U.S. patent application number 09/832065 was filed with the patent office on 2003-03-13 for self-assembling recombinant papilloma virus capsid proteins.
Invention is credited to Kirnbauer, Reinhard, Lowy, Douglas R., Schiller, John T..
Application Number | 20030050439 09/832065 |
Document ID | / |
Family ID | 25476357 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030050439 |
Kind Code |
A1 |
Lowy, Douglas R. ; et
al. |
March 13, 2003 |
Self-assembling recombinant papilloma virus capsid proteins
Abstract
Recombinant papillomavirus capsid proteins that are capable of
self-assembly into capsomer structures and viral capsids that
comprise conformational antigenic epitopes are provided. The
capsomer structures and viral capsids, consisting of the capsid
proteins that are expression products of a bovine, monkey or human
papillomavirus L1 conformational coding sequence proteins, can be
prepared as vaccines to induce a high-titer neutralizing antibody
response in vertebrate animals. The self assembling capsid proteins
can also be used as elements of diagnostic immunoassay procedures
for papillomavirus infection.
Inventors: |
Lowy, Douglas R.;
(Washington, DC) ; Schiller, John T.; (Silver
Spring, MD) ; Kirnbauer, Reinhard; (Bethesda,
MD) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
25476357 |
Appl. No.: |
09/832065 |
Filed: |
April 9, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09832065 |
Apr 9, 2001 |
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09316487 |
May 21, 1999 |
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09316487 |
May 21, 1999 |
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08484503 |
Jun 7, 1995 |
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5985610 |
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08484503 |
Jun 7, 1995 |
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08032869 |
Mar 16, 1993 |
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5437951 |
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08032869 |
Mar 16, 1993 |
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07941371 |
Sep 3, 1992 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2710/20034 20130101; Y10S 977/804 20130101; C12N 2710/20022
20130101; C12N 2800/30 20130101; C07K 2319/00 20130101; C12N 7/00
20130101; C12N 2710/20023 20130101; A01K 2227/10 20130101; A61P
35/00 20180101; Y10S 977/795 20130101; Y10S 977/927 20130101; Y10S
435/975 20130101; Y10S 977/918 20130101; A61K 2039/525 20130101;
A61P 31/12 20180101; A61K 2039/5258 20130101; A61P 31/20 20180101;
A61P 17/12 20180101; A61K 39/00 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed is:
1. HPV16 L1 polypeptide, assembled in a capsomeric structure
possessing conformational epitopes, made by expression of a
recombinant coding sequence in a human.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 08/484,503, filed Jun. 7, 1995, which is a
continuation of U.S. patent application Ser. No. 08/032,869, filed
Mar. 16, 1993, now U.S. Pat. No. 5,437,951, which is a
continuation-in-part of U.S. patent application Ser. No.
07/941,371, filed Sep. 3, 1992. These applications are hereby
incorporated by reference as if fully set forth herein. The
invention relates to recombinant viral proteins. It relates
particularly to recombinant viral proteins that are suitable for
use in the diagnosis, prophylaxis and therapy of viral
infections.
[0002] Papillomaviruses infect the epithelia of a wide variety of
species of animals, including humans, generally inducing benign
epithelial and fibro-epithelial tumors, or warts, at the site of
infection. Each species of vertebrate is infected by a distinct
group of papillomaviruses, each papillomavirus group comprising
several papillomavirus types. For example, more than 60 different
human papillomavirus (HPV) genotypes have been isolated.
Papillomaviruses are highly species specific infective agents; for
example, a bovine papillomavirus cannot induce papillomas in a
heterologous species, such as humans. Papillomavirus types ALSO
appear to be highly specific as immunogens in that a neutralizing
immunity to infection against one papillomavirus type does not
usually confer immunity against another type, even when the types
infect an homologous species.
[0003] In humans, genital warts, which are caused by human
papillomaviruses, represent a sexually transmitted disease. Genital
warts are very common, and subclinical, or inapparent HPV infection
is even more common than clinical infection. Some benign lesions in
humans, particularly those arising from certain papillomavirus
types, undergo malignant progression. For that reason, infection by
one of the malignancy associated papilloma virus types is
considered one of the most significant risk factors in the
development of cervical cancer, the second most common cancer of
women worldwide (zur Hausen, H., 1991; Schiffman, M. 1992). Several
different HPV genotypes have been found in cervical cancer, with
HPV16 being the most common type that is isolated from 50% of
cervical cancers.
[0004] Immunological studies demonstrating the production of
neutralizing antibodies to papillomavirus antigens indicate that
papillomavirus infections and malignancies associated with these
infections in vertebrate animals could be prevented through
immunization; however the development of effective papillomavirus
vaccines has been impeded by a number of difficulties.
[0005] First, it has not been possible to generate in vitro the
large stocks of infectious virus required to determine the
structural and immunogenic features of papillomavirus that are
fundamental to the development of effective vaccines. Cultured
cells express papillomavirus oncoproteins and other non-structural
proteins and these have been extensively studied in vitro, but
expression of the structural viral proteins, L1 and L2 (and the
subsequent assembly of infectious virus) occurs only in terminally
differentiated layers of infected epithelial tissues. Therefore,
the characterization of viral genes, proteins, and structure has
necessarily been assembled from studies of virus harvested from
papillomas. In particular, papillomavirus structure and related
immunity have been carried out in the bovine papillomavirus system
because large amounts of infectious virus particles can be isolated
from bovine papillomavirus (BPV) warts.
[0006] The information derived from studies of papillomavirus
structure to date indicates that all papillomaviruses are
non-enveloped 50-60 nm icosahedral structures (Crawford, L., et
al., 1963) which are comprised of conserved L1 major capsid protein
and less well conserved L2 minor capsid protein (Baker, C., 1987).
There is no sequence relationship between the two proteins. The
function and location of L2 in the capsid is unclear; however
immunologic data suggests that most of L2 is internal to L1.
[0007] Recently, high resolution cryoclectron microscopic analysis
of BPV1 and HPV1 virions has determined that the two viruses have a
very similar structure, with 72 pentameric capsomers, each capsomer
presumably composed of five L1 molecules, forming a virion shell
with T=7 symmetry (Baker, T., 1991). The location of the minor L2
capsid protein in the virion has not been determined, and it is not
certain whether L2 or other viral proteins are needed for capsid
assembly. Superficially, papillomavirus structure resembles that of
the polyoma 45 nm virion, which has the same symmetry and capsomere
number (Liddington, R., et al., 1991); however, the systems of
intracapsomer contact for polyomavirus and papillomavirus species
are different, and the major and minor capsid proteins of
polyomavirus are not genetically related to L1 and L2.
[0008] Bovine papillomavirus studies are facilitated by a
quantitative focal transformation infectivity assay developed for
BPV that is not available for HPV (Dvoretzky, I., et al., 1980),
and an understanding of immunity to papillomavirus has therefore
also been derived from the bovine papillomavirus system. Limited
studies using intact bovine papillomavirus demonstrated that the
non-cutaneous inoculation of infectious or formalin-inactivated BPV
virus was effective as a vaccine to prevent experimental BPV
infection in calves (Olson, C., et al., 1960; Jarrett, W., et al.,
1990). Unfortunately, BPV virions cannot be used to develop
vaccines against papillomavirus which infects other species, or
even vaccines against other bovine types, because of the great
specificity of these viruses, as well as concern for the oncogenic
potential of intact viral particles.
[0009] A significant conclusion of studies of papillomavirus
immunity is that the ability of antibodies to neutralize papilloma
virus appears to be related to their ability to react with
type-specific, conformationally dependent epitopes on the virion
surface. For example, rabbit antisera raised against infectious
BPV1 virions inhibits focal transformation of C127 cells (Doretzky,
I., et al., 1980), as well as the transformation of fetal bovine
skin grafts; whereas antisera raised against denatured virions does
not (Ghim, S., et al., 1991).
[0010] In contrast, neutralizing sera generated against bacterially
derived BPV L1 and L2 (Pilacinski, W. et al., 1984; Jin, X., et
al., 1989) and against in vitro synthesized cottontail rabbit
papillomavirus (CRPV) L1 and L2 (Christensen, N., et al., 1991;
Lin, Y-L, et al., 1992), neither of which has the structural
features of native virions, had low titers, and the use of
recombinant HPV L1 fusion peptides expressed in E. coli to detect
cellular immune reactivity has had only limited success (Hopfl, R.
et al., 1991). The results in the BPV system are consistent with
those of the HPV system, in which monoclonal antibodies that
neutralized HPV11 infection in a mouse xenograft assay recognized
native, but not denatured, HPV11 virions (Christensen, N., et al.,
1990).
[0011] There have been isolated attempts to produce papillomavirus
capsids in vitro. Zhou, J. et al. (1991) and (1992) produced
virus-like particles by cloning HPV L1 and L2 genes, and HPV L1 and
L2 genes in combination with HPV E3/E4 genes into a vaccinia virus
vector and infecting CV-1 mammalian cells with the recombinant
vaccinia virus. These studies were interpreted by Zhou to establish
that expression of HPV16 L1 and L2 proteins in epithelial cells is
necessary and sufficient to allow assembly of virion type
particles. Cells infected with doubly recombinant vaccinia virus
which expressed L1 and L2 proteins showed small (40 nm) virus-like
particles in the nucleus that appeared to be incompletely assembled
arrays of HPV capsomers. Expressing L1 protein alone, or L2 protein
alone, was expressed did not produce virus-like particles; cells
doubly infected with singly recombinant vaccinia virus containing
L1 and L2 genes also did not produce particles. No neutralizing
activity was reported.
[0012] Ghim et al., (1992) reported that when L1 from HPV1, a
non-genital virus type associated mainly with warts on the hands
and feet, was expressed in mammalian cells, the L1 protein
contained conformational epitopes found on intact virions. Ghim did
not determine if particles were produced, nor was it evaluated if
the L1 protein might induce neutralizing antibodies. Even more
recently, Hagansee, et al. (1993) reported that when L1 from HPV1
was expressed in human cells, it self-assembled into virus-like
particles. No neutralizing antibody studies were performed.
[0013] Studies in other virus systems, for example, parvovirus,
indicate that capsid assembly alone may not confer immunogenicity.
Parvovirus VP2, by itself, was able to self-assemble when expressed
in insect cells, but only particles containing both VP1 and VP2
were able to induce neutralizing antibodies (Kajigaya, S., et al.,
1991).
[0014] It would be advantageous to develop methods for producing
renewable papillomavirus reagents of any selected species and type
in cell culture. It would also be beneficial to produce such
papillomavirus reagents having the immunity conferring properties
of the conformed native virus particles that could be used as a
subunit vaccine.
[0015] It is therefore the object of the invention to provide these
recombinant conformed papillomavirus proteins, as well as methods
for their production and use.
SUMMARY OF THE INVENTION
[0016] The invention is directed to the diagnosis and prevention of
papillomavirus infections and their benign and malignant sequelae
by providing recombinant papillomavirus capsid proteins that self
assemble to form capsomer structures comprising conformational
epitopes that are highly specific and highly immunogenic.
Therefore, according to the invention there is provided a genetic
construct, comprising a papillomavirus L1 conformational coding
sequence, inserted into a baculovirus transfer vector, and
operatively expressed by a promoter of that vector. The
papillomavirus L1 conformational coding sequence can be isolated
from a bovine, monkey, or human gene. In a preferred embodiment,
the papillomavirus L1 conformational coding sequence is isolated
from a wild type HPV16 gene. In a particularly preferred
embodiment, the papillomavirus L1 conformational coding sequence is
Sequence ID No. 2. The genetic construct can further comprise a
papillomavirus L2 coding sequence.
[0017] According to another aspect of the invention there is
provided a non-mammalian eukaryotic host cell transformed by the
genetic constructs of the invention.
[0018] According to yet another aspect of the invention there is
provided a method for producing a recombinant papillomavirus capsid
protein, assembled into a capsomer structure or a portion thereof,
comprising the steps of (1) cloning a papillomavirus gene that
codes for an L1 conformational capsid protein into a transfer
vector wherein the open reading frame of said gene is under the
control of the promoter of said vector; (2) transferring the
recombinant vector into a host cell, wherein the cloned
papillomavirus gene expresses the papillomavirus capsid protein;
and (3) isolating capsomer structures, comprising the
papillomavirus capsid protein, from the host cell. In a preferred
embodiment, the cloned papillomavirus gene consists essentially of
the conformational L1 coding sequence, and the expressed protein
assembles into capsomer structures consisting essentially of L1
capsid protein. In another preferred embodiment, the cloning step
of the method further comprises the cloning of a papillomavirus
gene coding for L2 capsid protein, whereby said L1 and L2 proteins
are coexpressed in the host cell, and wherein the isolated capsomer
structures comprise L1 and L2 capsid proteins; provided that said
transfer vector is not a vaccinia virus when said host cell is a
mammalian cell. The conformational L1 coding sequence can be cloned
from a bovine, monkey, or human papillomavirus. According to a
preferred embodiment, the conformational L1 coding sequence is
cloned from a wild type HPV16 papillomavirus. In a particularly
preferred embodiment, the conformational L1 coding sequence is
Sequence ID No. 2. Also in a preferred embodiment, the host cell
into which the genetic construct is transfected is an insect cell.
Also preferred are embodiments wherein the transfer vector is a
baculovirus based transfer vector, and the papillomavirus gene is
under the control of a promoter that is active in insect cells.
Accordingly in this embodiment, the recombinant baculovirus DNA is
transfected into Sf-9 insect cells, preferably co-transfected with
wild-type baculovirus DNA into Sf-9 insect cells.
[0019] In an alternative embodiment of the method of the invention,
the transfer vector is a yeast transfer vector, and the recombinant
vector is transfected into yeast cells.
[0020] According to yet another aspect of the invention there is
provided a virus capsomer structure, or a portion thereof,
consisting essentially of papillomavirus L1 capsid protein,
produced by the method the invention. Alternatively, the virus
capsomer structure can consist essentially of papillomavirus L1 and
L2 capsid proteins, produced by the method of the invention. In a
particularly preferred embodiment, the virus capsomer structure
comprises papillomavirus L1 capsid protein that is the expression
product of an HPV16 L1 DNA cloned from a wild type virus.
[0021] The virus capsids or capsomer structures of the invention,
or portions or fragments thereof, can consist essentially of
papillomavirus L1 capsid protein. Alternatively, these capsids or
capsomer structures or their fragments can consist essentially of
wild type HPV16 papillomavirus L1 capsid protein.
[0022] The virus capsid structures according to any of the methods
of the invention comprise capsid proteins having immunogenic
conformational epitopes capable of inducing neutralizing antibodies
against native papillomavirus. The capsid proteins can be bovine,
monkey or human papillomavirus L1 proteins. In a preferred
embodiment, the papillomavirus L1 capsid protein is the expression
product of a wild type HPV16 L1 gene. In a particularly preferred
embodiment, the HPV16 L1 gene comprises the sequence of Sequence ID
No. 2.
[0023] According to yet another aspect of the invention there is
provided a unit dose of a vaccine, comprising a peptide having
conformational epitopes of a papillomavirus L1 capsid protein, or
L1 protein and L2 capsid proteins, in an effective immunogenic
concentration sufficient to induce a papillomavirus neutralizing
antibody titer of at least about 10.sup.3 when administered
according to an immunizing dosage schedule. In a preferred
embodiment, the vaccine comprises an L1 capsid protein which is an
HPV16 capsid protein. In a particularly preferred embodiment, the
vaccine comprises an L1 capsid protein that is a wild type HPV16 L1
protein.
[0024] Use of the L1 open reading frame (ORF) from a wild type
HPV16 papillomavirus genome, according to the methods of the
invention, particularly facilitates the production of preparative
amounts of virus-like particles on a scale suitable for vaccine
use.
[0025] According to yet another aspect of the invention, there is
provided a method of preventing or treating papillomavirus
infection in a vertebrate, comprising the administration of a
papillomavirus capsomer structure or a fragment thereof according
to the invention to a vertebrate, according to an
immunity-producing regimen. In a preferred embodiment, the
papillomavirus capsomer structure comprises wild type HPV16 L1
capsid protein.
[0026] The invention further provides a method of preventing or
treating papillomavirus infection in a vertebrate, comprising the
administration of the papillomavirus capsomer structure of the
invention, or a vaccine product comprising the capsomer structure
to a vertebrate, according to an immunity-producing regimen. In a
preferred embodiment, the papillomavirus vaccine comprises wild
type HPV16 L1 capsid protein.
[0027] Also within the scope of the invention is a method for
immunizing a vertebrate against papillomavirus infection,
comprising administering to the vertebrate a recombinant genetic
construct of the invention comprising a conformational
papillomavirus L1 coding sequence, and allowing said coding
sequence to be expressed in the cells or tissues of said
vertebrate, whereby an effective, neutralizing, immune response to
papillomavirus is induced. In a preferred embodiment, the
conformational papillomavirus L1 coding sequence is derived from
human papillomavirus HPV16. In a particularly preferred embodiment,
the human papillomavirus HPV16 is a wild type papillomavirus.
[0028] According to yet another aspect of the invention, there is
provided a method of detecting humoral immunity to papillomavirus
infection in a vertebrate comprising the steps of: (a) providing an
effective antibody-detecting amount of a papillomavirus capsid
peptide having at least one conformational epitope of a
papillomavirus capsomer structure; (b) contacting the peptide of
step (a) with a sample of bodily fluid from a vertebrate to be
examined for papillomavirus infection, and allowing papillomavirus
antibodies contained in said sample to bind thereto, forming
antigen-antibody complexes; (c) separating said complexes from
unbound substances; (d) contacting the complexes of step (c) with a
detectably labelled immunoglobulin-binding agent; and (e) detecting
anti-papillomavirus antibodies in said sample by means of the
labelled immunoglobulin-binding agent that binds to said complexes.
In a preferred embodiment of this aspect of the invention, the
peptide consists essentially of papillomavirus L1 capsid protein.
According to an alternative embodiment, the peptide consists
essentially of the expression product of a human papillomavirus
HPV16. In a particularly preferred embodiment, the peptide consists
essentially of the expression product of a wild type human
papillomavirus HPV 16 gene, for example, the peptide can consist
essentially of the expression product of Sequence ID No. 2.
[0029] According to yet another aspect of the invention, there is
provided a method of detecting papillomavirus in a specimen from an
animal suspected of being infected with said virus, comprising
contacting the specimen with antibodies having a specificity to one
or more conformational epitopes of the capsid of said
papillomavirus, wherein the antibodies have a detectable signal
producing label, or are attached to a detectably labelled reagent;
allowing the antibodies to bind to the papillomavirus; and
determining the presence of papillomavirus present in the specimen
by means of the detectable label.
[0030] According to yet another aspect of the invention, there is
provided a method of determining a cellular immune response to
papillomavirus in an animal suspected of being infected with the
virus, comprising contacting immunocompetent cells of said animal
with a recombinant wild type papillomavirus L1 capsid protein, or
combined recombinant L1 and L2 capsid proteins according to the
invention; and assessing cellular immunity to papillomavirus by
means of the proliferative response of said cells to the capsid
protein. In a preferred embodiment of this aspect of the invention,
the recombinant papillomavirus protein is introduced into the skin
of the animal.
[0031] According to yet another aspect of the invention there is
provided a papillomavirus infection diagnostic kit, comprising
capsomer structures consisting essentially of papillomavirus L1
capsid protein, or capsomer structures comprising papillomavirus L1
protein and L2 capsid proteins, or antibodies to either of these
capsomer structures, singly or in combination, together with
materials for carrying out an assay for humoral or cellular
immunity against papillomavirus, in a unit package container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows the expression of BPV L1 and HPV16 L1 by means
of recombinant virus as demonstrated by SDS-PAGE analysis of
lysates from infected insect cells.
[0033] FIG. 2 shows the conformation of purified recombinant BPV L1
and HPV16 L1 capsid proteins as demonstrated by electron
microscopy, compared with authentic BPV virions.
[0034] FIG. 3 shows the titers of neutralizing antisera induced in
animals inoculated with recombinant BPV L1 as compared to antisera
against intact and denatured BPV virions.
DETAILED DESCRIPTION OF THE INVENTION
[0035] We have discovered that the gene coding for the L1 major
capsid protein of BPV or HPV16, following introduction into host
cells by means of an appropriate transfer vector, can express L1 at
high levels, and that the recombinant L1 has the intrinsic capacity
to self-assemble into empty capsomer structures that closely
resemble those of an intact virion. Further, the self-assembled
recombinant L1 capsid protein of the invention, in contrast to L1
protein extracted from recombinant bacteria, or denatured virions,
has the efficacy of intact papillomavirus particles in the ability
to induce high levels of neutralizing antiserum that can protect
against papillomavirus infection. The high level of immunogenicity
of the capsid proteins of the invention implies strong antibody
binding properties that make them sensitive agents in serological
screening tests to detect and measure antibodies to conformational
virion epitopes. Their immunogenicity also indicates that the
capsid proteins of the invention can also be used as highly
effective vaccines or immunogens to elicit neutralizing antibodies
to protect a host animal against infection by papillomavirus. These
observations were recently published in Kirnbauer, et al., (1992),
and formed the basis of U.S. application Ser. No. 07/941,371.
[0036] We have now discovered that the capsid protein L1 expressed
by wild type HPV16 genomes isolated from benign papillomavirus
lesions, when expressed in the baculovirus system described, will
self-assemble with an efficiency heretofore unknown and comparable
to that of bovine papillovirus L1 capsid protein.
[0037] The HPV16 L1 Gene Sequences
[0038] The source of HPV16 L1 DNA, as disclosed in published
studies, for example, by Zhou, et al.(1991) was the prototype
clone, GenBank Accession No. K02718, that had been isolated from a
cervical carcinoma (Seedorf, et al., 1985). We have found that L1
from wild type HPV16 genome, which differs from the prototype
genome by a single point mutation, will self-assemble into
virus-like particles with an efficiency similar to that seen with
BPV L1 or BPV L1/L2. Compared with the self-assembly seen when L1
from the prototype HPV genome is used with L2, L1 from a wild-type
genome self-assembles at least 100 times more efficiently.
[0039] To provide genetic insight into the self-assembly efficiency
of different HPV16 L1 expression products, the open reading frames
from HPV16 L1 genes isolated from both benign lesions and lesions
associated with dysplasia or carcinoma were sequenced.
[0040] The analysis detected two errors in the published sequence
of the published L1 sequence of the prototype strain, as
follows:
[0041] (1) there should be an insertion of three nucleotides (ATC)
between nt 6902 and 6903, which results in the insertion of a
serine in the L1 protein; and
[0042] (2) there should be a deletion in the published prototype
sequence of three nucleotides (GAT), consisting of nt 6952-6954,
which deletes an aspartate from the L1 protein sequence. The
corrected nucleotide sequence of the prototype HPV16 L1 genome,
consisting of nt 5637-7155, is that of Sequence ID No. 1, listed
herein.
[0043] The numbering of the nucleotide bases in Sequence ID Nos. 1
and 2 is indexed to 1, and the numbering of nucleotide bases of the
published HPV sequence, that is from nt 5638-7156, corresponds to
those of the sequence listing from 1-1518. The sites referred to in
the original sequence can be thus readily identified by one skilled
in the art.
[0044] Three other HPV16 L1 genomes, clone 16PAT; and clones
114/16/2 and 114/16/11, were sequenced and those sequences compared
to that of the corrected prototype.
[0045] Clone 16PAT, kindly provided by Dennis McCance at the
University of Rochester School of Medicine, and cloned from a
dysplastic (pre-malignant) lesion of the cervix, expresses an L1
that does not self-assemble efficiently.
[0046] Clones 114/16/2 and 114/16/11, kindly provided by Matthias
Durst of the German Cancer Research Center in Heidelburg, were both
cloned from non-malignant lesions, and both expressed L1 protein
that self-assembled efficiently.
[0047] Comparison of Genetic Characteristics of HPV16 L1 associated
with Dysplasia, Malignant Progression and Benign Lesions
[0048] Clone 16PAT, isolated from papillomavirus infected
dysplastic lesions and the prototype HPV16, isolated from malignant
cervical carcinoma, both encode Histidine at nt 6242-6244, while
clones 2 and 11, isolated from benign papillomavirus infected
lesions (like isolates of many other papillomavirus) encode
Aspartate at this site.
[0049] It appears that this single amino acid difference between
the prototype, malignancy-associated HPV16 species, and the HPV16
species from benign lesions accounts for the difference in
self-assembly efficiency. It is likely that among closely related
HPV types, Aspartate at this locus may be necessary for efficient
self-assembly, and that the substitution of Histidine for Aspartate
impairs this ability in the capsid protein. The impairment in
capsid assembly in malignancy-associated viruses, associated with
loss of the conformational epitopes required for the production of
neutralizing antibodies, may also be linked to a lowered
immunogenicity which would allow the papillomavirus to escape
immune control.
[0050] Accordingly, HPV16 L1 genes that express capsid protein that
self-assembles efficiently can be obtained by
[0051] (1) isolation of the wild type HPV16 L1 open reading frame
from benign lesions of papillomavirus infection; or
[0052] (2) carrying out a site specific mutation in the prototype
sequence at nt 6242-6244 to encode Aspartate.
[0053] Recombinant Capsid Protein
[0054] The method of the invention provides a means of preparing
recombinant capsid particles for any papillomavirus. Particles
consisting of either L1 or L2 capsid protein alone, or consisting
of both L1 and L2 capsid proteins together can be prepared. L1/L2
capsid protein particles are more closely related to the
composition of native papillomavirus virions, but L2 does not
appear to be as significant as L1 in conferring immunity, probably
because most of L2 is internal to L1 in the capsid structure.
Although L1 can self-assemble by itself, in the absence of L2,
self-assembled L1/L2 capsid protein particles are more closely
related to the composition of native papillomavirus virions.
Accordingly, particles comprising L1 alone are simpler, while those
comprising L1/L2 may have an even more authentic structure. Both
self-assembled L1 and L1/L2 particles induce high-titer
neutralizing antibodies and may therefore be suitable for vaccine
production. Particles comprising L1 capsid protein expressed by a
wild type HPV genome, either as L1 alone or L1/L2 together, are
particularly preferred.
[0055] Production of the recombinant L1, or combined L1/L2, capsid
particles is carried out by cloning the L1 (or L1 and L2) gene(s)
into a suitable vector and expressing the corresponding
conformational coding sequences for these proteins in a eukaryotic
cell transformed by the vector. It is believed that the ability to
form a capsid-like structure is intimately related to the ability
of the capsid protein to generate high-titer neutralizing antibody,
and that in order to produce a capsid protein that is capable of
self-assembling into capsid structures having conformational
epitopes, substantially all of the capsid protein coding sequence
must be expressed. Accordingly, substantially all of the capsid
protein coding sequence is cloned. The gene is preferably expressed
in a eukaryotic cell system. Insect cells are preferred host cells;
however, yeast cells are also suitable as host cells if appropriate
yeast expression vectors are used. Mammalian cells similarly
transfected using appropriate mammalian expression vectors can also
be used to produce assembled capsid protein, however, cultured
mammalian cells are less advantageous because they are more likely
than non-mammalian cells to harbor occult viruses which might be
infectious for mammals.
[0056] According to a preferred protocol, a baculovirus system is
used. The gene to be cloned, substantially all of the coding
sequence for bovine papillomavirus (BPV1) or human papillomavirus
(HPV16) L1 capsid protein, or human papillomavirus HPV16 L1 and L2,
is inserted into a baculovirus transfer vector containing flanking
baculovirus sequences to form a gene construct, and the recombinant
DNA is co-transfected with wild type baculovirus DNA into Sf-9
insect cells as described in Example 1, to generate recombinant
virus which, on infection, can express the inserted gene at high
levels. The actual production of protein is made by infecting fresh
insect cells with the recombinant baculovirus; accordingly, the L1
capsid protein and the L1 and L2 capsid proteins are expressed in
insect cells that have been infected with recombinant baculovirus
as described in Example 2.
[0057] In the procedure of Example 1, the complete L1 gene of BPV1
was amplified by polymerase chain reaction (PCR; Saiki, R., et al.,
1987) and cloned into AcMNPV (Autographa californica nuclear
polyhedrosis virus) based baculovirus vector (Summers, M. et al.,
1987). The L1 open reading frame was put under the control of the
baculovirus polyhedrin promoter. After co-transfection of the L1
clone with the wild type (wt) baculovirus DNA into Sf-9 insect
cells (ATCC Accession No. CRL 1711) and plaque purification of
recombinant clones, high titer recombinant virus was generated.
Extracts from cells infected with wt AcMNPV or BPV1 L1 recombinant
viruses (AcBPV-L1) (Example 2) were analyzed by polyacrylamide gel
electrophoresis. After Coomassie blue staining, a unique protein of
the predicted size, 55 kilodaltons, was detected in extracts from
the cultures infected with the AcBPV1-L1 virus (FIG. 1A). The
identity of this protein as BPV L1 was verified by immunoblotting
(FIG. 1B), using a BPV L1 specific monoclonal antibody (Nakai, Y.,
et al., 1986).
[0058] To test the hypothesis that papillomavirus L1 has the
ability to self-assemble into virus-like particles when
overexpressed in heterologous cells, electron micrographs of thin
sections from AcBPV-L1 infected cells were examined for the
presence of papillomavirus-like structures. Cells infected with the
BPV recombinant virus contained many circular structures of
approximately 50 nm which were preferentially localized in the
nucleus; these structures were absent from wild type baculovirus
infected cells. These results suggested that self assembly of L1
into virus-like particles had occurred, since in vivo
papillomavirus virion assembly takes place in the nucleus and the
diameter of the virions has been reported as 55 nm.
[0059] Following expression of the conformed capsid protein in the
host cell, virus particles are purified from lysates of infected
cells as described in Example 4. To obtain further evidence that
the L1 protein had self-assembled, virus-like particles were
isolated from the infected insect cells by means of gradient
centrifugation (FIG. 2).
[0060] High molecular mass structures were separated from lysates
of L1 recombinant or wild type infected cells by centrifugation
through a 40% sucrose cushion and the pelleted material was
subjected to CsCl density gradient centrifugation. Fractions were
collected and tested for reactivity to the BPV L1 specific
monoclonal antibody by immunoblotting.
[0061] L1 positive fractions from the gradient were adsorbed onto
carbon film grids, stained with 1% uranyl acetate and examined by
transmission electron microscopy. The positive fractions contained
numerous circular structures that exhibited a regular array of
capsomers (FIG. 2A). Consistent with previous reports of the
density of empty BPV virions (Larsen, P., et al., 1987), the
density of the CsCl fraction containing the peak of the virus-like
particles was approximately 1.30 gm/ml. Most were approximately 50
nm in diameter, although smaller circles and partially assembled
structures were also seen. The larger particles were very similar
in size and subunit structure to infectious BPV virions that had
been stained and photographed concurrently (FIG. 2B). These
particles were not observed in preparations from mock infected or
wt AcMNPV infected cells. These results indicate that BPV L1 has
the intrinsic capacity to assemble into virus-like particles in the
absence of L2 or other papillomavirus proteins. In addition,
specific factors limited to differentiating epithelia or mammalian
cells are not required for papillomavirus capsid assembly.
[0062] To determine if the ability to self-assemble in insect cells
is a general feature of papillomavirus L1, we also expressed the L1
of HPV16, the HPV type most often detected in human genital
cancers, via an analogous recombinant baculovirus. A protein of the
expected 58 kd size was expressed at high levels in the insect
cells infected with the HPV16-L1 recombinant virus (FIG. 1A) and it
reacted strongly with an HPV16 L1 monoclonal antibody (which also
reacted weakly with BPV L1; FIG. 1C). After CsCl gradient
purification, immunoreactive fractions were examined by electron
microscopy and found to contain 50 nm papillomavirus-like particles
(FIG. 2C). Although somewhat fewer completely assembled particles
were seen in the human system in comparison to the BPV L1
preparations, possibly due to the lower levels of expression or
greater extent of HPV16 L1 degradation (FIG. 1), the results
conclusively indicate that the L1 of the HPV16 and presumably the
L1 proteins of other types, have the intrinsic capacity to assemble
into virion-type structures. Preparations of recombinant
papillomavirus capsid particles for Rhesus monkey PV have also been
carried out as described in the Examples.
[0063] Recombinant Conformed Capsid Proteins as Immunogens
[0064] Subunit vaccines, based on self-assembled major capsid
proteins synthesized in heterologous cells, have been proved
effective in preventing infections by several pathogenic viruses,
including human hepatitis B (Stevens, C., et al., 1987).
[0065] Studies demonstrating that infectious or formalin
inactivated BPV is effective as a vaccine, while BPV transformed
cells are ineffective, suggest that viral capsid proteins, rather
than early gene products, elicit the immune response. Other data in
the scientific literature indicates that L1 protein extracted from
bacteria was partially successful in eliciting an immune response
despite the low titers of neutralizing antibodies. Accordingly, the
BPV L1 that was expressed and assembled into virus-like particles
in insect cells was studied for its ability to induce neutralizing
antisera in rabbits. Two types of preparations were tested: whole
cell extracts of L1 recombinant or wild type infected Sf-9 cells
and partially purified particles isolated by differential
centrifugation and ammonium sulfate precipitation. Following a
primary inoculation, the rabbits received two biweekly booster
inoculations.
[0066] The rabbit sera were tested for the ability to inhibit BPV
infection of mouse C127 cells, as measured by a reduction in the
number of foci induced by a standard amount of BPV virus (a
representative assay is shown in FIG. 3). The immune sera generated
by inoculation with baculovirus derived L1 were able to reduce the
infectivity of the BPV virus by 50% at a dilution of at least
1:11,000 (a titer of 11,000; Table 1), whereas the preimmune sera
from the same rabbits did not inhibit focal transformation at a
dilution of 1:20, the lowest dilution tested. Both the crude
preparations and partially purified particles were effective in
inducing high titer neutralizing antisera, with 290,000 being the
highest titer measured. This was the same as the neutralizing titer
of the positive control antiserum raised against infectious BPV
virions. In comparison, the highest titer generated in a previous
study using bacterially derived L1 was 36 (Pilancinski, W., et al.,
1984). The serum from the rabbit inoculated with the extract from
the wild type baculovirus infected cells was unable to inhibit
infectivity at a dilution of 1:20, indicating that the neutralizing
activity was L1 specific. Disruption of the partially purified L1
particles, by boiling in 1% SDS, abolished the ability of the
preparation to induce neutralizing antibodies (Table 1). The
demonstration that L1 can self-assemble into virion-like particles
that elicit neutralizing antisera titers at least three orders of
magnitude higher than previous in vitro-produced antigens suggests
the recombinant L1 capsid proteins has the potential to induce
effective long term protection against naturally transmitted
papillomavirus. In view of these results, it appears that the L1
particles assembled in insect cells mimic infectious virus in the
presentation of conformationally dependent immunodominant epitopes.
These results also establish that L2 is not required for the
generation of high titer neutralizing antibodies. The reported weak
neutralizing immunogenicity of bacterially derived L1 may occur
because it does not assume an appropriate conformation or has not
assembled into virion like structures. Also, multiple
electrophoretic variants of L1 have been detected in virions
(Larsen, P., et al., 1987). Some of these modified species, which
are probably absent in the bacterially derived L1, may facilitate
the generation of neutralizing antibodies.
[0067] The ability of recombinant L1 (or L2) papillomavirus capsid
proteins such as those disclosed herein to induce high titer
neutralizing antiserum makes them suitable for use as vaccines for
prophylaxis against communicable papillomatosis. Examples of
populations at risk that could benefit from immunization are bovine
herds, which are susceptible to papilloma warts; all humans for
non-genital types of HPV infection; and sexually active humans for
genital HPV types of infection.
[0068] Therapeutic vaccination can be useful for productive
papillomavirus lesions, which usually express L1 (and L2) capsid
proteins. Such lesions are most likely to occur in benign
infections, such as warts or laryngeal papillomatosis. Laryngeal
papillomatosis in newborns is usually contracted by the infant
during passage through the birth canal where infectious
papillomavirus is present in vaginal secretions. Therapeutic
vaccination of infected pregnant women against the papillomavirus
can induce neutralizing IgG antibody capable of passing through the
placental barrier and into the circulation of the fetus to provide
prophylactic passive immunity in the infant against this type of
papillomavirus infection. Additional infant-protecting mechanisms
are provided by maternal IgA which is secreted into the vaginal
fluid and into breast milk. Jarrett (1991) demonstrates some
therapeutic efficacy for L2 in treating BPV-induced warts.
Malignant tumors typically do not express L1 or L2, and the
efficacy of vaccination with recombinant L1 or L2 in conditions
such as cervical cancer, is uncertain.
[0069] Protective immunity against both benign and malignant
papillomavirus disease can be induced by administering an effective
amount of recombinant L1 capsid protein to an individual at risk
for papillomavirus infection. A vaccine comprising the capsid
protein can be directly administered, either parenterally or
locally, according to conventional immunization protocols. In an
alternative embodiment, the conformational coding sequence of L1
can be cloned into a transfer vector, for example, a semliki forest
virus vector (which produces a mild transient infection), the
recombinant virus introduced into the cells or tissues of the
recipient where the immunizing capsid protein is then expressed.
Vaccinia virus can also be used as a vehicle for the gene.
[0070] Recombinant Conformed Capsid Proteins as Serological
Screening Agents
[0071] Published serologic studies of human immune response to
papillomavirus virion proteins have principally utilized
bacterially derived L1 and L2 capsid proteins, and the results have
not correlated well with other measures of HPV infection (Jenison,
S., et al., 1990). BPV papillomavirus immunity studies described
above indicate that papilloma-virus virion proteins extracted from
bacteria do not present the conformationally dependent epitopes
that appear to be type-specific and recognized by most neutralizing
antibodies. Compared with such assays that primarily recognize
linear epitopes, a serological test using self-assembled L1
particles is likely to be a more accurate measure of the extent of
anti-HPV virion immunity in the human population. The recombinant
L1 capsid proteins disclosed herein, presenting conformational
epitopes, can therefore be used as highly specific diagnostic
reagents to detect immunity conferring neutralizing antibody to
papilloma virus in binding assays of several types. The procedures
can be carried out generally as either solid phase or solution
assays that provide a means to detect antibodies in bodily fluids
that specifically bind to the capsid protein in antigen-antibody
pairs. Examples of procedures known to those skilled in the art for
evaluating circulating antibodies are solution phase assays, such
as double-antibody radioimmunoassays or enzyme immunoassays, or
solid phase assays such as strip radioimmunoassay based on Western
blotting or an enzyme-linked immunoabsorbent assay (ELISA) as
disclosed in U.S. Pat. No. 4,520,113 to Gallo et al., or
immunochromatographic assays as disclosed in U.S. Pat. No.
5,039,607 to Skold et al. A preferred ELISA method for the
detection of antibodies is that disclosed in Harlow, E., and Lane,
D. in Antibodies: A Laboratory Manual Cold Spring Harbor, N.Y.,
1988, pp. 563-578.
[0072] The recombinant L1 or L1/L2 capsid proteins disclosed herein
can also be used to measure cellular immunity to papillomavirus by
means of in vivo or in vitro assays, for example, antigen-induced
T-cell proliferative responses as described by Bradley, L., 1980,
and particularly cellular responses to viral antigens, as described
in U.S. Pat. No. 5,081,029 to Starling. Cellular immunity to
papillomavirus can also be determined by the classical in vivo
delayed hypersensitivity skin test as described by Stites, D.,
1980; or in a preferred method, according to Hopfl, R., et al.,
1991, by the intradermal injection of recombinant HPV L1 fusion
proteins.
[0073] The capsid proteins of the invention can also be used as
immunogens to raise polyclonal or monoclonal antibodies, according
to methods well known in the art. These papillomavirus-specific
antibodies, particularly in combination with labelled second
antibodies, specific for a class or species of antibodies, can be
used diagnostically according to various conventional assay
procedures, such as immunohistochemistry, to detect the presence of
capsid proteins in samples of body tissue or bodily fluids.
[0074] The genetic manipulations described below are disclosed in
terms of their general application to the preparation of elements
of the genetic regulatory unit of the invention. Occasionally, the
procedure may not be applicable as described to each recombinant
molecule included within the disclosed scope. The situations for
which this occurs will be readily recognized by those skilled in
the art. In all such cases, either the operations can be
successfully performed by conventional modifications known to those
skilled in the art, e.g. by choice of an appropriate alternative
restriction enzyme, by changing to alternative conventional
reagents, or by routine modification of reaction conditions.
Alternatively, other procedures disclosed herein or otherwise
conventional will be applicable to the preparation of the
corresponding recombinant molecules of the invention. In all
preparative methods, all starting materials are known or readily
preparable from known starting materials. In the following
examples, all temperatures are set forth in degrees Celsius; unless
otherwise indicated, all parts and percentages are by weight.
[0075] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
invention to its fullest extent. The following preferred
embodiments are therefore to be construed as merely illustrative
and not limiting the remainder of the disclosure in any way
whatsoever.
EXAMPLE 1
[0076] Full length L1, or L1 and L2 open reading frames (ORF) were
amplified by PCR using the cloned prototypes of BPV1 DNA (Chen, E.,
et al., 1982), GenBank Accession No. X02346 or HPV16 DNA (Seedorf,
K., et al., 1985), GenBank Accession No. K02718; or wild type HPV16
DNA (Sequence ID No. 2) as templates. Unique restriction sites were
incorporated into the oligonucleotide primers (underlined).
[0077] BPV1-L1 primer sequence (Sequence ID No. 3):
[0078] 5'-CCGCTGAATTCAATATGGCGTTGTGGCAACAAGGCCAGAAGCTGTAT-3'
(sense) and (Sequence ID No. 4):
[0079] 5'-GCGGTGGTACCGTGCAGTTGACTTACCTTCTGTTTTACATTTACAGA-3'
(antisense);
[0080] HPV16-L1 primer sequence (Sequence ID No. 5):
[0081] 5'-CCGCTAGATCTAATATGTCTCTTTGGCTGCCTAGTGAGGCC-3' (sense); and
(Sequence ID No. 6):
[0082] 5'-GCGGTAGATCTACACTAATTCAACATACATACAATACTTACAGC-3'
(antisense).
[0083] L1 coding sequences begin at the 1st methionine codon (bold)
for BPV1 and the 2nd methionine for HPV16. BPV1-L1 was cloned as a
5'-EcoRI to 3'-KpnI fragment and HPV16-L1 as a 5'-BglII to 3'-BglII
fragment into the multiple cloning site downstream of the
polyhedrin promoter of the AcMNPV based baculovirus transfer vector
pEV mod (Wang, X., et al. 1991) and verified by sequencing through
the AcMNPV/L1 junction. A quantity of 2 .mu.g of CsCl-purified
recombinant plasmid was cotransfected with 1 .mu.g wild type AcMNPV
DNA (Invitrogen, San Diego, Calif.) into Sf-9 cells (ATCC) using
lipofectin (Gibco/BRL, Gaithersburg, Md.) (Hartig, P., et al.,
1991) and the recombinant baculoviruses plaque-purified as
described (Summers, M., et al., 1987).
EXAMPLE 2
Expression of L1 Proteins or L1/L2 Proteins in Insect Cells
[0084] Sf-9 cells were either mock infected (mock) or infected at a
multiplicity of infection of 10 with either wt AcMNPV (wt) or
AcBPV-L1 (B-L1), AcHPV16-L1 (16-L1), or AcHPV16-L1 (16-L1) and
AcHPV16-L2 (16-L2) recombinant virus. After 72 hours, cells were
lysed by boiling in Laemmli buffer and the lysates subjected to
SDS-PAGE in 10% gels. Proteins were either stained with 0.25%
Coomassie blue (FIG. 1A) or immunoblotted and probed with BPV L1
mAb AU-1 (Nakai, Y., et al., 1986)(FIG. 1B) or HPV16L1 mAb CAMVIR-1
(McLean, C., et al., 1990)(FIG. 1C) and .sup.125I-labeled Fab
anti-mouse IgG (Amersham). P designates polyhedrin protein.
EXAMPLE 3
Production of Antisera
[0085] Rabbits were immunized by subcutaneous injection either with
whole cell Sf-9 lysates (3.times.10.sup.7 cells) prepared by one
freeze/thaw cycle and 20.times. dounce homogenization (rabbit #1,2,
and 8) or with 200 .mu.g of L1 protein partially purified by
differential centrifugation and 35% ammonium sulfate precipitation
(#3,4,6, and 7), in complete Freund's adjuvant, and then boosted
twice at two week intervals, using the same preparations in
incomplete Freund's adjuvant.
EXAMPLE 4
Purification of Particles and Transmission Electron Microscopic
(EMK) Analysis
[0086] 500 ml of Sf-9 cells (2.times.10.sup.6/ml) were infected
with AcBPV-L1 (FIG. 2A) or AcHPV16-L1 (FIG. 2C) or or AcHPV16-L1/L2
(16-L1/L2) recombinant baculoviruses. After 72 hr, the harvested
cells were sonicated in PBS for 60 sec. After low speed
clarification, the lysates were subjected to centrifugation at
110,000 g for 2.5 hr through a 40% (wt/vol) sucrose/PBS cushion
(SW-28). The resuspended pellets were centrifuged to equilibrium at
141,000 g for 20 hr at room temperature in a 10-40% (wt/wt)
CsCl/PBS gradient. Fractions were harvested from the bottom and
analyzed by SDS-PAGE. Immunoreactive fractions were dialyzed
against PBS, concentrated by Centricon 30 (Millipore)
ultrafiltration, and (for HPV16-L1) pelleted by centrifugation for
10 min at 30 psi in a A-100 rotor in an airfuge (Beckman). BPV1
virions (FIG. 2B) were purified from a bovine wart (generously
provided by A. B. Jenson) as described (Cowsert, L., et al., 1987).
Purified particles were adsorbed to carbon coated TEM grids,
stained with 1% uranyl acetate and examined with a Philips electron
microscope EM 400T at 36,000.times. magnification. Results are
shown in FIG. 2. [The bar=50 nm].
EXAMPLE 5
BPV1 Neutralization Assay
[0087] Serial dilutions of sera obtained 3 wk after the second
boost were incubated with approximately 500 focus forming units of
BPV1 virus for 30 min, the virus absorbed to C127 cells for 1 hr
and the cells cultured for 3 weeks (Dvoretzky, I., et al., 1980).
The foci were stained with 0.5% methylene blue/0.25% carbol
fuchsin/methanol. The results are shown in FIG. 3 and are discussed
below. The antisera and dilutions used are indicated below the
plates. Anti-AcBPV-L1 was obtained from rabbit #1 and anti-wt
AcMNPV from rabbit #8 (Table 1). The normal rabbit serum negative
control is designated "nrs"; anti-BPV-1 virion was raised against
native BPV virions in a previous study (Nakai, Y., et al., 1986);
and Dako is the commercially available (Dako Corp., Santa Barbara,
Calif.) rabbit antiserum raised against denatured BPV virions.
EXAMPLE 6
Serum Neutralizing Titer against BPV1
[0088] Assays were carried out as in Example 5. Rabbits #1, 2, and
8 were inoculated with crude whole cell Sf-9 lysates, and rabbits
#3,4,6, and 7 with partially purified L1 protein (Table 1). Rabbits
#6 and 7 were immunized with L1 protein preparations that had been
denatured by boiling in 1% SDS. At least two bleeds, taken 3-6
weeks after the second boost, were tested for each rabbit and found
to have the same titer. The titer of the preimmune sera from each
of the rabbits was less than 20, the lowest dilution tested.
1TABLE 1 serum neutralization titer Antigen rabbit against BPV1*
AcBPV-L1 1 11,000 " 2 97,000 " 3 290,000 " 4 97,000
BPV1-virions.dagger. 5 290,000 AcBPV-L1/SDS 6 <2 " 7 <2 wt
AcMNPV 8 <20 *reciprocal of dilution that caused 50% focus
reduction .dagger. provided by A. B. Jenson (Nakai, Y., et al.,
1986).
[0089] The invention may be embodied in other specific forms
without departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive, and the scope of the invention
is, therefore, indicated by the appended claims rather than by the
foregoing description. All modifications which come within the
meaning and range of the lawful equivalency of the claims are to be
embraced within their scope.
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Sequence CWU 1
1
* * * * *