 VLP
unfolded.sup.a IgG titers.sup.b #4 serum.sub.(week 27) 60,000 100
#5 serum.sub.(week 27) 80.000 200 #6 serum.sub.(week 27) 20,000 100
Camvir.sup.c 16,000 80,000 IgA titers #4 Vaginal Washes.sub.(week
19) 40 <1 #5 Vaginal Washes.sub.(week 19) 20 <1 #6 Vaginal
Washes.sub.(week 19) 40 1 .sup.aELISA plates were coated with VLP
in Carbonate buffer pH 9.5 .sup.bTiters are expressed as the
reciprocal of the highest sample dilution that yielded an
OD.sub.492 four times that of preimmune sample .sup.cAnti-HPV16 L1
monoclonal IgG (35 .mu.g/ml), 30) used as positive control
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