U.S. patent application number 10/367367 was filed with the patent office on 2004-06-24 for optimization of gene sequences of virus-like particles for expression in insect cells.
This patent application is currently assigned to Novavax, Inc.. Invention is credited to Robinson, Robin A..
Application Number | 20040121465 10/367367 |
Document ID | / |
Family ID | 27739582 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121465 |
Kind Code |
A1 |
Robinson, Robin A. |
June 24, 2004 |
Optimization of gene sequences of virus-like particles for
expression in insect cells
Abstract
Codon optimized polynucleotides for optimal expression of
recombinant proteins in eukaryotic cells are provided. The codon
optimized polynucleotides encode a viral capsid protein that self
assembles into a virus-like particle. The virus-like particle is
expressed extracellularly and exhibits conformational antigenic
epitopes capable of raising neutralizing antibodies. Pharmaceutical
compositions, vaccines, and diagnostic test kits containing the
gene products of the codon-optimized polynucleotides are also
provided.
Inventors: |
Robinson, Robin A.;
(Dickerson, MD) |
Correspondence
Address: |
Ralph A. Loren
LAHIVE & COCKFIELD, LLP
28 State Street
Boston
MA
02109
US
|
Assignee: |
Novavax, Inc.
Columbia
MD
21046
|
Family ID: |
27739582 |
Appl. No.: |
10/367367 |
Filed: |
February 14, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60356119 |
Feb 14, 2002 |
|
|
|
60356161 |
Feb 14, 2002 |
|
|
|
60356118 |
Feb 14, 2002 |
|
|
|
60356133 |
Feb 14, 2002 |
|
|
|
60356157 |
Feb 14, 2002 |
|
|
|
60356156 |
Feb 14, 2002 |
|
|
|
60356123 |
Feb 14, 2002 |
|
|
|
60356113 |
Feb 14, 2002 |
|
|
|
60356154 |
Feb 14, 2002 |
|
|
|
60356135 |
Feb 14, 2002 |
|
|
|
60356126 |
Feb 14, 2002 |
|
|
|
60356162 |
Feb 14, 2002 |
|
|
|
60356150 |
Feb 14, 2002 |
|
|
|
60356151 |
Feb 14, 2002 |
|
|
|
60356152 |
Feb 14, 2002 |
|
|
|
Current U.S.
Class: |
435/456 ;
435/235.1; 435/348 |
Current CPC
Class: |
C12N 2510/02 20130101;
C12N 7/00 20130101; C12N 2720/12322 20130101; A61K 2039/5258
20130101; C12N 2710/14143 20130101; C12N 2710/20023 20130101; C07K
14/005 20130101; C12N 2710/20022 20130101 |
Class at
Publication: |
435/456 ;
435/235.1; 435/348 |
International
Class: |
C12N 015/86; C12N
007/01; C12N 005/06 |
Claims
What is claimed is:
1. A codon optimized polynucleotide encoding a viral capsid protein
that self assembles into a virus-like particle that exhibits
conformational antigenic epitopes capable of raising neutralizing
antibodies, wherein the virus-like particle is expressed from a
host cell extracellularly.
2. The codon optimized polynucleotide of claim 1 comprising at
least one of the following characteristics: (a) an increased number
of nucleotide sequences that are utilized at high levels in insect
cells, (b) a ratio of GC nucleotide pairs to AT nucleotide pairs of
approximately 1:1, (c) a minimum number of palindromic and
stem-loop DNA structures, and (d) a minimum number of transcription
and post-transcription repressor elements.
3. The codon optimized polynucleotide of claim 1 comprising a
polynucleotide from an enveloped virus, or a non-enveloped
virus.
4. The codon optimized polynucleotide of claim 3, wherein the
enveloped virus and the non-enveloped virus comprise rotavirus,
calicivirus, hepatitis E virus, papillomavirus, influenza virus,
hepatitis C virus, or retrovirus.
5. The codon optimized polynucleotide of claim 4, wherein the
papillomavirus is a human papillomavirus.
6. The codon optimized polynucleotide of claim 5, wherein the human
papillomavirus comprises genotypes HPV-16, HPV-18, HPV-45, HPV-31,
HPV-33, HPV-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV-42, HPV-43,
HPV-44, or a combination thereof,
7. The codon optimized polynucleotide of claim 5, wherein the human
papillomavirus comprises SEQ ID No. 1, 6, or a polynucleotide
having a sequence that is substantially homologous to SEQ ID No.
1.
8. A vector comprising the codon optimized polynucleotide of claim
1 operatively linked to an eukaryotic or a prokaryotic regulatory
control element, capable of replication in prokaryotic host,
eukaryotic host, or both.
9. The vector of claim 8, wherein the vector is a baculovirus
vector.
10. A host cell comprising the vector of claim 8.
11. A pharmaceutical composition for treating, ameliorating, or
preventing a papillomavirus related disease or disorder comprising
a multiplicity of virus-like particles that exhibit conformational
antigenic epitopes, wherein the virus-like particles are expressed
from a host cell extracellularly, and an acceptable carrier or
diluent.
12. The pharmaceutical composition of claim 11, wherein the
papillomavirus comprises a human papillomavirus.
13. The pharmaceutical composition of claim 12, wherein the human
papillomavirus comprises genotypes HPV-16, HPV-18, HPV-45, HPV-31,
HPV-33, HPV-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV-42, HPV-43,
HPV-44, or a combination thereof,
14. The pharmaceutical composition of claim 11, wherein the
virus-like particles comprise HPV L1 VLPs.
15. A pharmaceutical composition comprising: a) a polypeptide which
is encoded by a polynucleotide molecule comprising SEQ ID No. 1, or
a polynucleotide molecule having a sequence that is substantially
homologous to SEQ ID No. 1; b) a polynucleotide molecule comprising
SEQ ID No. 1, or a polynucleotide having a sequence that is
substantially homologous to SEQ ID No. 1; c) a vector carrying a
polynucloetide molecule comprising SEQ ID No. 1, or a
polynucleotide molecule having a sequence that is substantially
homologous to SEQ ID No. 1; d) a host cell genetically transformed
with a polynucloetide molecule comprising SEQ ID No. 1, or a
polynucleotide molecule having a sequence that is substantially
homologous to SEQ ID No. 1; and a pharmaceutically acceptable
carrier or diluent.
16. A vaccine composition to induce immunity against a
papillomavirus infection in humans comprising a multiplicity of
virus-like particles that exhibit conformational antigenic
epitopes, wherein the virus-like particles are expressed from a
host cell extracellularly, and an adjuvant.
17. The vaccine composition of claim 15, wherein the immunity is
humoral immunity, cell mediated immunity, or both.
18. The vaccine composition of claim 16, wherein the papillomavirus
comprises human papillomavirus genotypes HPV-16, HPV-18, HPV-45,
HPV-31, HPV-33, HPV-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV-42,
HPV-43, HPV-44, or a combination thereof,
19. The vaccine composition of claim 16 comprising a monovalent or
a multivalent formulation.
20. The vaccine composition of claim 16, wherein the virus-like
particles comprise HPV L1 VLPs.
21. A vaccine composition comprising: a) a polypeptide which is
encoded by a polynucleotide molecule comprising SEQ ID No. 1, or a
polynucleotide molecule having a sequence that is substantially
homologous to SEQ ID No. 1; b) a polynucleotide molecule comprising
SEQ ID No. 1, or a polynucleotide having a sequence that is
substantially homologous to SEQ ID No. 1; c) a vector carrying a
polynucleotide molecule comprising SEQ ID No. 1, or a
polynucleotide molecule having a sequence that is substantially
homologous to SEQ ID No. 1; or d) a host cell genetically
transformed with a polynucleotide molecule comprising SEQ ID No. 1,
or a polynucleotide molecule having a sequence that is
substantially homologous to SEQ ID No. 1; and an adjuvant.
22. A diagnostic test kit for detection of papillomavirus infection
comprising a multiplicity of virus-like particles that exhibit
conformational antigenic epitopes, wherein the virus-like particles
are expressed from a host cell extracellularly, and a detection
agent comprising a detectable label.
23. The diagnostic test kit of claim 22, wherein the papillomavirus
infection is caused by one or more human papillomavirus
genotypes.
24. The diagnostic test kit of claim 22, wherein the virus-like
particles comprise HPV L1 VLPs.
25. A method for preparing a codon optimized polynucleotide
comprising one or more of the following steps: (a) replacing codons
that are underutilized in insect cells with codons that are
utilized at high levels in insect cells, to create an
initially-modified nucleotide sequence; (b) modifying the
initially-modified nucleotide sequence by choosing a preferred
codon for the initially modified sequence, wherein: (i) the ratio
of GC nucleotide pairs to AT nucleotide pairs in the
further-modified nucleotide sequence trends toward approximately
1:1; (ii) the number of palindromic and stem-loop DNA structures in
the further-modified nucleotide sequence is minimized; and (iii)
the number of transcription and post-transcription repressor
elements are minimized.
26. The method of claim 25, wherein the polynucleotide comprises a
human papillomavirus gene.
27. A method of treating, ameliorating, or preventing a
papillomavirus related disease or disorder comprising,
administering to an individual in need thereof an effective amount
of the pharmaceutical composition of claim 11.
28. The method of claim 27 wherein the papillomavirus infection is
caused by one or more human papillomavirus genotypes.
29. A method of protecting an individual against a papillomavirus
infection comprising, administering to the individual a
prophylactically effective amount of the vaccine of claim 16.
30. The method of claim 29, wherein the papillomavirus infection is
caused by one or more human papillomavirus genotypes.
Description
[0001] This application claims benefit under 37 U.S.C. .sctn.
119(e) based on U.S. Provisional Application Nos. 60/356,119,
60/356,161, 60/356,118, 60/356,133, 60/356,157, 60/356,156,
60/356,123, 60/356,113, 60/356,154, 60/356,135, 60/356,126,
60/356,162, 60/356,150, 60/356,151, and 60/356,152, each filed Feb.
14, 2002, the entire contents of each of which are incorporated
herein by reference.
I. FIELD OF THE INVENTION
[0002] The present invention relates to the field of viral
vaccines, therapeutics, and diagnostics, compositions and methods
for the detection, protection and treatment of human papillomavirus
(HPV) infections and associated dysplasia. In particular, the
invention relates to novel polynucleotide molecules encoding
recombinant HPV gene products having increased antigenicity and
immunogenicy in mammals.
II. BACKGROUND OF THE INVENTION
[0003] Cervical cancer results in over 200,000 deaths per year
worldwide (Parkin et al., 1990; Pisani et al., 1990). The greatest
burden of disease is in developing countries, where cervical cancer
is the most frequent female malignancy and comprises 25% of all
female cancers. Cervical dysplasia makes up 7% of all female
cancers and causes greater than 5000 deaths per year in the U.S.
(Shah and Howley, 1996). Through clinical studies, epidemiologists
have identified human papillomavirus (HPV) as the major cause of
cervical cancer and cervical dysplasia. (Walboomers et al., 1999).
On a worldwide basis, most cervical cancers contain the genes of
"high-risk" HPVs (genotypes 16, 18, 31, and 45) (Bosch et al.,
1995; Walboomers et al., 1999). The nucleotide sequences of human
and animal papillomavirus genomes are accessible in GenBank.
[0004] HPV-16 is found in approximately 50% of cervical cancers,
and HPV-18, HPV-31, and HPV-45 account for an additional 25-30% of
HPV-positive tumors. Though early detection of HPV-induced cervical
neoplasia is possible with Papanicolau (PAP) smears and
cervicoscopy, screening programs in developing countries are only
now emerging. In the United States, where the widespread
availability of PAP screening and other methods have been
associated with a reduction in the incidence of cervical cancer,
the annual economic loss in the U.S. is still estimated at $5
billion (Kirnbauer et al., 1993). Effective HPV vaccines would
reduce the prevalence of worldwide cervical cancer and reduce the
cost of screening and treating premalignant cervical disease.
[0005] Prophylactic viral vaccines that efficiently prevent
infection or modify disease have a successful record as
cost-effective approaches to prevent and manage viral diseases.
Human papillomaviruses are DNA tumor viruses that encode several
viral oncogenes. Two of these viral oncogenes, E6 and E7, are
conserved and expressed in human genital warts, dysplasia, and
tumors, and may be required for maintenance of the tumorigenic
phenotype. These features raise theoretical arguments against a HPV
E6 and/or E7 subunit protein or DNA vaccine consisting of these
viral proteins alone. Wild type and intact versions of these viral
genes and their gene products in the context of a vaccine may
disrupt normal host cell gene regulation by increasing the levels
of Rb and p53 proteins and facilitate cell transformation. Subunit
protein viral vaccines utilizing virus-like particles (VLPs),
analogous to the hepatitis B virus vaccine derived from yeast, have
been developed as prophylactic vaccines to prevent viral infections
and diseases including HPV infections (Schiller and Lowy, 1996;
Cook et al., 1999; Harro et al., 2001).
[0006] Papillomaviruses encode the major capsid gene, L1, whose
gene products are able to self-assemble into virus-like particles
in the absence of other viral gene products (Kirnbauer et al.,
1992; Hagensee and Galloway, 1993; Kirnbauer et al., 1993).
Recombinant papillomavirus L1 VLPs display several properties that
are advantageous for vaccines. These features include the
following: (1) similar size and morphology as natural
papillomavirus virions as shown by electron microscopy, (2)
immunodominant and conformational epitopes present on natural
virions as determined by immunodetection assays with neutralizing
monoclonal antibodies, and (3) elicitation of high titers of
type-specific neutralizing antibodies as seen in sera of vaccines
(Kirnbauer et al., 1992; 1993).
[0007] Several trials of preventive papillomavirus vaccine
candidates using L1 VLPs purified from insect cells have been
conducted in animals using the cutaneous cottontail rabbit
papillomavirus (CRPV) disease model, the oral mucosal bovine
papillomavirus 4 (BPV4), and canine oral papillomavirus (COPV)
models in cattle and dogs, respectively. Three subcutaneous
injections of CRPV L1 VLPs given without adjuvant, or combined with
alum or Freund's adjuvant, protected rabbits for at least one year
against persistent infection and subsequent carcinoma after
high-dose CRPV challenge (Breitburd et al., 1993; Christensen et
al., 1996). Similarly, calves and dogs given two intramuscular
injections of BPV4 L1 VLPs (with alum) and COPV L1 VLPs (without
adjuvant), respectively, were protected from subsequent oral
mucosal challenge (Suzich et al., 1995; Kirnbauer et al., 1996). In
the CRPV and COPV models, passive transfer of serum or IgG from
animals immunized with the L1 VLPs protected animals challenged
with the homologous virus, indicating that neutralizing antibodies
were sufficient to confer protection (Breitburd et al., 1993;
Suzich et al., 1995).
[0008] Recombinant peptides or proteins encoded and expressed by
custom synthesized genes often require further modifications. These
peptides have often lost their ability to fold and show no
disulfide bond formation. Thus proteins frequently are not stable
in the presence of endogenous bacterial proteases, and tend to
aggregate into inactive complexes. Consequently, recombinant
peptides often suffer from low yield and demonstrate reduced
antigencity and immunogencity as compared with native peptides.
[0009] Purification of heterologous recombinant proteins from
baculovirus-infected insect cells demonstrated that host
contaminant proteins were best separated from the recombinant
protein using an ion exchange step as the first step in the
protocol (Robinson et al., 1998). Purification of
baculovirus-derived HPV L1 VLPs (Kirnbauer et al, 1993; Suzich et
al., 1995) and yeast-derived intracellular HPV L1 VLPs (Cook et
al., 1999) were described previously.
[0010] The invention as disclosed and described herein, overcomes
the prior art problems with HPV therapies through the generation of
novel synthetic polynucleotides that encode HPV capsid genes
encoding HPV capsid proteins capable of assembly into VLPs. The
capsid proteins of the invention retain their optimum native
folding and exhibit conformational presentation of epitopes that
elicit antigen-neutralizing antibodies. Large scale production and
purification of HPV-L1 VLPs and HPV chimeric VLPs and their
manufacturing for vaccines and other pharmaceutical products are
also disclosed.
III. SUMMARY OF THE INVENTION
[0011] This invention is directed toward the prevention, treatment,
and diagnosis of papillomavirus infections and associated benign
and neoplastic diseases in humans. In particular, the invention
discloses novel synthetic polynucleotides capable of expressing
highly immunogenic HPV VLP and HPV chimeric VLP products.
[0012] According to one aspect of the invention, there is provided
a codon-optimized polynucleotide encoding a viral capsid protein
that self assembles into virus-like particles exhibiting
conformational antigenic epitopes capable of raising neutralizing
antibodies wherein the virus-like particles are expressed from a
host cell extracellularly.
[0013] The viral capsid protein is from an enveloped virus, or a
non-enveloped virus. Preferably, the virus comprises rotavirus,
calicivirus, hepatitis E virus, papillomavirus, influenza virus,
hepatitis C virus, retrovirus, or a combination thereof. More
preferably the virus is a human papillomavirus.
[0014] In one embodiment, the codon-modified polynucleotide
comprises SEQ ID No. 1, or a polynucleotide having a sequence that
is substantially homologous to SEQ ID No. 1.
[0015] In another aspect, the invention provides pharmaceutical
compositions for treating, ameliorating, or preventing a
papillomavirus related disease or disorder comprising a
multiplicity of virus-like particles that exhibit conformational
antigenic epitopes, wherein the virus-like particles are expressed
from a host cell extracellularly, and an acceptable carrier or
diluent.
[0016] In one embodiment, the pharmaceutical composition comprises:
(a) a polypeptide which is encoded by a polynucleotide molecule
comprising SEQ ID No. 1, or a polynucleotide having a sequence that
is substantially homologous to SEQ ID No. 1, (b) a polynucleotide
molecule comprising SEQ ID No. 1, or a polynucleotide having a
sequence that is substantially homologous to SEQ ID No. 1; (c) a
vector carrying a polynucloetide a molecule comprising SEQ ID No.
1, or a polynucleotide having a sequence that is substantially
homologous to SEQ ID No. 1; or (d) transfected, or generally
transformed with a polynucleotide molecule comprising SEQ ID No. 1,
or a polynucleotide having a sequence that is substantially
homologous to SEQ ID No. 1; and a pharmaceutically acceptable
carrier or diluent.
[0017] In yet another aspect, the invention provides a vaccine
composition to induce immunity against a papillomavirus infection
in humans comprising a multipliticty of virus-like particles that
exhibit conformational antigenic epitopes, wherein the virus-like
particles are expressed from a host cell extracellularly, and an
adjuvant.
[0018] The vaccine provides humoral immunity, cell-mediated
immunity, or both. The vaccine protects against papillomavirus
infections that are caused by one or more human papillomavirus
genotypes.
[0019] In another aspect, the invention provides a diagnostic test
kit for detection of papillomavirus infection comprising a
multiplicity of virus-like particles that exhibit conformational
antigenic epitopes, wherein the virus-like particles are expressed
from a host cell extracellularly, and a detection agent comprising
a detectable label. Preferably, the diagnostic test kit detects
papillomavirus infections that are caused by one or more human
papillomavirus genotypes.
[0020] In yet another aspect, the invention provides a vector
comprising the codon-optimized polynucleotides of the invention
operatively linked to an eukaryotic or a prokaryotic regulatory
control element, capable of replication in a prokaryotic host,
eukaryotic host, or both. Transformed cells carrying the vector are
also disclosed.
[0021] In yet another aspect, the invention provides a method for
preparing a codon-optimized polynucleotide comprising one or more
of the following steps: (a) replacing codons that are underutilized
in insect cells with codons that are utilized at high levels in
insect cells, to create an initially-modified nucleotide sequence;
(b) modifying the initially-modified nucleotide sequence by
choosing a preferred codon for the initially modified sequence,
wherein: (i) the ratio of GC nucleotide pairs to AT nucleotide
pairs in the further-modified nucleotide sequence trends toward
approximately 1:1; (ii) the number of palindromic and stem-loop DNA
structures in the further-modified nucleotide sequence is
minimized; and (iii) the number of transcription and
post-transcription repressor elements are minimized.
[0022] In another aspect, the invention provides a method of,
treating, ameliorating, or preventing a papillomavirus-related
disease or disorder comprising administering to an individual in
need thereof an effective amount of the pharmaceutical composition
of the invention.
IV. BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A-1C show the alignment of two wild-type HPV-16
papilloma virus L1 polynucleotide sequences with a codon-optimized
HPV-16 L1 polynucleotide of the invention. The aligned sequences
are: HPV-16 L1 wild-type sequence from GenBank record Accession No.
K0278 ("l1gbseq"; SEQ ID No. 12), HPV-16 wild type clone NVAX
("l1nvax"; SEQ ID No. 11), and HPV-16 codon-optimized L1
("l1optmzd"; SEQ ID No. 1). The sequences were aligned using the
Gene Runners program (Hastings Software) available through the
website maintained by the National Center for Biotechnology
Information (NCBI). Nucleotides which differ between the aligned
sequences are boxed.
[0024] FIG. 2 shows a schematic flowchart of the steps in the
weaning selection process of an insect cell line capable of growing
in serum-free media as suspension cell cultures.
[0025] FIG. 3 shows a schematic flowchart of the steps in the
protein secretion selection process of an insect cell line capable
of growing in serum-free media as suspension cell cultures and of
enhanced expression of extracellular recombinant proteins and
virus-like particles.
[0026] FIG. 4 shows a photomicrograph of a confluent monolayer of
Sf-9S insect cells grown in serum-free insect cell media (Sf-900 II
SFM, GIBCO) visualized by inverted phase-contrast microscopy at
400.times. magnification using Kodachrome 100 color film
(Kodak).
[0027] FIG. 5 shows a schematic flowchart of the basic steps in the
production or manufacturing of purified HPV VLP products.
[0028] FIG. 6 shows a schematic flowchart of the steps in upstream
processing of baculovirus infected insect cell suspensions for
production of recombinant HPV L1 VLPs.
[0029] FIG. 7 shows a schematic flowchart of the steps in upstream
processing of baculovirus-infected insect cell suspensions for
production of recombinant HPV chimeric VLPs.
[0030] FIG. 8A shows a schematic flowchart of the downstream
processing method for purification of recombinant HPV VLPs by
continuous flow ultracentrifugation using linear sucrose
gradients.
[0031] FIG. 8B shows a schematic flowchart of the downstream
processing method for purification of recombinant HPV VLPs by
column chromatography using ion exchange and affinity binding
matrices.
[0032] FIG. 8C shows a schematic flowchart of the downstream
processing method for purification of recombinant HPV VLPs by
ultracentrifugation using discontinuous sucrose step gradients.
[0033] FIG. 9 shows a stained protein gel of the products of the
invention (i.e., baculovirus-derived recombinant HPV-16 L1 VLPs
purified according to the methods of the present invention.
[0034] FIG. 10A shows proteins detected chromogenically on
membranes by Western blot analysis of recombinant HPV-16 L1 VLPs
purified according to the methods of the present invention and
bound to polyclonal antisera to HPV-16 L1 protein (1:10,000).
[0035] FIG. 10B shows proteins detected chromogenically on
membranes by Western blot analysis of recombinant HPV-16 L1 VLPs
purified according to the methods of the present invention and
bound to polyclonal antisera to Sf-9S insect cell proteins
(1:500).
[0036] FIG. 10C shows proteins detected chromogenically on
membranes by Western blot analysis of recombinant HPV-16 L1 VLPs
purified according to the methods of the present invention and
bound to polyclonal antisera to AcMNPV wild-type baculovirus
(1:500).
[0037] FIG. 11 shows a graph of the binding results of H16.V5
murine monoclonal antibody to conformational epitopes on untreated
and Triton X-100-treated recombinant HPV-16 L1 VLPs purified and
treated according to the methods of the present invention as
measured by enzyme linked immunoadsorbent assay (ELISA)
analysis.
[0038] FIG. 12 shows a chromatogram of a product of the invention,
recombinant HPV-16 L1 VLPs purified according to the methods of the
present invention, analyzed by analytical size exclusion
chromatography.
[0039] FIG. 13 shows an electron micrograph of baculovirus-derived
recombinant HPV-16 L1 VLPs purified according to the methods of the
present invention, stained negatively with uranyl acetate, and
magnified 36,000.times.. The bar scale is 50 nm.
V. DETAILED DESCRIPTION OF THE INVENTION
[0040] The invention, as disclosed and described herein, provides
compositions and methods for detecting, preventing, ameliorating,
or treating papillomavirus related diseases and disorders. The
pharmaceutical composition of the invention contains recombinant
viral proteins that self assemble into virus-like particles (VLPs)
exhibiting conformational antigenic epitopes capable of raising
neutralizing antibodies. The VLPs of the invention are expressed
from a host cell extracellulary, intracellularly, or both.
[0041] Definitions
[0042] The definitions used in this application are for
illustrative purposes and do not limit the scope of the
invention.
[0043] As used in herein, "virus-like particles" or "VLPs" refers
to virus particles that self-assemble into intact virus structures
comprised of capsid proteins such as papillomavirus L1 capsid
proteins. VLPs are morphologically and antigenically similar to
authentic virions, but do not contain genetic information
sufficient to replicate and thus are non-infectious. VLPs are
produced in suitable insect host cells (i.e., yeast, mamalian, and
insect host cells), wherein upon isolation and further purification
under suitable conditions, are purified as intact VLPs.
[0044] As used herein, "chimeric VLP" refers to recombinant
papillomavirus L1 capsid protein, or peptide fragment thereof, that
encapsulates other papillomavirus gene products or heterologous
gene products during self-assembly into virus-like particles. For
example, gene products containing the HPV L2, E2, E6, and/or E7 and
which become encapsulated into the HPV L1 VLPs are considered
herein as chimeric VLPs.
[0045] As used herein, "L2 fusion protein" refers to a protein, or
a peptide fragment thereof, encoded by a papillomavirus L2
scaffolding gene fused to papillomavirus or other viral genes
including heterologous gene(s).
[0046] As used herein, "heterologous viral capsid genes" refers to
viral genes encoding the major structural virion component from
different viruses, for example, the rotavirus VP2, VP6, HPV-16 L2,
and HPV-16 L1 genes.
[0047] As used herein, "protein" is used interchangeably with
polypeptide, peptide and peptide fragments.
[0048] As used herein, "polynucleotide" includes cDNA, RNA, DNA/RNA
hybrid, anti-sense RNA, ribozyme, genomic DNA, synthetic forms, and
mixed polymers, both sense and antisense strands, and may be
chemically or biochemically modified to contain non-natural or
derivatized, synthetic, or semi-synthetic nucleotide bases. Also,
included within the scope of the invention are alterations of a
wild type or synthetic gene, including but not limited to deletion,
insertion, substitution of one or more nucleotides, or fusion to
other polynucleotide sequences, provided that such changes in the
primary sequence of the gene do not alter the expressed peptide
ability to elicit protective immunity.
[0049] As used herein, "gene products" include any product that is
produced in the course of the transcription, reverse-transcription,
polymerization, translation, post-translation and/or expression of
a nucleotide molecule. Gene products include, but are not limited
to, proteins, polypeptides, peptides, or peptide fragments.
[0050] As used herein, "L1 protein" refers to the structural
protein of papillomavirus L1 capsid genes and constitutes the major
portion of the papillomavirus ("PV") capsid structure. This protein
has reported application in the preparation of HPV vaccines and
diagnostic reagents.
[0051] As used herein, "L2 protein" refers to the structural
scaffolding protein of papillomavirus, which constitutes a minor
portion of the papillomavirus capsid structure and facilitates the
assembly of papillomavirus particles within cell nuclei.
[0052] As used herein, "L2/E7 protein" refers to a fusion protein,
or a fragment thereof, encoded by a papillomavirus L2 scaffolding
gene fused to a papillomavirus E7 transforming gene that may have
one or more mutations.
[0053] As used herein, "L2/E7/E2 protein" refers to a fusion
protein, or a fragment thereof, encoded by a papillomavirus L2
scaffolding gene fused to (a) papillomavirus E2 transactivation
gene that may have mutations and (b) a papillomavirus E7
transforming gene. The fused gene includes one or more mutated
genes.
[0054] As used herein, "L2/E6 protein" refers to a fusion protein,
or a peptide fragment thereof, encoded by a papillomavirus L2
scaffolding gene fused to a papillomavirus E6 transforming gene
that may have one or more mutations.
[0055] As disclosed herein, "mutation" includes substitutions,
transversions, transitions, transpositions, reversions, deletions,
insertions, or other events that may have improved desired
activity, or a decreased undesirable activity of the gene. Mutation
encompasses null mutations in natural virus isolates or in
synthesized genes that may change the primary amino acid sequences
of the expressed protein but do not affect the self-assembly of
capsid proteins, and antigenicity or immunogenicity of VLPs or
chimeric VLPs.
[0056] As disclosed herein, "substantially homologous sequences"
include those sequences which have at least about 50%, homology,
preferably at least about 60-70%, more preferably at least about
70-80% homology, and most preferably at least about 95% or more
homology to the codon optimized polynucleotides of the
invention.
[0057] As used herein "vaccine" refers to compositions that result
in both active and passive immunizations. Both polynucleotides and
their expressed gene products are used as vaccines.
[0058] As used herein "biologically active fragments" refer to
fragments exhibiting activity similar, but not necessarily
identical, to an activity of the viral polypeptide of the present
invention. The biologically active fragments may have improved
desirable activity, or a decreased undesirable activity.
[0059] As used herein "polypeptides" include any peptide or protein
comprising two or more amino acids joined to each other by peptide
bonds. As used herein, the term refers to both short chains, which
also commonly are referred to in the art as peptides, oligopeptides
and oligomers, for example, and to longer chains, which generally
are referred to in the art as proteins, of which there are many
types. "Polypeptides" include, for example, biologically active
fragments, substantially homologous polypeptides, oligopeptide,
homodimers, heterodimers, variants of the polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, agonists,
antagonists, or antibody of the polypeptide, among others. The
polypeptides of the invention are natural peptides, recombinant
peptides, synthetic peptides, or a combination thereof.
[0060] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments," "immunogenic
fragments," or "antigenic fragments" refer to fragments of viral
proteins which are preferably at least 5 to about 15 amino acids or
more in length, and which retain some biological activity or
immunological activity of the viral protein.
[0061] As used herein, "purity" refers to the amount of intact VLPs
present in a final product of the invention.
[0062] As used herein, "yield" refers to the amount of purified
intact VLPs as a function of the wet weight or the number of
initial cells infected with recombinant baculoviruses expressing
VLPs. For example, a preferred yield of intact VLPs is greater than
about 10 mg of VLPs per 10.sup.9 host cells.
[0063] As used herein, "antigenic characteristic(s)" refers to the
ability of HPV VLPs to bind or cross-react with antisera generated
against wild-type HPV virions of the same genotype. Antisera
generated by immunization of animals or humans with HPV VLPs
produced according to the present invention contains immunoglobulin
molecules that share binding sites of native HPV virions with
antisera from humans infected with HPV of the same genotype.
[0064] 1. Sf-9S Cell Line
[0065] According to one aspect of the invention described herein,
there is provided a novel cell line designated as Sf-9S, which was
deposited as cell line ATCC PTA-4047 on Feb. 4, 2002, under the
Budapest Treaty, with the American Type Culture Collection (ATCC),
located at 10801 University Boulevard, Manassas, Va. 20110. The
Sf-9S cell line is derived from the parent S. frugiperda Sf-9 cell
line (ATCC CRL-1771) and established by clonal selection based on
serum-independent growth. This cell line is used as a host cell
substrate in a single cell suspension maintained at a large
manufacturing scale. The Sf-9S cell line of the invention is
capable of enhanced expression of recombinant gene products. The
designations "Sf-9S" and "ATCC PTA-4047" are used herein
interchangeably, and refer to the same cell line.
[0066] According to one embodiment of the invention, there is
provided a process for developing cell lines from the parent cell
line Sf-9S. The first step of this process involves progressive
weaning of cells from serum-containing media to serum-free media.
Master and working cell banks of the cell line are constructed and
qualified according to safety, identity, and biological criteria or
specifications. Prior to commencement of the clonal selection
process, master and working cell banks of the parental cell line
Sf-9 cells are cultivated as monolayer cultures for at least 10,
preferably at least 20, and more preferably at least 30 passages in
Grace's insect media (Life Technologies, Grand Island, N.Y. 14072)
supplemented with 10% fetal bovine serum (Life Technologies, Grand
Island, N.Y. 14072). A master cell bank of Sf-9 cells is stored in
conditioned serum-containing media at -70.degree. C. and in liquid
nitrogen. A working cell bank is established from a single cryovial
of the Sf-9 master cell bank and cultivated in serum-containing
insect media for multiple cell passages. The clonal selection
process, according to the invention, includes several rounds as
demonstrated in FIG. 2.
[0067] The method of clonal selection according to the invention
described herein includes generally weaning a plurality of cells
from serum-dependence to obtain at least one cell that can grow in
serum-free medium.
[0068] According to another aspect, the invention provides a
process for producing a cell line comprising one or more of the
following steps: (a) plating a plurality of cells in wells
containing serum-containing medium, one cell per well; (b)
culturing the cell in each separate well; (c) identifying each well
with replicating cells; (d) culturing the replicating cells into
replica-plating wells; (e) changing the medium in each identified
well with replicating cells by increasing the proportion of
serum-free medium to serum-containing medium; (f) repeating
identifying, culturing, and medium-changing of steps (c)-(e) until
the medium for each well is approximately 100% serum-free; (g)
harvesting the cells from each serum-free well; and (h) culturing
the harvested cells in suspension. Suspension cultures of harvested
cells that grow to a predetermined cell density for multiple
passages are designated serum-free cell clones.
[0069] According to another embodiment, the method of clonal
selection of the invention includes at least one of the following
steps. First, cell clones capable of growing in commercial
serum-free media as suspension cultures are isolated from monolayer
cultures of parent Sf-9 cells dependent on serum-containing media
by sequential weaning of parent cells from serum-containing media.
According to one embodiment depicted in FIG. 2, Sf-9 parent cells
are prepared at step 201 for sequential serum-weaning. In this
embodiment, monolayer and suspension cultures of Sf-9 cells are
grown at about 26-28.degree. C. in a dry environment. Shaker
suspension cultures are agitated at about 100-150 rpm in a standard
orbital or platform shaker incubator and stir flask suspension
cultures are stirred at about 25-75 rpm on a standard laboratory
magnetic stirrer.
[0070] In step 202, cell aliquots are dispensed from a cell
suspension (one cell per aliquot) of the parental cell line in
serum-containing media into wells of 96-well dishes at a ratio of
one aliquot per well. Following cell attachment, cell acclimation
to wells, and exclusion of wells with no cells and wells with more
than one cell, in step 202 the media is changed from
serum-containing media (100%) to a media mixture comprised of 75%
serum-containing media and 25% serum-free media. In step 203, cells
are cultured in "75/25" media mixture for approximately one to two
weeks. Wells that initially contain only one cell per well and
demonstrate cell growth and replication (i.e., four to five cells)
after step 203 are subjected in step 204 to another media change to
a mixture comprising 50% serum-containing media and 50% serum-free
media. In step 205, cells in the "50/50" media mixture of step 204
are allowed to grow for approximately another one to two weeks. The
media mixture is changed again in step 206, to a mixture comprising
25% serum-containing media and 75% serum-free media. In step 207,
cells are allowed to grow and replicate in the new "25/75" media
mixture of step 206. After another two to four weeks, in step 208
the media is changed in wells containing growing cells to a final
media comprising serum-free media (100%).
[0071] During each step of the weaning process depicted in FIG. 2,
a majority of the cells, for example about 95% of the cells or
more, do not survive the reduction in serum. While not wanting to
be bound by this theory, it is believed that this high level of
cell death creates a selective pressure to permit development of a
new cell phenotype. In step 209, cells from wells that demonstrate
continuous cell growth and replication are harvested by vigorous
aspiration with serum-free media. In step 210, the harvested cells
are seeded into larger culture flasks (i.e., 75 or 150 cm.sup.2
T-flasks), and, in step 211, the suspension cultures are grown.
When greater than 4.times.10.sup.6 cells with a viability >95%
is obtained, cells are harvested in step 211 and seeded into shaker
or stir flasks as suspension cultures with a starting cell density,
for example, about 0.2-0.5.times.10.sup.6 cells/ml and a minimal
ratio, for example, about 2.5 for total vessel capacity to total
volume of culture media. In step 212, cell clones that grow
exponentially to a saturation cell density of greater than
6.times.10.sup.6 cells/ml in serum-free media are selected,
expanded, and frozen.
[0072] Finally, one cell clone is selected, passaged for at least
10, preferably at least 20, and more preferably more than 30 times
as a suspension culture in serum-free media at a split ratio of at
least 1:10, and established as a cell line. This serum-independent
cell line is used to establish a master cell bank and subsequent
working cell banks.
[0073] 1.1 Sf-9S Cells
[0074] According to another aspect of the invention described
herein, there are provided host cells that express one or more
recombinant gene products with an enhanced yield. Insect host cells
include, for example, Lepidopteran insect cells, and particularly
preferred are Spodoptera frugiperda, Bombyx mori, Heliothis
virescens, Heliothis zea, Mamestra brassicas, Estigmene acrea or
Trichoplusia insect cells. Non-limiting examples of insect cell
lines include, for example, Sf21, Sf9, High Five (BT1-TN-5B1-4),
BT1-Ea88, Tn-368, mb0507, Tn mg-1, and Tn Ap2, among others.
[0075] In addition to the serum-weaning process described above,
the Sf-9S cells of the present invention have undergone a
recombinant peptide secretion selection process. An example of the
process of the recombinant peptide secretion selection, according
to the invention, is demonstrated in FIG. 3. The Sf-9S cells
express extracellularly a foreign recombinant protein with an
enhanced yield.
[0076] According to one embodiment of the invention, the cells are
infected with a recombinant Baculovirus vector to express
recombinant proteins or polypeptides of medical, pharmaceutical, or
veterinary importance. Baculoviruses including Autographa
californica multinucleocapsid nuclear polyhedrovirus (AcMNPV) are
propagated in cell lines derived from larval tissues of insects of
the Lepidopteran insect family. General methods for handling and
preparing baculovirus vectors and baculovirus DNA, as well as
insect cell culture procedures, are outlined for example in
O'Reilly et al., 1994; Vaughn, J. 1999, supra; Frieson et al. 1986.
In: The Molecular Biology of Baculoviruses, Doerfler et al., Eds.
Springer-Verlag, Berlin, pages 31-49; Kool et al, 1993. Arch.
Virol. 130: 1-16, incorporated herein by reference in their
entirety.
[0077] In one embodiment, polynucleotide molecules, including
chimeric and heterologous polynucleotides, which encode a foreign
peptide of interest, are inserted into the baculovirus genome
operably coupled to or under the control of the polyhedrin or other
Baculovirus promoters. The recombinant baculovirus vector is then
used to infect a host cell. The foreign peptide or protein is
expressed upon culture of the cells infected with the recombinant
virus.
[0078] In another embodiment, the invention provides a method for
producing a selected foreign protein in an insect cell. The method
comprises preparing infected insect cells that express at least a
first recombinant viral protein, and infecting the cell with a
baculovirus comprising an expression vector that encodes a second
recombinant viral protein. The first, or the second viral proteins,
or both are, for example, viral capsid proteins including
heterologous peptides and chimeric peptides. The cells produced
according to the method disclosed herein produce substantially high
yields of recombinant baculoviruses expressing the desired
recombinant peptides.
[0079] The insect cells of the invention have passed through a
recombinant peptide secretion selection. As described herein, the
process of recombinant peptide secretion selection includes one or
more of the following steps. Cells from a serum-weaned clone are
infected with a first baculovirus expressing a first recombinant
protein. Cells capable of secreting high levels of the first
recombinant protein are selected further for infection with a
second baculovirus expressing a second recombinant protein. Cells
from a clone that secretes high levels of both recombinant proteins
independently are passaged further to establish the Sf-9S cell line
of the present invention.
[0080] According to a preferred embodiment, the first recombinant
protein or the second recombinant protein, or both, is a viral
capsid protein that self-assembles into virus-like particles. In a
more preferred embodiment, the virus-like particles are derived
from viral capsid proteins of an enveloped virus, or a
non-enveloped virus, including, but not limited to, an influenza
virus, a hepatitis C virus, a retrovirus such as a human
immunodeficiency virus, a calicivirus, a hepatitis E virus, a
papillomavirus, or a combination thereof. In a most preferred
embodiment of the invention, the virus-like particles are derived
from human papillomavirus.
[0081] According to a preferred embodiment of the invention, the
Sf-9S cells support intracellular, and preferably extracellular,
expression of recombinant proteins and macromolecules. More
preferably, infected Sf-9S cells extracellularly express viral
capsid proteins that self assemble into VLPs. Virus-like particles
typically self assemble in the cell and remain intracellular;
therefore isolation of these particles requires processes of cell
disruption and protein solublilization with the accompanying risks
of VLP disruption, proteolysis and contamination of the end
product. Accordingly, the infected cells of the invention that
afford self-assembly of viral capsid antigens into VLPs and
facilitate secretion of VLPs extracellularly are highly
desirable.
[0082] An example of a process for recombinant peptide secretion
selection as depicted in FIG. 3 is described below. FIG. 3
demonstrates that additional rounds of clonal selection are used to
obtain cells capable of enhanced secretion of recombinant proteins.
In step 301, cell aliquots from a cell suspension (one cell per
aliquot) of the parent serum-free cell clone (i.e., a cell line
from one of the serum-free cell clones selected in step 213 of FIG.
2) are replica-plated into each well of 96-well plates at a ratio
of one cell per well. In step 302, wells containing a single cell
from the original seeding are identified and grown to confluency.
Upon confluency, cells from wells identified as single cell wells
are subcultured into replica plates in step 303. Cells in replica
plates are grown at step 304 to confluency and infected with a
first recombinant baculovirus expressing first virus capsid
proteins that are capable of self-assembly into virus-like
particles.
[0083] During baculovirus infection, in step 305 the infected cells
and extracellular media are harvested by centrifugation to isolate
infected cells and extracellular media, heat-denatured under
reduced conditions (>75.degree. C. for 5 minutes in 1% sodium
dodecyl sulfate (SDS) and 10 mM .beta.-mercaptoethanol), and
analyzed by SDS-PAGE and Western blot analyses with antisera to
viral capsid proteins. In step 306, cells in replica plates that
contain cell clones exhibiting extracellular VLPs at levels higher
than control Sf-9 cells are infected with a second baculovirus
expressing the second viral capsid proteins that self-assemble into
virus-like particles. The infected cells and extracellular media
from the second selection round are isolated in step 307 by
centrifugation and analyzed by SDS-PAGE and Western blot analyses.
The first and second viral capsid proteins are the same or
different proteins and include, for example, rotavirus VP2, VP6,
and HPV-16 L1, HPV-L2 proteins, among others.
[0084] The test results from the first and second rounds of
selection (i.e. virus infections producing VLPs) are examined in
step 308. The cell clone exhibiting the highest levels of
extracellular VLPs from both virus infections is chosen in step
310. From the replica plate, cells of the selected cell clone
exhibiting highest extracellular VLP levels are passaged repeatedly
in suspension culture with serum-free insect cell media to
establish a cell line. The cell line supports high levels of
extracellular VLP production upon infection with recombinant
baculoviruses expressing viral capsid proteins that self-assemble
into virus-like particles. Thus, in one embodiment, the clone
selected in step 310 is processed again according to steps 304-309
with recombinant baculovirus expressing HPV-16 L1 capsid proteins.
The cell clone that produces the highest levels of extracellular
VLPs for both sets of viral capsid proteins is chosen in step 311
to establish a cell line capable of producing extracellular
VLPs.
[0085] Master cell banks of Sf-9S cells are established, for
example, from a single cell passage of the new cell line grown in
suspension culture of serum-free medium and stored at -70.degree.
C. in liquid nitrogen in a cryopreservation freezing media
containing fresh serum-free media, conditioned serum-free media,
and dimethyl sulfoxide. Working cell banks are developed, for
example, from single cryovials of the master cell bank, subjected
to safety and biological testing for qualification as a host cell
substrate for manufacturing of recombinant protein products, and
stored at -70.degree. C. in liquid nitrogen in cryovials in
cryopreservation freezing media as described above.
[0086] The Sf-9S cell line of the present invention demonstrate one
or more of the following properties: (1) they replicate in
serum-free media; (2) they are genetically distinct from parent
Sf-9 parent cell line; (3) they grow as single cells in suspension
cultures; (4) they demonstrate cell division rate of approximately
18-24 hours; (5) they demonstrate high cell viability (more than
95%) upon continuous cell culture for more than one year; (6) they
constitute a cell substrate for Autographa californica
baculoviruses to produce high-titered virus stocks (more than
10.sup.7 plaque forming units (pfu)/ml); (7) they are suitable for
recombinant protein expression and production from baculovirus
vectors; (8) they are suitable host cell substrates for agarose
plaque assays to titer baculovirus stocks; (9) they are compliant
with recognized identity and safety guidelines; (10) they are
suitable cell substrates for large-scale manufacturing of human and
animal biological products including vaccines, therapeutics, and
diagnostic reagents; (11) they are suitable cell substrates for
transfection of genes in recombinant baculovirus transfer vectors
and/or bacmids to produce recombinant baculoviruses, and (12) they
produce high levels of extracellular VLPs from baculoviruses
expressing viral capsid proteins that self-assemble into VLPs of
non-enveloped viruses such as rotaviruses, caliciviruses, hepatitis
E virus, and human papillomaviruses and of enveloped viruses such
as influenza virus, hepatitis C virus, and human immunodeficiency
virus.
[0087] In FIG. 4, a confluent monolayer of Sf-9S cells grown in
serum-free insect cell media is shown at 400.times. magnification
using a phase-contrast microscope. The cuboidal and fibroblastic
cell morphologies of the cell line are displayed. The cell
morphology of Sf-9S cells changes from fibroblastic to cuboidal, as
the monolayer becomes confluent.
[0088] Safety testing of the Sf-9S cell line produced according to
the present invention and deposited at the ATCC may be performed in
accordance with United States federal regulatory guidelines and
include microbial sterility, mycoplasma and spiroplasma growth,
endotoxins, adventitious agents (in vitro and in vivo assays), and
electron microscopic examination for type C endogenous retrovirus
particles. The cell identity of the Sf-9S cell line was shown by
karyology and isotype enzyme analyses, to be S. frugiperda insect
species with the typical polyploid chromosomal pattern distinct
from mammalian cells.
[0089] 1. Expression Systems
[0090] The expression vector of the invention is a baculovirus
vector. For baculovirus vectors and baculovirus DNA, as well as
insect cell culture procedures, see, for example in O'Reilly et al.
1994, incorporated herein by reference in its entirety. The
baculovirus vector construct of the invention preferably contains
additional elements, such as an origin of replication, one or more
selectable markers allowing amplification in the alternative hosts,
such as yeast cells and insect cells.
[0091] Host cells are infected, transfected, or genetically
transformed to incorporate codon-optimized polynucleotides and
express polypeptides of the present invention. The recombinant
vectors containing a polynucleotide of interest are introduced into
the host cell by any of a number of appropriate means, including
infection (where the vector is an infectious agent, such as a viral
or baculovirus genome), transduction, transfection, transformation,
electroporation, microprojectile bombardment, lipofection; or a
combination thereof. A preferred method of genetic transformation
of the host cells, according to the invention described herein, is
infection.
[0092] In certain embodiments, there are provided baculovirus
vectors that contain cis-acting control regions effective for
expression in a host operatively linked to the polynucleotide to be
expressed. Appropriate trans-acting factors are either supplied by
the host, supplied by a complementing vector or supplied by the
vector itself upon introduction into the host. Host cells are
infected with baculovirus vectors comprising codon optimized
polynucleotides to express polypeptides.
[0093] The polynucleotides are introduced alone or with other
polynucleotides. Such other polynucleotides are introduced
independently, co-introduced or introduced joined to the
polynucleotides of the invention. Thus, for instance, a
polynucleotides (i.e., L1 gene) is transfected into host cells with
another, separate polynucleotide (i.e., L2 or fusion L2 genes)
using standard techniques for co-transfection and selection. In
another embodiment, the polynucleotides encoding L1 capsid protein
and the polynucleotides encoding L2 protein or an L2 fusion protein
are present on two mutually compatible baculovirus expression
vectors which are each under the control of their own promoter.
[0094] 3. Codon-Optimized Polynucleotides Encoding HPV
Polypeptides
[0095] This invention also encompasses nucleic acid sequences that
correspond to, and code for the HPV polypeptides. Nucleic acid
sequences are synthesized using automated systems well known in the
art. Either the entire sequence is synthesized or a series of
smaller oligonucleotides are made and subsequently ligated together
to yield the full-length sequence. Alternatively, the nucleic acid
sequence is derived from a gene bank using oligonucleotides probes
designed based on the N-terminal amino acid sequence and well known
techniques for cloning genetic material.
[0096] In addition, the codon-optimized polynucleotides comprising
unusual bases, such as inosine, or modified bases, such as
tritylated bases of 8-amino adenine bases, to name just a few are
polynucleotides, the term is used herein. It will be appreciated
that a great variety of modifications have been made to DNA and RNA
that serve many useful purposes known to those of skill in the art.
The term "codon-optimized polynucleotide", as it is employed
herein, embraces such chemically, enzymatically or metabolically
modified forms of polynucleotide.
[0097] The codon-optimized polynucleotides of the present invention
encode, for example, the coding sequence for the mature
polypeptide, the coding sequence for the mature polypeptide and
additional coding sequences, and the coding sequence of the mature
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences. Examples
of additional coding sequences include, but are not limited to,
sequences encoding a leader or secretory sequence, such as a pre-,
pro-, or prepro-protein sequences. Examples of additional
non-coding sequences include, but are not limited to, introns and
non-coding 5' and 3' sequences, such as the transcribed,
non-translated sequences that play a role in transcription and mRNA
processing, including splicing and polyadenylation signals, for
example, for ribosome binding and stability of mRNA.
[0098] The codon-modified polynucleotides also encode a polypeptide
which is the mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to the
mature polypeptide (when the mature form has more than one
polypeptide chain, for instance). Such sequences may play a role in
processing of a protein from precursor to a mature form, may
facilitate protein trafficking, may prolong or shorten protein
half-life or may facilitate manipulation of a protein for assay or
production, among other things. The additional amino acids may be
processed away from the mature protein by cellular enzymes.
[0099] In sum, a codon-optimized polynucleotide of the present
invention encodes, for example, a mature protein, a mature protein
plus a leader sequence (which may be referred to as a preprotein),
a precursor of a mature protein having one or more prosequences
which are not the leader sequences of a preprotein, or a
preproprotein, which is a precursor to a proprotein, having a
leader sequence and one or more prosequences, which generally are
removed during processing steps that produce active and mature
forms of the polypeptide.
[0100] According to one embodiment of the invention, there are
provided codon-optimized polynucleotides that encode one or more
foreign proteins. In this embodiment, the codon optimization of the
invention is based on the following criteria: (1) abundance of
aminoacyl-tRNAs for a particular codon in Lepidopteran species of
insect cells for a given amino acid as described by Levin and
Whittome (2000), (2) maintenance of GC-AT ratio in L1 gene sequence
is approximately 1:1, (3) minimal introduction of palindromic or
stem-loop DNA structures, and (4) minimal introduction of
transcription and post-transcription repressor element
sequences.
[0101] The optimized genes sequence is synthesized in vitro, for
example, as overlapping oligonucleotides, cloned, and expressed in
a host cell. Cloning and expression of the codon modified viral
genes were achieved following the methods known in the art and
exemplified at Examples 3 and 4 herein.
[0102] In a preferred embodiment of the invention, polynucleotides
encoding a viral gene, for example HPV genes, are optimized for
expression in a baculovirus-infected insect cell, comprising one or
more of the following steps (a) replacing nucleotide sequences of
codons in the gene that are underutilized in insect cells of
Lepidopteran species with sequences of preferred codons in insect
cells; and (b) for each amino acid encoded by this modified
nucleotide sequence, if a plurality of codons for the same amino
acid is preferred in insect cells, then the nucleotide sequence of
the modified gene is changed further by selecting a codon from
preferred codons for a amino acid so that (i) the ratio of GC
nucleotides to AT nucleotides in the sequence trends toward 1:1;
(ii) the number of palindromic and stem-loop structures is
minimized unless indicated otherwise for functional activity; and
(iii) the number of transcription and/or post-transcription
repressor elements in the sequence is minimized.
[0103] This method was used to develop the codon-optimized
polynucleotides encoding HPV L1 (used to generate L1 VLPs), and HPV
L2 (including wildtype L2), L2/E7, L2/E7/E2, and L2/E6 (used to
generate chimeric VLPs). The nucleic acid sequences of the codon
optimized HPV L1, HPV L2, and HPV L2 fusion genes are represented
herein as HPV L1 (SEQ ID NO. 1), HPV L2 (SEQ ID NO. 2), HPV L2/E7
(SEQ ID NO. 3), HPV L2/E7/E2 (SEQ ID NO. 4), and HPV L2/E6 (SEQ ID
NO. 5), respectively.
[0104] The method of codon optimization of the invention, as
described herein, is used to, inter alia, optimize the expression
of variety of enveloped and non-enveloped viral genes expressed in
insect cells.
[0105] The codon-optimized polynucleotides of the invention include
"variant(s)" of polynucleotides, or polypeptides as the term is
used herein. Variants include polynucleotides that differ in
nucleotide sequence from another reference polynucleotide.
Generally, differences are limited so that the nucleotide sequences
of the reference and the variant are closely similar overall and,
in many regions, identical. As noted below, changes in the
nucleotide sequence of the variant amy be silent. That is, they may
not alter the amino acids encoded by the polynucleotide. Where
alterations are limited to silent changes of this type, a variant
will encode a polypeptide with the same amino acid sequence as the
reference.
[0106] Changes in the nucleotide sequence of the variant may alter
the amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Such nucleotide changes may result in amino acid
substitutions, additions, deletions, fusions and truncations in the
polypeptide encoded by the reference sequence. According to a
preferred embodiment of the invention, there are no alterations in
the amino acid sequence of the polypeptide encoded by the codon
optimized polyneucleotide of the invention, as compared with the
amino acid sequence of the wild type peptide.
[0107] The present invention further relates to polynucleotides
that hybridize to the herein-described sequences. The term
"hybridization under stringent conditions" according to the present
invention is used as described by Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press
1.101-1.104, 1989. Preferably, a stringent hybridization according
to the present invention is given when after washing for an hour
with 1%. SSC and 0.1% SDC at 50.degree. C., preferably at
55.degree. C., more preferably at 62.degree. C., most preferably at
68.degree. C. a positive hybridization signal is still observed. A
polynucleotide sequence which hybridizes under such washing
conditions with the nucleotide sequence shown in any sequence
disclosed herein or with a nucleotide sequence corresponding
thereto within the degeneration of the genetic code is a nucleotide
sequence according to the invention.
[0108] The codon-optimized polynucleotides of the invention include
polynucleotide sequences that have at least about 50%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99% or more nucleotide sequence
identity to the codon optimized polynucleotides or a
transcriptionally active fragment thereof. To determine the percent
identity of two amino acid sequences or two nucleic acid sequences,
the sequences are aligned for optimal comparison purposes (i.e.,
gaps can be introduced in the sequence of a first amino acid or
nucleic acid sequence for optimal alignment with a second nucleic
acid sequence). The amino acid residue or nucleotides at
corresponding amino acid or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position.
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical overlapping positions/total # of
positions.times.100). In one embodiment, the two sequences are the
same length.
[0109] The determination of percent identity between two sequences
also can be accomplished using a mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268,
modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci.
USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST
and XBLAST program of Altschul, et al., 1990, J. Mol. Biol.
215:403-410. BLAST nucleotide searches can be performed with the
NBLAST program program, score=100, wordlength=12 to obtain
nucleotide sequences homologous to a nucleic acid molecules of the
invention. The BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to a protein molecule of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., 1997, Nucleic Acids
Res. 25:3389-3402.
[0110] Alternatively, PSI-Blast can be used to perform an iterated
search which detects distant relationships between molecules (Id.).
When utilizing BLAST, Gapped BLAST and PSI-Blast programs, the
default parameters of the respective programs (i.e., XBLAST and
NBLAST program can be used (see, HTTP://WWW.NCBI.NLM.NIH.GOV).
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated
into the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences of a PAM 120 weight
residue table, a gap length penalty of 12 and a gap penalty of 4
can be used. In an alternate embodiment, alignments can be obtained
using the NA_MULTIPLE_ALIGNMENT 1.0 program, using a GapWeight of 5
and a GapLengthWeight of 1.
[0111] 4. Recombinant HPV Polypeptides
[0112] In general, as used herein, the term polypeptide encompasses
variety of modifications, particularly those that are present in
polypeptides expressed by polynucleotides in a host cell. It will
be appreciated that polypeptides often contain amino acids other
than the 20 amino acids commonly referred to as the 20 naturally
occurring amino acids, and that many amino acids, including the
terminal amino acids, may be modified in a given polypeptide,
either by natural processes, such as processing and other
post-translational modifications, or by chemical modification
techniques.
[0113] It will be appreciated, that polypeptides are not always
entirely linear. For instance, polypeptides may be branched as a
result of ubiquitination, and they may be circular, with or without
branching, generally as a result of posttranslational events,
including natural processing event and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by entirely synthetic
methods, as well.
[0114] Modifications occur anywhere in a polypeptide, including the
peptide backbone, the amino acid side chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, occur in a
natural or synthetic polypeptides and such modifications may be
present in polypeptides of the present invention, as well. In
general, the nature and extent of the modifications are determined
by the host cell's post-translational modification capacity and the
modification signals present in the polypeptide amino acid
sequence. It will be appreciated that the same type of modification
may be present in the same or varying degrees at several sites in a
polypeptide.
[0115] The recombinant foreign polypeptide according to the
invention includes truncated and/or N-terminally or C-terminally
extended forms of the polypeptide, analogs having amino acid
substitutions, additions and/or deletions, allelic variants and
derivatives of the polypeptide, so long as their sequences are
substantially homologous to the native antigenic viral
polypeptide.
[0116] Specifically, as will be appreciated by those skilled in the
art, the recombinant viral polypeptides of the invention include
those polypeptides having slight variations in amino acid sequences
or other properties. Such variations may arise naturally as allelic
variations, as disclosed above, due to genetic polymorphism, for
example, or may be produced by human intervention (i.e., by
mutagenesis of cloned DNA sequences), such as induced point,
deletion, insertion and substitution mutants. Minor changes in
amino acid sequence are generally preferred, such as conservative
amino acid replacements, small internal deletions or insertions,
and additions or deletions at the ends of the molecules.
[0117] Substitutions may be designed based on, for example, the
model of Dayhoff, et al., Atlas of Protein Sequence and Structure,
Nat'l Biomed. Res. Found. Washington, D.C., 1978. These
modifications can result in changes in the amino acid sequence,
provide silent mutations, modify a restriction site, or provide
other specific mutations. The recombinant viral polypeptides may
comprise one or more selected antigenic determinants of the viral
polypeptide peptides, possess catalytic activity exhibited by their
native protein or alternatively lack such activity.
[0118] The conserved and variable sequence regions of a viral
polypeptide and the homology thereof can be determined by
techniques known to the skilled artisan, such as sequence alignment
techniques. For example, the determination of percent identity
between two sequences can also be accomplished using a mathematical
algorithm, as described above.
[0119] 4.1 Virus-Like Particles (VLPs)
[0120] Virus-like particles (VLPs) are the expressed product of the
codon-optimized polynucleotides of the invention. The capsid
protein encoded by the codon-optimized polynucleotide of the
invention is capable of self assembly into virus-like particles
that exhibit conformational antigenic epitopes capable of eliciting
neutralizing antibodies in a subject.
[0121] Encompassed within the scope of the invention are VLPs
comprising capsid protein of non-enveloped and enveloped viruses,
including rotaviruses, caliciviruses, hepatitis E virus, and human
papillomaviruses, influenza virus, hepatitis C virus, and
retrovirus, including human immunodeficiency virus. Preferably, the
VLPs comprise Papillomavirus L1 capsid protein.
[0122] Also encompassed within the scope of the invention are VLPs
derived from different species and genotypes of papillomaviruses.
Papillomaviruses of the invention are, for example, from human,
simian, bovine, or other origins. Preferably, the papillomavirus of
the invention is a human papillomavirus (HPV). More than 100
different human papillomavirus (HPV) genotypes have been isolated.
Human papillomavirus genotypes include, but are not limited to,
HPV-16, HPV-18, and HPV-45 for high-risk cervical cancers, HPV-31,
HPV-33, HPV-35, HPV-51, and HPV-52 for intermediate-risk cervical
cancers, and HPV-6, HPV-11, HPV-42, HPV-43, and HPV-44 for low-risk
cervical cancer and anogenital lesions (Bosch et al., 1995;
Walboomers et al., 1999). HPV genotypes are also disclosed in PCT
publication No. WO 92/16636 (Boursnell et al., 1992), incorporated
herein by reference in its entirety. HPV-16 is a preferred genotype
of the invention.
[0123] 4.2. Chimeric VLPs
[0124] Chimeric VLPs refer to viral capsid proteins that
encapsulate other viral proteins or heterologous gene products. A
preferred chimeric VLP according to the invention is a
papillomavirus L1 capsid protein, or peptide fragment thereof,
which encapsulate other papillomavirus gene products or
heterologous gene products during self-assembly into virus-like
particles. For example, gene products containing the HPV L2, E2,
E6, and/or E7 gene products become encapsulated into the HPV L1
VLPs and are considered herein as chimeric VLPs.
[0125] 4.2.1. Fusion Proteins
[0126] As one of skill in the art will appreciate, and as discussed
above, the HPV polypeptide of the invention can be fused to
heterologous polypeptide sequences. For example, the HPV L2
polypeptide of the present invention (including fragments or
variants thereof) may be fused to one or more additional HPV
polypeptide or other non-enveloped virus or enveloped virus
polypeptides.
[0127] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33,
1997; Harayama, Trends Biotechnol. 16(2):76-82, 1998; Hansson, et
al., J. Mol. Biol. 287:265-76, 1999; and Lorenzo and Blasco,
Biotechniques 24(2):308-13, 1998 (each of these patents and
publications are hereby incorporated by reference in its entirety).
DNA shuffling involves the assembly of two or more DNA segments by
homologous or site-specific recombination to generate variation in
the polynucleotide sequence.
[0128] In another embodiment, polynucleotides of the invention, or
the encoded polypeptides, may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion
or other methods prior to recombination. In another embodiment, one
or more components, motifs, sections, parts, domains, fragments,
etc., of a polynucleotide encoding a polypeptide of the invention
may be recombined with one or more components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous
molecules.
[0129] Nucleic acids encoding the above fusion polypeptides can be
recombined with a gene of interest as an epitope tag (i.e., the
hemagglutinin ("HA") tag or flag tag) to aid in detection and
purification of the expressed polypeptide. For example, a system
described by Janknecht et al. allows for the ready purification of
non-denatured fusion proteins expressed in human cell lines. (See,
for example, Janknecht et al., Proc. Natl. Acad. Sci. USA
88:8972-897, 1991). In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that the open
reading frame of the gene is translationally fused to an
amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix-binding domain for the fusion protein. Extracts
from cells infected with the recombinant vaccinia virus are loaded
onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged
proteins can be selectively eluted with imidazole-containing
buffers.
[0130] The cloning and expression of the L2 and L2 fusion genes can
be achieved following the methods known in the art. One example of
such methods is exemplified at Example 4 herein.
[0131] 5. Production, Isolation and Purification of Recombinant
VLPs
[0132] The present invention, as disclosed and described herein,
provides methods for production, isolation and purification of
recombinant viral gene products that are capable of self-assembly
into intact virus-like particles exhibiting conformational
antigenic epitopes. The virus-like particles of the invention can
be prepared as pharmaceutical compositions or 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.
[0133] 5.1. Production of VLPs
[0134] The present invention encompasses process for producing a
recombinant virus-like particle including, for example, a
virus-like particle of an enveloped virus or a non-enveloped virus,
by infecting a permissive insect cell with a recombinant
baculovirus that encodes viral capsid and/or envelope genes of one
or more viruses. In one embodiment, the invention provides methods
for harvesting and purifying HPV VLPs, including HPV chimeric VLPs
from infected insect cells or other host cells. The
baculovirus-infected cell expresses viral capsid and/or envelope
proteins that self-assemble into virus-like particles. The VLPs are
expressed intracellularly, extracellularly, or both.
[0135] In a preferred embodiment of the invention, the VLPs are
produced extracellularly.
[0136] According to another embodiment of the invention, there is
provided a method for the production of intracellular and
extracellular HPV VLPs. In this embodiment as depicted step 505 of
FIG. 5, production of intracellular and extracellular HPV-16 L1
VLPs begins with high multiplicity infection of log phase Sf-9S
cells with an aliquot of a working virus stock, as depicted in step
504 of FIG. 5, of baculoviruses expressing HPV L1 capsid proteins.
The virus infection can be monitored daily by the trypan blue
exclusion method for cytopathic effects, cell viability, and cell
density and by SDS-PAGE and Western blot analyses of recombinant
HPV L1 capsid proteins in infected cells and extracellular media.
At peak recombinant HPV L1 gene expression, infected cells and
extracellular media containing intracellular and extracellular HPV
L1 VLPs, respectively, are harvested and processed to obtain
purified HPV L1 VLP products as outlined in step 506 of FIG. 5 and
in depicted in more detail in FIG. 6 described below.
[0137] 5.2. Production of Chimeric VLPs
[0138] According to yet another embodiment of the invention, there
is provided a method for production of chimeric VLPs. In a
preferred embodiment, the chimeric VLPs are HPV chimeric VLPs. In
one embodiment, as depicted in FIG. 5, production of intracellular
HPV chimeric VLPs begins with high multiplicity infection of log
phase Sf-9S insect cells in suspension cultures containing
serum-free insect cell medium with aliquots of HPV L1 and L2 fusion
working virus stocks (i.e., recombinant baculoviruses expressing L1
capsid protein and L2 fusion protein, respectively).
[0139] The ratio of co-infecting viruses is approximately at least
about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,
1:12, 1:13, 1:14, 1:15, or more. In a preferred embodiment the
ratio of co-infecting viruses is approximately at least about 1:3,
1:5 or 1:10 (L1 to L2 virus). The virus infection is monitored
daily by the trypan blue exclusion method for cytopathic effects,
cell viability, and cell density and by SDS-PAGE and Western blot
analyses of recombinant HPV L1 and L2 fusion proteins in infected
cells. At peak recombinant HPV L1 and L2 fusion gene expression,
infected cells containing intracellular HPV chimeric VLPs are
harvested and processed to obtain purified HPV chimeric VLPs
products as outlined in FIG. 5 and described in more detail below
with reference to FIG. 7. This co-infection process may be used to
manufacture recombinant papillomavirus chimeric VLPs of various
viral genotypes including, but not limited to, those identified
above associated with HPV infections and cancers.
[0140] 5.3 Upstream Processing of L1 VLPs
[0141] The present invention also includes methods for upstream
processing of transformed host cells including yeast, insect, and
mammalian cells expressing recombinant VLPs. The recombinant VLPs
are preferably L1 VLPs. More preferably the recombinant VLPs are
HPV L1 VLPs.
[0142] In one embodiment, the process for purifying recombinant
extracellular HPV L1 VLPs includes at least one of the steps of
harvesting a cell suspension containing recombinant extracellular
HPV L1 VLPs to produce a harvested supernatant, clarifying the
harvested supernatant, concentrating the clarified supernatant, and
dialyzing the concentrated supernatant.
[0143] In another embodiment, the process for purifying
intracellular recombinant HPV L1 VLPs includes at least one of the
steps of harvesting a cell suspension comprising infected cells
containing intracellular recombinant papillomavirus L1 VLPs, and
disrupting the harvested cells (which may have been resuspended in
a buffer containing protease inhibitors) by, for example,
sonication to produce crude infected cell lysates containing
recombinant HPV L1 VLPs. The crude infected cell lysates is then
clarified by, for example, centrifugation to produce a clarified
supernatant containing recombinant HPV L1 VLPs. The clarified
supernatant is then concentrated by, for example, ultrafiltration
to produce a concentrate containing recombinant HPV L1 VLPs, and
dialyzing concentrates, for example, against high salt buffers by
ultrafiltration to produce a diafiltered crude product containing
recombinant HPV L1 VLPs. The crude product is further processed by
downstream processing as described below.
[0144] One example of the upstream processing of L1 VLPs is shown
by FIG. 6. Recombinant HPV L1 VLPs from baculovirus-infected insect
cells (step 601) or from other cells expressing recombinant HPV
VLPs are harvested by, for example, low-speed centrifugation (step
602). In step 603 of the embodiment as depicted in FIG. 6,
extracellular supernatants are separated from cell pellets by, for
example, aspiration following centrifugation and, at step 604, are
clarified by, for example, centrifugation to remove large cell
debris from extracellular recombinant HPV L1 VLPs. At step 605 of
the embodiment depicted in FIG. 6, cell pellets containing
intracellular recombinant HPV VLPs are discarded or resuspended in
multifold cell volumes of buffer solution such as
phosphate-buffered saline solution to produce a cell suspension. In
step 606 of the embodiment depicted in FIG. 6, resuspended cells
containing intracellular recombinant HPV L1 VLPs are disrupted
with, for example, several pulses of sonication to produce crude
cell lysates without proteosome disruption. The cell sonicates
containing HPV L1 VLPs are monitored for cell disruption by, for
example, the trypan blue exclusion method. In step 607 of the
embodiment depicted in FIG. 6, crude cell lysates containing
intracellular HPV L1 VLPs are clarified by, for example,
centrifugation to remove large cellular debris.
[0145] In another embodiment, clarified supernatants from
supernatant media and cell lysates are combined as depicted in step
608 and concentrated multifold by, for example, ultrafiltration
using hollow fiber filters (step 609). The concentrates are
dialyzed against multiple volumes of buffer by, for example,
ultrafiltration using hollow fiber filters (step 610).
[0146] Biological products including mock and/or wild type
baculovirus-infected insect cells as described above may be used to
prepare cell lysates containing host cells and/or baculovirus
proteins. The proteins in the cell lysates are solubilized by, for
example, sonication to disrupt cells and clarified by
centrifugation as described above and mixed with Freund's or other
adjuvant to produce immunogens.
[0147] Immunogenicity of the immunogens obtained above can be
determined according to methods known in the art. For example, the
immunogens can be administered at least once by intramuscular,
subcutaneous, or intranasal routes into animals. After testing sera
or immune cells from immunized animals for antigen specificity, the
sera or immune cells from immunized animals were isolated following
vaccination. The antibody titer specific for host cell and/or
baculovirus proteins were determined by immunodetection methods
such as ELISA or Western blot assays. The titered antisera can were
usec in immunodetection assays such as Western blot analysis to
determine the level of host cell and/or baculovirus present in
recombinant protein products derived from baculovirus-derived
sources. T cell assays such as lumphocute proliferation assays and
ELISPOT assays, which are used to determine the abundance of CD8+
cytotoxic T cells and the level of accompanying lymphokines and
cytokines produced as a result of sensitization with the VLPs in
this invention following immunization.
[0148] 5.4 Upstream Processing of Chimeric VLPs
[0149] In another embodiment of the invention, methods for upstream
processing of expressed VLPs are provided. In a preferred
embodiment, the invention discloses the upstream processing of HPV
VLPs and chimeric VLPs derived from insect cells co-infected with
recombinant baculoviruses expressing L1 and L2 fusion genes.
[0150] In a specific embodiment, the upstream processing of
chimeric VLPs generally includes one or more of the steps of
harvesting a cell suspension comprising co-infected cells
containing intracellular recombinant HPV chimeric VLPs,
resuspending the harvested infected cells in a buffer containing a
protease inhibitor, disrupting the resuspended infected cells by,
for example, sonication, to produce crude infected cell lysates
containing recombinant HPV chimeric VLPs, clarifying the crude
infected cell lysates by, for example, centrifugation, to produce a
clarified supernatant containing recombinant HPV chimeric VLPs,
concentrating the clarified supernatants by, for example,
ultrafiltration, to produce a concentrate containing recombinant
HPV chimeric VLPs, and dialyzing concentrates against high salt
buffers by, for example, ultrafiltration, to produce a diafiltered
crude product containing recombinant HPV chimeric VLPs. The crude
product thus obtained goes through downstream processing.
[0151] In a preferred embodiment, upstream processing of the
invention is performed following steps of the method depicted in
FIG. 7. Recombinant HPV chimeric VLPs are harvested from
transformed cells, for example, baculovirus-infected insect cells,
by, for example, low-speed centrifugation (step 702). At step 703,
supernatant media are separated from cell pellets by, for example,
aspiration following centrifugation. At step 704 supernatants media
are discarded and cell pellets containing intracellular recombinant
papillomavirus chimeric VLPs were resuspended in multiple cell
volumes of a buffer solution containing protease-inhibitors that
block the activity of at least one of the following protease
classes: serine, aspartate, cysteine, and metallo. In step 705,
resuspended cells containing intracellular recombinant HPV chimeric
VLPs are disrupted with, for example, several pulses of sonication
to produce crude cell lysates without proteosome disruption that
may cause proteolysis of chimeric L2 fusion proteins. The cell
sonicates containing HPV chimeric VLPs are monitored for cell
disruption by the trypan blue exclusion method. In step 706, crude
cell lysates containing intracellular HPV chimeric VLPs were
clarified by, for example, centrifugation to remove large cellular
debris. Clarified supernatants from cell lysates are concentrated
multifold by ultrafiltration using hollow fiber filters (step 707).
The concentrates are dialyzed against multiple volumes of buffer
containing protease inhibitors by ultrafiltration using hollow
fiber filters (step 708).
[0152] 5.5 Downstream Processing of VLPs
[0153] In yet another embodiment of the invention, methods for
downstream processing of expressed VLPs are provided. In a
preferred embodiment, the invention discloses the downstream
processing of HPV VLPs harvested from cells.
[0154] Downstream processing as depicted in FIGS. 5 and 8A-8C
includes one or more of the following steps: a linear sucrose
gradient scheme (FIG. 8A), a chromatographic scheme (FIG. 8B), or a
sucrose step gradient scheme (FIG. 8C). The product of these
purification schemes yields recombinant HPV VLPs that are
formulated to inactivate residual baculovirus contaminants by one
or more of the following treatments: detergent treatment to remove
process excipients; by ultrafiltration, to provide a buffer
solution that promotes VLP stability; by diafiltration, to remove
any microbial contaminants by terminal filtration.
[0155] 5.5.1. Downstream Processing of VLPs: Linear Sucrose
Gradient Scheme
[0156] The methods of the present invention encompasses downstream
processing of crude materials containing recombinant VLPs by one or
more of the following steps: sedimenting the recombinant VLPs
through a first linear sucrose gradient (for example, by continuous
flow ultracentrifugation), collecting fractions from the first
gradient, identifying fractions containing recombinant VLPs,
pooling fractions containing recombinant VLPs, rebanding pooled
fractions through a second linear sucrose gradient, collecting
fractions from a second gradient, and pooling fractions containing
recombinant VLPs from the second gradient. In an embodiment, only
one sedimentation step is used.
[0157] In one specific embodiment depicted in FIG. 8A, diafiltered
concentrates (step 610 of FIG. 6 and step 708 of FIG. 7) or other
materials containing VLPs are purified by continuous flow
rate-zonal ultracentrifugation on linear sucrose gradients, based
primarily on the mass and density of recombinant VLPs in sucrose
(FIGS. 5 and 8A). For example, diafiltrates containing recombinant
HPV VLPs are loaded under pressure onto approximately 0-65% linear
sucrose gradients in a vertical rotor accelerating at high speed in
a continuous flow ultracentrifuge. The gradient is resolved by
ultracentrifugation at high speed until recombinant HPV VLPs
separate from baculovirus particles (step 802). Gradient materials
from the first round of sucrose gradients are monitored by
ultraviolet light during collection in a fraction collector.
Gradient fractions from the first round of linear sucrose gradients
are analyzed by SDS-PAGE and Western blot analysis using antisera
against papillomavirus L1 capsid proteins and/or L2 fusion
proteins. Peak fractions containing HPV VLPs or their component
proteins are pooled, diluted multifold with buffer solution, and
are subjected to a second round of ultracentrifugation on linear
sucrose gradients (step 803).
[0158] In another embodiment, gradient materials from the second
round of sucrose gradients are monitored by ultraviolet light
during collection in a fraction collector. Gradient fractions from
the second round of ultracentrifugation may be analyzed also by
SDS-PAGE and Western blot analysis using antisera against
papillomavirus L1 capsid proteins and/or L2 fusion proteins. Peak
fractions containing HPV VLPs or their component proteins are
pooled. The purified recombinant HPV VLPs in the pooled fractions
are formulated as recombinant HPV VLP products.
[0159] 5.5.2. Downstream Processing of VLPs: Chromatographic
Scheme
[0160] The chromatographic method for downstream processing of
VLPs, and preferably HPV VLPs includes at least one of the
following three steps: adsorptive cation exchange chromatography
using a pH gradient, affinity chromatography using heparin-like
matrices for binding VLPs, and displacement anion exchange
chromatography.
[0161] In the first chromatography step, diafiltered concentrates
(step 610 of FIG. 6 and step 708 of FIG. 7) or other materials
containing HPV papillomavirus VLPs, are loaded in the initial
chromatographic step (step 805 of FIG. 8B) onto a chromatography
column containing a strong cation exchange chromatography resin
with an exposed amino group such as Streamline SP (Amersham
Biosciences) that is equilibrated with multiple volumes of loading
buffer at low salt and a pH between 4.5 to 6.0. This step separates
recombinant HPV VLPs from the bulk majority of host contaminant
proteins and other molecules based on the isoelectric charge of HPV
L1 proteins.
[0162] Following binding of diafiltrates or other materials
containing recombinant papillomavirus VLPs to the charged resin,
the bound column is washed with multiple volumes of loading buffer.
In one embodiment, bound recombinant papillomavirus VLPs are eluted
from the column resin using a low salt pH step gradient from 6.0 to
8.0. Elution fractions are analyzed by SDS-PAGE and Western blot
analysis using antisera against papillomavirus L1 capsid proteins.
Purification of chimeric VLPs by this chromatographic scheme has
not been tried to date. In another embodiment, peak fractions
containing VLPs or their component proteins are pooled.
[0163] In the second chromatography step (FIG. 8, step 806), the
pooled eluates from step 805 resulting from the cation exchange
chromatography step or other materials containing recombinant
papillomavirus VLPs are dialyzed against multiple volumes of
affinity loading buffer by diafiltration. The dialysate is loaded
onto a column containing heparin agarose or other molecules having
an exposed carboxy group such as, for example, heparin sulfate
glycans, glycoaminoglycans, .alpha..sub.6.beta..sub.1 integrin,
.alpha..sub.6.beta..sub.4 integrin, syndecan 1, Matrex Cellufine
Sulfate (American Biosciences), or other heparin-like resins
equilibrated with affinity loading buffer. Affinity chromatography
using heparin serves as receptors for papillomavirus as binding
matrices and affords high levels of specific and selective
purification of recombinant HPV VLPs.
[0164] The bound column is washed with multiple volumes of affinity
loading buffer. Bound proteins including recombinant papillomavirus
VLPs, are eluted from the column resin using a linear salt gradient
from approximately 300 mM to 2 M. Elution fractions are analyzed by
SDS-PAGE and Western blot analysis using antisera against
papillomavirus L1 capsid proteins and/or L2 fusion proteins. Peak
fractions containing VLPs or their component proteins are
pooled.
[0165] In the third chromatography step, pooled eluates from the
affinity chromatography step (step 806), that contain recombinant
papillomavirus VLPs are dialyzed against multiple volumes of anion
loading buffer by dialfiltration. The removal of small molecular
weight molecules and residual host contaminant proteins in pooled
affinity eluates or other material containing recombinant HPV VLPs
is provided by anion exchange chromatography (FIG. 8 step 807)
using a displacement polymer as a final polishing step based on the
isoelectric point of HPV L1 proteins. The dialysate is loaded onto
a column containing a strong anion exchange chromatography resin
with an exposed carboxyl group such as, for example, Q Sepharose
Fast Flow (FF) (Amersham Biosciences), Toyopearl Super Q-650 M
(Tosoh Biosep), Q Sepharose FF, or Fractogel TMAE (USB)
equilibrated with multiple volumes of anion loading buffer.
[0166] Bound proteins including recombinant HPV VLPs are displaced
from the anion column resin with a linear gradient from
approximately 0 to 5 mg/ml dextran sulfate (5000 MW). Elution
fractions are analyzed by, for example, SDS-PAGE and Western blot
analysis using antisera against papillomavirus L1 capsid proteins.
If required, peak fractions containing recombinant HPV VLPs or
their component proteins are pooled and dialyzed by ultrafiltration
against multiple volumes of high salt buffer to remove dextran
sulfate.
[0167] 5.5.3 Downstream Processing of VLPs: Sucrose Step Gradient
Scheme
[0168] In yet another alternate embodiment of the present
invention, diafiltered concentrates (step 610 of FIG. 6 and step
708 of FIG. 7) or other materials containing VLPs are purified by
rate-zonal ultracentrifugation on discontinuous sucrose step
gradients based primarily on mass and density of recombinant VLPs
in sucrose.
[0169] The methods of the present invention encompasses downstream
processing of recombinant VLPs and preferably recombinant HPV VLPs
from crude materials by pelleting crude materials containing
recombinant HPV VLPs through a sucrose cushion, resuspending the
pelleted recombinant HPV VLPs, banding resuspended recombinant HPV
VLPs by ultracentrifugation on discontinuous linear step gradients,
collecting at least one bands containing recombinant HPV VLPs, and
dialyzing banded material by diafiltration to remove sucrose.
[0170] In one embodiment depicted in FIG. 8C, diafiltrates
containing recombinant HPV VLPs are loaded onto approximately 25%
sucrose cushions in a swinging bucket rotor accelerating at high
speed in an ultracentrifuge (step 809 of FIG. 8). The pellets at
the bottom of the sucrose cushion are collected, while the sucrose
cushion and load material are discarded. The sucrose cushion
pellets are solubilized in buffer and loaded onto sucrose step
gradients containing multiple steps comprising approximately 25 to
65% sucrose. The sucrose step gradients are resolved by
ultracentrifugation in a swinging bucket rotor at high speed until
recombinant HPV VLPs are separated from baculovirus particles (step
810). Gradient materials from the first round of sucrose step
gradients are monitored by ultraviolet light during collection in a
fraction collector. Gradient fractions are analyzed by, for
example, SDS-PAGE and Western blot analysis using antisera against
papillomavirus L1 capsid proteins and/or L2 fusion proteins.
[0171] Peak fractions containing HPV VLPs or their component
proteins are pooled, diluted multifold with buffer solution, and
optionally subjected to a second round of ultracentrifugation on
sucrose step sucrose gradients (step 811). Gradient materials from
the second round of sucrose step gradients are monitored by
ultraviolet light during collection in a fraction collector.
Gradient fractions from the second round of ultracentrifugation are
analyzed by, for example, SDS-PAGE and Western blot analysis using
antisera against papillomavirus L1 capsid proteins and/or L2 fusion
proteins. Peak fractions containing HPV VLPs or their component
proteins are pooled. In an embodiment, the purified recombinant HPV
VLPs in the pooled fractions are formulated as recombinant HPV VLP
products.
[0172] 5.6. Formulation of Papillomavirus VLP Products
[0173] According to the present invention as depicted in FIG. 5,
pooled fractions or other material containing recombinant VLPs from
the purification schemes described above may contain recombinant
baculovirus particles, which are inactivated by treatment with, for
example, a nonionic detergent, surfactants, ultraviolet light, or a
combination thereof. In an embodiment of the present invention, a
nonionic detergent, and a surfactant, such as Triton X-100, is
added to the product containing recombinant HPV VLPs at a final
concentration of approximately >0.1%. The recombinant
papillomavirus VLP mixture with detergent is incubated for at least
approximately one hour to inactivate residual baculoviruses.
[0174] In an another embodiment, the recombinant HPV VLPs are
irradiated with one or more rounds of ultraviolet (UV) light
<300 nm and then incubated in a nonionic detergent, or both.
Multiple log reduction of the baculovirus is afforded by these
treatments which may have additive or synergistic effect. The
process for inactivating residual baculovirus products are also
used for other recombinant protein products, including recombinant
protein products comprising VLPs of virus types identified above.
In addition, VLP products treated according to this process are
dialyzed against a buffer in order to refold the conformational
epitopes of the VLPs in the product.
[0175] In yet another embodiment, following the baculovirus
inactivation treatment(s), recombinant HPV VLP products are
dialyzed by ultrafiltration against multiple volumes of high salt
buffer containing approximately >0.5 M sodium chloride at
approximately neutral pH to remove process excipients such as
sucrose, Triton X-100 detergent, and other molecules. Diafiltrates
containing recombinant HPV VLP bulk products are filtered
aseptically through a 0.2 .mu.m membrane at ambient temperature to
remove microbial contaminants. To maintain high levels of intact
VLPs in the final bulk products, the filtered recombinant HPV VLPs
are dispensed directly into sterilized 316 L stainless steel tanks,
silanized borosilicate glass bottles, or polyethylene plastic
bioprocess bags and stored at 2-8.degree. C. for <six (6)
months, or at <-70.degree. C. for 2 years.
[0176] Bulk recombinant HPV VLP products made according to the
present invention are formulated alone or with adjuvants such as,
for example, Novasomes.TM. and micelle nanoparticles, among others.
For monovalent products, bulk products containing one genotype of
recombinant HPV VLPs are diluted with buffer solution to the
appropriate antigen concentration such as 100 .mu.g/ml, mixed with
an adjuvant, adjusted for final pH and salt concentrations,
filtered aseptically through 0.2 .mu.m membranes, and dispensed
into silanized borosilicate vials. For multivalent products, equal
molar antigen concentrations of bulk products representing more
than one genotype of recombinant HPV VLPs are formulated and
processed into final container products as described above for
monovalent products. Final container products are stored at
2-8.degree. C. for <6 months or <-70.degree. C. for extended
time such as two years or less. Following qualification of final
container products for purity, strength, identity, potency, and
safety, final container products are used as pharmaceutical
composition, prophylactic vaccines, or diagnostic reagents.
[0177] In one embodiment, prophylactic vaccines for the prevention
of anal genital warts are formulated as mixtures of at least HPV-6
and/or HPV-11 L1 or chimeric VLPs. Prophylactic vaccines for the
prevention of HPV-induced cervical cancer are formulated as
mixtures of at least HPV-16, HPV-18, HPV-31, and/or HPV-33 L1 or
chimeric VLPs. Therapeutics for treatment of anal genital warts are
formulated as mixtures of at least HPV-6 and/or HPV-11 chimeric
VLPs. Pharmaceutical compositions for treatment of HPV-induced
cervical cancer are formulated as mixtures of at least HPV-16,
HPV-18, HPV-31, and HPV-33 chimeric VLPs.
[0178] These and other products comprising recombinant VLPs made
according to the present invention are administered by various
parenteral and local routes including but not limited to
intramuscular, intradermal, intranasal, or oral, according to
conventional protocols. Reagents used for diagnosis of HPV
infections and associated neoplasia may be formulated as
type-specific products capable of detecting antibodies for one or
more genotypes of HPV.
[0179] 5.7. Characterization of VLP in Final Bulk Products
[0180] Immunological identification of recombinant products made
according to the present invention is afforded by, for example,
Western blot analysis using polyclonal sera for HPV L1 capsid
antigens (linear epitopes) or by enzyme linked immunoadsorbent
assay (ELISA) using monoclonal antisera for PV L1 conformational
epitopes specific for neutralizing antibodies. For Western blot
analyses, aliquots (2 .mu.g) of recombinant proteins from crude
lysates, purified intermediates, or purified VLPs and control L1
capsid proteins are heat denatured (5-10 min. at 95-99.degree. C.)
under reduced conditions with .beta.-mercaptoethanol (10 mM) and
loaded onto 4-12% NuPAGE (Novex) protein gels (FIG. 9) or
equivalent polyacrylamide gels.
[0181] Proteins are resolved by gel electrophoresis in MES buffer
under reduced conditions. Control proteins include recombinant PV
L1 capsid proteins verified for authenticity, host cell proteins,
and/or AcMNPV baculovirus proteins. Protein molecular weight
markers are, for example, SeeBlue pre-stained standards (Novex)
including proteins with molecular weights of 188 kilodaltons (kD),
62 kD, 49 kD, 38 kD, 28 kD, 18 kD, 14 kD, 6 kD, and 3 kD. For
protein gels, the electrophoresced proteins are visualized by
staining with Colloidal Coomassie Blue reagent (Novex). The
molecular weights of the L1 proteins are 50-65 kD depending on the
species and genotype of papillomavirus capsid gene. The purity of
purified recombinant HPV L1 VLPs purified by the invention is
expected to be 95% or more as determined by scanning densitometry.
No more than 5% of the purified recombinant HPV L1 VLP product is
expected to be proteolytic breakdown products.
[0182] For Western blot analysis (FIGS. 10A-10C, as also described
with reference to Example 21, below), proteins are transferred by
electroblotting in methanol from unstained protein gels containing
L1 capsid proteins and control proteins to nitrocellulose or
polyvinyldifluoride membranes. Bound membranes are reacted with
primary antisera including antisera to PV L1 capsid proteins,
polyclonal sera to host cell proteins, and/or polyclonal sera #3 to
AcMNPV wild type baculovirus proteins. Bound primary antibodies are
reacted with secondary antisera comprised of anti-IgG conjugated to
alkaline phosphatase. The bound secondary antibodies are detected
by reacting with the chromogenic substrate such as NBT/BCIP
(InVitrogen) or the chemiluminescent substrate Lumi-Phos (In
Vitrogen). The anti (.alpha.)-papillomavirus L1 sera is expected to
detect protein bands with molecular weights of about 50 to 65 kD
depending on the species and genotype of papillomavirus L1 capsid
gene. Less than 5% of the recombinant PV L1 VLP products purified
by the invention is expected to be degradation breakdown products.
Less than 5% reactivity is expected to be seen using antisera to
host or vector proteins.
[0183] The potency of recombinant HPV VLPs according to the present
invention are ascertained by, for example, ELISA testing using
antibodies specific for conformational epitopes on papillomavirus
L1 capsid proteins that elicit neutralizing antibodies. In one
embodiment of the present invention, ELISA testing of recombinant
HPV-16 L1 VLP bulk products is performed using murine monoclonal
antibody H16.V5 (Christensen et al., 1996). Dilutions of VLPs
(antigen) and control proteins such as VLPs, denatured VLPs, and
heterologous proteins are bound to wells of ELISA plates, and a
constant amount of monoclonal antibody is added to each well.
[0184] Antigen-antibody binding occurs for at least two time
durations, such as one minute and 2.5 hours. Antigen-antibody
complexes are washed successively with wash buffer to remove
nonspecific antigens. A secondary antibody comprised of anti-murine
immunoglobulins conjugated to an enzyme such as horseradish
peroxidase is added to each well of the ELISA plate. Detection of
antigen-antibody complexes is afforded by the addition of a
chromogenic substrate such as NBT/BCIP (InVitrogen).
[0185] As depicted in FIG. 11, and also described with reference to
Example 20, below, the L1 proteins of Triton-treated recombinant
HPV VLPs made according to the methods of the present invention are
not degraded, as determined by SDS-PAGE and Western blot analyses.
Triton-treated recombinant HPV VLPs remain as intact VLPs, as
determined by analytical size exclusion chromatography and/or
electron microscopy. The conformational epitopes of Triton-treated
recombinant HPV VLPs made according to the methods of the present
invention are restored by diafiltration against 0.5 M sodium
chloride buffers, as determined by ELISA analysis using monoclonal
antibodies raised against neutralizing epitopes of L1 antigens such
as H16.V5 monoclonal antibody.
[0186] The amount of total protein purified by the methods of the
present invention are determined by one of several calorimetric
methods such as the bicinchoninic acid (BCA) assay or other protein
quantitation assay by one skilled in the art of protein chemistry.
The absolute amount of protein are determined by acid hydrolysis
and amino acid determination. The results are compared with those
results from calorimetric assays to adjust the relative
amounts.
[0187] The amount of intact VLPs present following purification of
recombinant HPV VLPs according to the present invention are
ascertained by analytical size exclusion chromatography and/or
electron microscopy. Size exclusion chromatography (SEC) are used
to assess the relative amount of VLPs in production lots of HPV
VLPs and and the relative amount of other viral VLPs made according
to the present invention. In one embodiment, the pre-poured column
used for SEC HPLC is an analytical size-exclusion HPLC column such
as a TSK-GEL G6000PWXL column (Tosoh Biosep) that is used with, for
example, a fractionation range of more than 1,000,000 daltons to
approximately 20,000 daltons. The test sample is applied to the
column with a resolution for intact VLPs at approximately 15-16
minutes and monomeric proteins at approximately 24-26 minutes.
[0188] Other macromolecules such as capsomeres of pentameric HPV L1
structures are resolved at 19-20 minutes when present. An
analytical HPLC system such as a Waters 6000 HPLC system using
Millennium computer software provides the mechanics and programs
necessary for sample injection, buffer transfer, column
development, UV monitoring, fraction collection, and protein data
management. Data is presented in a graphic format with protein
absorbance as a function of column development in minutes, as
exemplified by FIG. 12 and also described with reference to Example
20, below. Confirmation of SEC HPLC results on viral VLPs is
obtained by negative-stain electron microscopy (EM). Purified
recombinant HPV VLPs are adsorbed onto carbon coated transmission
electron microscopy (TEM) grids, stained with 1% uranyl acetate,
and examined with a Philips electron microscope at 36,000.times.
magnification. Results are shown in FIG. 13, also described with
reference to Example 20, below. The size of the HPV-16 L1 VLPs is
estimated about 40-55 nm.
[0189] 6. Pharmaceutical Compositions
[0190] The present invention also provides pharmaceutical
compositions comprising a therapeutically effective amount of one
or more recombinant viral gene products, VLPs, agonists,
antagonists, or a biologically active fragment of a viral gene
product. The recombinant papillomavirus gene products preferably
comprise HPV VLPs. More preferably, VLPs are HPV L1 VLPs, or
chimeric VLPs. Administration of the pharmaceutical compositions of
the invention, including vaccines, results in a detectable change
in the physiology of a recipient subject, preferably by enhancing a
humoral or cellular immune response to one or more papillomavirus
antigens.
[0191] A multivalent vaccine of the present invention can confer
protection to one or more genotypes of papillomavirus. The present
invention thus concerns and provides a means for preventing or
attenuating infection by at least one papillomavirus genotype. As
used herein, a vaccine is said to prevent or attenuate a disease if
its administration to an individual results either in the total or
partial attenuation (i.e., suppression) of a symptom or condition
of the disease, or in the total or partial immunity of the
individual to the disease.
[0192] The "protection" provided need not be absolute, i.e., the
papillomavirus infection need not be totally prevented or
eradicated, provided that there is a statistically significant
improvement relative to a control population. Protection can be
limited to mitigating the severity or rapidity of onset of symptoms
of the disease.
[0193] The pharmaceutical preparations of the present invention,
suitable for inoculation or for parenteral or oral administration,
are in the form of sterile aqueous or non-aqueous solutions,
suspensions, or emulsions, and can also contain auxiliary agents or
excipients that are known in the art. The pharmaceutical
composition of the invention can further comprise immunomodulators
such as cytokines which accentuate the immune response. (See, i.e.,
Berkow et al., eds.: The Merck Manual, Fifteenth Edition, Merck and
Co., Rahway, N.J., 1987; Goodman et al., eds., Goodman and Gilman's
The Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon
Press, Inc., Elmsford, N.Y., 1990; Avery's Drug Treatment:
Principles and Practice of Clinical Pharmacology and Therapeutics,
Third Edition, ADIS Press, LTD., Williams and Wilkins, Baltimore,
Md., 1987; and Katzung, ed. Basic and Clinical Pharmacology, Fifth
Edition, Appleton and Lange, Norwalk, Conn., 1992, which references
and references cited therein, are entirely incorporated herein by
reference as they show the state of the art.
[0194] As would be understood by one of ordinary skill in the art,
when a composition of the present invention is provided to an
individual, it can further comprise at least one of salts, buffers,
adjuvants, or other substances which are desirable for improving
the efficacy of the composition. Adjuvants are substances that can
be used to specifically augment at least one immune response.
Normally, the adjuvant and the composition are mixed prior to
presentation to the immune system, or presented separately.
[0195] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions.
[0196] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol,
mannitol, sorbitol, trehelose, and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like.
[0197] The pharmaceutical composition of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with anions such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with cations such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0198] Adjuvants can be generally divided into several groups based
upon their composition. These groups include lipid micelles, oil
adjuvants, mineral salts (for example, AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH4(SO.sub.4)), silica, kaolin,
polynucleotides (for example, poly IC and poly AU nucleic acids),
and certain natural substances, for example, wax D from
Mycobacterium tuberculosis, substances found in Corynebacterium
parvum, or Bordetella pertussis. Preferred adjuvant of the
invention includes, for example, Freund's adjuvant (DIFCO), alum
adjuvant (Alhydrogel), MF-50 (Chiron) Novasomes.TM., or micelles,
among others.
[0199] A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient patient. Such
an agent is said to be administered in a "therapeutically or
prophylactically effective amount" if the amount administered is
physiologically significant.
[0200] The pharmaceutical composition of the invention is
administration through various routes, including, subcutaneous,
intravenous, intradermal, intramuscular, intraperitoneal,
intranasal, transdermal, or buccal routes. Subcutaneous
administration is preferred. Parenteral administration are
achieved, for example, by bolus injection or by gradual perfusion
over time.
[0201] A typical regimen for preventing, suppressing, or treating a
disease or condition which can be alleviated by a cellular immune
response by active specific cellular immunotherapy, comprises
administration of an effective amount of the composition as
described above, administered as a single treatment, or repeated as
enhancing or booster dosages, over a period up to and including one
week to about 48 months.
[0202] According to the present invention, an "effective amount" of
a composition is an amount sufficient to achieve a desired
biological effect, in this case at least one of cellular or humoral
immune response to a papillomavirus genotype. It is understood that
the effective dosage will be dependent upon the age, sex, health,
and weight of the recipient, kind of concurrent treatment, if any,
frequency of treatment, and the nature of the effect desired. The
most preferred dosage will be tailored to the individual subject,
as is understood and determinable by one of skill in the art,
without undue experimentation.
[0203] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims. The contents of
all references, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
Example 1
ESTABLISHMENT OF SERUM-FREE SF-9 INSECT CELL LINE
[0204] A new insect cell line designated Sf-9S was derived from the
parent S. frugiperda Sf-9 cell line (ATCC CRL-1771) by several
rounds of selective processes based on serum-independent growth and
enhanced expression of secreted recombinant proteins from
baculovirus vectors. Specifically, Sf-9 cells were cultivated to
passage 38 in Grace's insect media (Life Technologies, Grand
Island, N.Y. 14072) supplemented with 10% fetal bovine serum (Life
Technologies, Grand Island, N.Y. 14072) as monolayer cultures in
T-75 flasks (Corning, Inc., Corning, N.Y.). The master cell bank of
Sf-9 cells was stored at passage 38 in serum-containing media at
-70.degree. C. and in liquid nitrogen. A working cell bank was
established from a single cryovial of the Sf-9 master cell bank and
cultivated in serum-containing insect media for an additional five
(5) passages.
[0205] Initially, cell clones capable of growing in commercial
serum-free media as suspension cultures were isolated from
monolayer cultures of parent Sf-9 cells dependent on
serum-containing media by sequential weaning of parent cells from
serum-containing media. This process involved the plating of cell
aliquots (200 .mu.l) from a cell suspension (one cell per 200
.mu.l) of the parent cell line in serum-containing media onto
96-well dishes at a ratio of 200 .mu.l per well. Following
attachment of cells and inspection of wells for wells with more
than one cell, the media was changed from serum-containing media
(100%) to a media mixture comprised of 75% serum-containing media
and 25% serum-free media. After one to two weeks in culture, the
media was changed from wells that initially contained only one cell
per well and demonstrated cell growth and replication (i.e. four to
five cells).
[0206] The second media mixture was comprised of 50%
serum-containing media and 50% serum-free media. The cells were
allowed to grow for another one to two weeks. The media was changed
from wells containing cells that continued to grow and replicate.
The third media mixture was comprised of 25% serum-containing media
and 75% serum-free media. The cells were allowed to grow and
replicate. After another two to four weeks, the media was changed
from wells containing cells that continued to grow and replicate.
The final media was comprised of serum-free media (100%). During
each round of the weaning process, more than 90% of the cells did
not survive the reduction in serum. This high level of cell death
created a selective pressure to permit development of a new cell
phenotype. Cells from wells that demonstrated continuous cell
growth and replication were harvested and seeded into larger
culture vessels. When a total cell density of >4.times.10.sup.6
cells was obtained, cells were seeded into shaker flasks (50 ml) as
10 ml suspension cultures with a starting cell density of
0.2-0.5.times.10.sup.6 cells/ml. Eight (8) clones that grew
exponentially to a saturation cell density of >6.times.10.sup.6
cells/ml in serum-free media was selected, expanded, and frozen.
One of the clones was established as a serum-free independent cell
line.
Example 2
ESTABLISHMENT OF TRANSFORMED SF-9S CELL LINE
[0207] In a second selection process, one of the serum-free cell
clones developed in Example 1 was chosen to select cell clones that
may produce enhanced levels of recombinant extracellular proteins
and VLPs from several viruses including rotaviruses and human
papillomaviruses by successive rounds of clonal selection of cells
infected with recombinant baculoviruses and expressing
extracellular self-assembled VLPs.
[0208] This process involved the plating of cell aliquots (200
.mu.l) from a cell suspension (one cell per 200 .mu.l) of the
parent cell clone (#23) in serum-free media onto 96-well dishes at
a ratio of 200 .mu.l per well. From wells containing a single cell
in the original seeding, cells were grown to confluency and
subcultured into six replica-plates (96-well). The first round of
selection was performed when a total cell density of
2-4.times.10.sup.3 cells/well was obtained; the cells were infected
with recombinant baculoviruses encoding human rotavirus virus VP2
and VP6 capsid genes. After three days of baculovirus infection,
the infected cells and extracellular media were harvested by
centrifugation. Infected cells were solubilized by adding 250 .mu.l
of 1% sodium dodecyl sulfate (SDS) and 10 mM .beta.-mercaptoethanol
(.beta.-ME). SDS and .beta.-ME were added to extracellular
supernatants to final concentrations of 1% and 10 mM, respectively.
Aliquots (10 .mu.l) of solubilized cell lysates and extracellular
media were heat-denatured (99.degree. C. for 10 min.) under reduced
conditions and analyzed by SDS-PAGE and Western blotting using
antisera to rotaviruses.
[0209] After review of the test results from the first virus
infection, twenty four (24) cell clones demonstrating the highest
levels of extracellular recombinant rotavirus VLPs were identified,
seeded into 96-well plates, grown to confluency, and infected with
a second recombinant baculovirus encoding HPV-16 L1 capsid protein.
At three days post-infection, infected cells and extracellular
supernatants were produced by centrifugation of infected cell
suspensions from the plate.
[0210] Infected cells and extracellular supernatants were analyzed
by SDS-PAGE and Western blot analyses using polyclonal anti-HPV-16
L1 sera. The test results of both viral infections were reviewed
and compared. One cell clone (#12) that produced the high levels of
extracellular VLPs from rotavirus and HPV capsid proteins was
chosen to establish a cell line capable of producing extracellular
VLPs. To establish a cell line from the selected cells, cells from
an uninfected replica plate were amplified at 28.degree. C. and 150
rpm in a platform shaker incubator into a suspension culture using
Sf-900 II serum free media (GIBCO). The amplified cell culture was
diluted to a seeding cell density of 0.25.times.10.sup.6 cells/ml,
grown in 100 ml of Sf-900 II SFM within a 500 ml shaker flask, and
subcultured at a split ratio of 1:20 for forty three passages.
After continuous passaging, the cell line was established and was
passaged three more times to establish a master cell bank.
Example 3
CLONING CODON-OPTIMIZED HPV-16 L1 GENES AND ESTABLISHMENT OF
RECOMBINANT BACULOVIRUS STOCKS
[0211] A HPV-16 L1 prototype (GenBank Accession No. K02718) and
modified in U.S. Pat. No. 5,985,610, was optimized for codon usage
in insect cells of the Lepidopteran family. The HPV-16 L1 gene was
optimized (FIG. 1A) in this embodiment of the present invention for
codon usage based on the following criteria: (1) abundance of
aminoacyl-tRNAs for a particular codon in Lepidopteran species of
insect cells for a given amino acid as described by Levin and
Whittome (2000); (2) maintenance of GC-AT ratio in L1 gene sequence
at approximately 1:1; (3) minimal introduction of palindromic or
stem-loop DNA structures, and (4) minimal introduction of
transcription and post-transcription repressor element
sequences.
[0212] The optimized gene sequence was synthesized in vitro as
overlapping oligonucleotides, cloned into a subcloning plasmid
vector, and then cloned into a bacmid transfer vector (i.e., Luckow
et al., 1993), according to procedures known in the art (i.e.,
Summers and Smith, 1987). The bacmid transfer vector pFASCTBAC1
with HPV-16 L1 gene was used to transform competent E. coli DH10BAC
cells and produce recombinant bacmid DNA. The recombinant bacmid
DNA with the L1 gene was transfected into insect cells to produce
recombinant baculoviruses encoding L1 genes.
[0213] In particular, a restriction fragment (Bam HI/Sal I
restriction fragment (1572 bp) containing a HPV-16 L1 gene (K
strain)) containing a HPV L1 capsid gene from a natural virus
isolate or synthesized gene is ligated to a bacmid transfer vector,
such as pFASTBAC-1 (see, for example, Luckow et al., 1993), at the
multiple cloning site, which contains a Tn7 transposable element
surrounded by the transcription promoter and
polyadenylation/transcription termination elements of the
polyhedrin (polh) gene from a wild type AcMNPV genome. Competent E.
coli DH10BAC cells, which contain bacmid DNA (an AcMNPV baculovirus
genome with a Tn7 transposable element within the polyhedrin
locus), are transformed with the bacmid transfer vector containing
the HPV L1 gene.
[0214] Recombinant bacmids are produced by site-directed
recombination between the respective Tn7 transposable elements of
the transfer vector and the bacmid genome resulting in the
production of recombinant bacmid genomes encoding the optimized L1
gene in the E. coli hosts. The recombinant bacmid DNA is isolated
for example by miniprep DNA isolation and transfected into Sf-9S
insect cells to produce recombinant baculoviruses encoding the L1
genes.
[0215] The progeny recombinant baculoviruses (.about.10.sup.4
plaque forming units) are plaque-purified (3.times.) and selected
for high expression of the HPV-16 L1 gene product, as determined by
SDS-PAGE and Western blot analyses using rabbit polyclonal antisera
specific for the HPV16 L1 gene product (Pharmingen). A HPV-16 L1
master virus stock is prepared in Sf-9S insect cells, as described
in Example 2, from one of the plaque-purified clones expressing
high levels of recombinant HPV-16 L1 proteins that self-assemble
into virus-like particles and is qualified for safety and
biological properties as described below. Working virus stocks of
HPV-16 L1-expressing baculoviruses are prepared by infection of
Sf-9S insect cells at a multiplicity of infection of 0.1 pfu/cell
with the qualified HPV-16 L1 master virus stock and are
characterized as described below to qualify for recombinant HPV-16
L1 VLP product manufacturing.
Example 4
CLONING CODON-OPTIMIZED HPV-16 CHIMERIC GENES AND ESTABLISHMENT OF
CHIMERIC RECOMBINANT BACULOVIRUS STOCKS
[0216] HPV-16 L2 fusion genes are optimized for codon usage in
insect cells as described above for L1 genes (FIGS. 1C-1E). The
L2/E7/E2 fusion gene sequence is synthesized in vitro as
overlapping oligonucleotides, cloned into a subcloning plasmid
vector, and then cloned into a bacmid transfer vector according to
procedures known in the art (i.e., Luckow et al., 1993; Summers and
Smith, 1987).
[0217] For example, a baculovirus transfer vector with a L2 fusion
gene is co-transfected into insect cells with linearized wild-type
baculovirus genomic DNA to produce recombinant baculoviruses
encoding L2 fusion genes. Alternatively, the bacmid transfer vector
with the L2 fusion gene is used to transform competent E. coli
DH10BAC cells and produce recombinant bacmid DNA. The
codon-optimized gene is cloned into a bacmid transfer vector (i.e.,
Luckow et al., 1993).
[0218] In particular, a Bam HI/Kpn I restriction DNA fragment (2834
bp) containing a HPV-16 L2/E7/E2 fusion gene was ligated with T4
DNA ligase to a Bam HI/Kpn I digest of the bacmid transfer vector
pFASTBAC-1 (Luckow et al., 1993) at the multiple cloning site.
Competent E. coli DH10BAC cells were transformed with the bacmid
transfer vector containing the HPV-16 L2/E7/E2 fusion gene.
Recombinant bacmids were produced by site-directed recombination
between the respective Tn7 transposable elements of the transfer
vector and the bacmid genome resulting in the production of
recombinant bacmid genomes encoding the optimized HPV L1 gene in
the E. coli hosts.
[0219] The recombinant bacmid DNA was isolated by miniprep DNA
isolation and transfected into Sf-9S insect cells to produce
recombinant baculoviruses encoding the HPV-16 L2/E7/E2 L2 fusion
genes. The recombinant baculoviruses were plaque-purified
(3.times.) in Sf-9S insect cells and selected for high expression
of the HPV-16 L2, E7, and E2 gene products, as determined by
SDS-PAGE and Western blot analyses using antisera specific for each
peptide within the L2 fusion gene product (HPV-16 L2, E7, and E2
peptides). A master virus stock of baculoviruses expressing HPV-16
L2/E7/E2 fusion proteins was prepared in Sf-9S insect cells from
one of the plaque-purified clones expressing high levels of
recombinant HPV L2 fusion proteins and was qualified for safety and
biological properties. Working virus stocks of baculoviruses
expressing HPV-16 L2/E7/E2 fusion proteins were prepared in Sf-9S
insect cells at a multiplicity of infection of 0.1 pfu/cell with
the qualified master virus stock and were tested.
Example 5
CHARACTERIZATION AND QUALIFICATION OF L1 AND CHIMERIC RECOMBINANT
BACULOVIRUS STOCKS
[0220] Recombinant baculovirus stocks for each of the HPV L1 and L2
fusion viruses were established. Master and working virus stocks
were established from high expression virus clones and
characterized for safety and biological properties. Safety
properties of master and working virus stocks included microbial
sterility, adventitious agent presence, endotoxin level,
spiroplasma, and mycoplasma contaminants, and the like. Biological
properties included genetic identity, virus titer, viral
replication competence, and recombinant protein production
competence. The genetic identity of the master virus stock was
determined, for example, by DNA sequence analysis of both strands
of bacmid DNA encoding the HPV L1 or L2 fusion genes and flanking
sequences. The virus titer of master and working virus stocks were
determined by an agarose plaque assay using insect cells and serial
dilutions of the virus stock.
[0221] Viral replication competency was evaluated by passage of an
aliquot of the virus stocks in insect cells at low multiplicity of
infection. Subsequent determination of the virus titer for the
progeny virus passage was performed by agarose plaque assay.
Recombinant protein expression competency was evaluated, for
example, by infection of insect cells with an aliquot of the virus
stocks and subsequent determination of the relative abundance of
recombinant proteins such HPV L1 and L2 fusion proteins per total
cell protein in infected cells by SDS-PAGE analysis.
Example 6
Virus Infection for HPV-16 L1 VLPs
[0222] Recombinant HPV-16 L1 VLPs expressed in baculovirus-infected
Sf-9S cells were purified from intracellular and extracellular
crude lysates. Sf-9S insect cells from Example 2 were thawed from a
single cryovial of the working cell bank frozen at -70.degree. C.
in Sf-900 II SFM insect cell media (GIBCO) at a concentration of
1.0.times.10.sup.7 cells/ml. Thawed cells were seeded into 50 ml of
Sf-900 II SFM insect cell media and cultured as suspension cultures
in 500 ml shaker flasks in a platform shaker incubator at
28.degree. C. with an agitation speed of 125 rpm.
[0223] After the cell density reached 6.times.10.sup.6 cells/ml and
a cell viability of more than 95%, the culture was seeded into two
(2) liter flasks in a final volume of 800 ml of insect serum-free
media per flask at a starting seed density of 0.5.times.10.sup.6
cells/ml. The cells were cultured in a platform shaker incubator at
28.degree. C. with an agitation speed of 100-125 rpm. When the cell
density reached 2-3.times.10.sup.6 cells/ml, the insect cells were
infected with a recombinant baculovirus encoding the HPV-16 L1
capsid gene (K strain) from the polh locus made according to
Example 3. The virus infection was established at a MOI of 3 pfu
per cell. The virus infection was carried out for six days in a
platform shaker incubator at 28.degree. C. with an agitation speed
of 125 rpm. The infected cells were harvested by centrifugation (at
1,500.times.g and 2-8.degree. C. for 10 minutes) after the
following conditions were met: cell viability was less than 25%,
and L1 gene products were in culture fluids and within infected
cells.
Example 7
Virus Infection for HPV-16 Chimeric VLPs
[0224] Production of intracellular HPV-16 L2/E7/E2 chimeric VLPs
began with high multiplicity infection of log phase Sf-9S insect
cells (1.5.times.10.sup.6 cells/ml) in suspension shaker flask
cultures (2 L) containing serum-free insect cell medium (800 ml;
HyQ SFM media, HyClone) made according to Example 2 with aliquots
of HPV-16 L1 and L2/E7/E2 working virus stocks prepared according
to Example 4. The ratio of co-infecting viruses was approximately
1:10 (L1 to L2 virus). The virus infection was monitored daily by
the trypan blue exclusion method for cytopathic effects, cell
viability, and cell density and by SDS-PAGE and Western blot
analyses of recombinant HPV L1 and L2 fusion proteins in infected
cells. At three days post-infection when peak recombinant HPV-16 L1
and L2/E7/E2 fusion gene expression occurred, infected cells
containing intracellular HPV-16 chimeric VLPs were harvested by
centrifugation at 1500.times.g and 2-8.degree. C. for 5 minutes and
processed to obtain purified HPV-16 chimeric VLP products.
Example 8
PREPARATION OF CRUDE CELL LYSATES FOR HPV-16 CHIMERIC VLPS
[0225] Recombinant HPV-16 chimeric VLPs including recombinant
HPV-16 L2/E7/E2 fusion proteins encapsulated into HPV-16 L1 VLPs
produced according to Example 6 were harvested from modified Sf-9
insect cells infected with recombinant baculoviruses encoding the
HPV-16 L1 capsid gene (K strain) and HPV-16 L2/E7/E2 fusion gene.
Infected cells were harvested by low-speed centrifugation at
1,500.times.g and 2-8.degree. C. for 10 minutes. Infected cell
pellets containing intracellular recombinant HPV-16 chimeric VLPs
were resuspended in phosphate-buffered saline II solution (1.54 mM
KH.sub.2PO.sub.4, 2.71 mM Na.sub.2HPO.sub.4.7H.sub.2O, and 154 mM
NaCl (pH 7.2)) at a ratio of 10 ml buffer per gram of cell pellet.
Protease inhibitors corresponding to serine, cysteine, and
aspartate classes of proteases were added to the following final
concentrations: (PMSF, 1 mM; Aprotinin, 1 .mu.g/ml; Leupeptin, 10
.mu.g/ml; Pepstatin, 5 .mu.g/ml). The resuspended cells containing
intracellular recombinant papillomavirus VLPs were disrupted by
mild sonication in phosphate-buffered saline solution with two (2)
pulses at 200-300 watts and 2-8.degree. C. with a Branson Model 250
sonifier equipped with a 1/8" probe. The result of sonication was a
crude cell lysate containing intact intracellular recombinant
HPV-16 chimeric VLPs with minimal disruption of cellular
proteosomes and degradation of HPV-16 L2/E7/E2 fusion proteins.
Example 9
CLARIFICATION OF HPV-16 L1 VLP CRUDE CELL LYSATES AND
SUPERNATANTS
[0226] Crude cell lysates made according to Example 7 containing
intracellular recombinant HPV-16 L1 VLPs were clarified by
centrifugation at 12,000.times.g and 2-8.degree. C. for 60 minutes
to remove large cellular debris and membranes. Clarified
supernatants were collected by aspiration, and cellular pellets
were discarded. Crude media supernatants containing extracellular
recombinant HPV-16 L1 VLPs were clarified by centrifugation at
12,000.times.g and 2-8.degree. C. for 60 minutes to remove large
cellular debris and membranes. Clarified supernatants were
collected by aspiration, and cellular pellets were discarded.
Example 10
Clarification of HPV-16 Chimeric VLP Crude Cell Lysates
[0227] Crude cell lysates made according to Example 8 were purified
as follows. The cell lysate containing intracellular recombinant
HPV-16 chimeric VLPs harvested from insect cells infected with
recombinant baculoviruses encoding HPV-16 L1 and L2/E7/E2 fusion
proteins were clarified by centrifugation at 12,000.times.g and
2-8.degree. C. for 60 minutes to remove large cellular debris and
membranes. Clarified supernatants were collected by aspiration, and
cellular pellets were discarded.
Example 11
CONCENTRATION AND DIAFILTRATION OF HPV-16 VLP CLARIFIED
SUPERNATANTS
[0228] Concentration and diafiltration steps of the present
invention involved ultrafiltration of clarified supernatants made
according to Example 9. The clarified supernatant contained
intracellular and extracellular recombinant HPV-16 L1 VLPs. These
VLPs were expressed from infection of Sf-9S insect cells with
recombinant baculovirus encoding the L1 capsid gene of HPV-16 (K
strain). Clarified supernatants containing intracellular and
extracellular recombinant HPV-16 L1 VLPs were concentrated ten fold
by ultrafiltration using an Amicon M-12 Proflux Tangential Flow
Ultrafiltration System equipped with a hollow fiber ultrafiltration
cartridge (A/G Technologies Model UFP-500-C-55A). Concentrates
containing intracellular and extracellular recombinant HPV-16 L1
VLPs were diafiltered against eight volumes of cation exchange
loading buffer solution containing 20 mM sodium phosphate (pH 5.7)
and 10 mM sodium chloride by ultrafiltration using an Amicon M-12
Proflux Tangential Flow Ultrafiltration System equipped with a
hollow fiber ultrafiltration cartridge (A/G Technologies Model
UFP-500-C-55A) at an initial flow rate of 0.8 L/min. and an
inlet/outlet pressure of 8 psi.
Example 12
CATION EXCHANGE CHROMATOGRAPHY OF HPV-16 VLP CONCENTRATED
DIALYSATES
[0229] Diafiltered concentrates containing intracellular and
extracellular recombinant HPV-16 VLPs made according to Example 11
were loaded onto a chromatography column containing Streamline SP
Adsorptive resin (Amersham Biosciences), a strong cation exchange
chromatography resin, at a flow rate of 0.5 L/hr and a ratio of 1
ml resin per 1 gram of diafiltrate. The chromatographic column was
developed with a Waters 6000 HPLC System. The SP resin was
equilibrated with a cation binding buffer (50 mM sodium phosphate
(pH 5.7) and 10 mM sodium chloride). Following binding of
diafiltrates containing intracellular and extracellular recombinant
HPV-16 VLPs, the column was rinsed with five (5) volumes of cation
binding buffer. Bound proteins were eluted as 1 ml fractions with
UV-monitoring at 214 mm from the column resin at a flow rate of 0.5
L/hr using a step pH gradient from 6.0 to 8.0 in 20 mM sodium
phosphate with 10 mM sodium chloride. Fractions containing
intracellular and extracellular recombinant HPV-16 VLPs eluted in
the 7.0-7.5 steps, as determined by SDS-PAGE and Western blot
analysis of elution fraction samples using antisera against
papillomavirus L1 capsid proteins. Those fractions containing
HPV-16 L1 VLPs were pooled.
Example 13
AFFINITY CHROMATOGRAPHY OF HPV-16 VLP CATION EXCHANGE
CHROMATOGRAPHY ELUATES
[0230] Pooled eluates from the cation exchange chromatography that
contain intracellular and extracellular recombinant HPV-16 L1 VLPs
made according to Example 12 were dialyzed against 100 volumes of
affinity loading buffer (20 mM sodium phosphate (pH 5.7), 2 mM
EGTA, and 300 mM sodium chloride) for 8-16 hrs. The dialyzed
material was loaded onto a column containing heparin agarose
(Amersham Biosciences) at a flow rate of 1 ml/min. The ratio of
packed heparin agarose to protein was 1 ml of resin per 0.5 grams
of protein. Bound proteins including recombinant HPV-16 L1 VLPs
were eluted as 1 ml fractions with UV-monitoring at 214 nm from the
column resin at a flow rate of 1 ml/min. using a linear salt
gradient from 300 mM to 2 M. Fractions containing recombinant
HPV-16 L1 VLPs eluted in salt fractions from 500-700 mM, as
determined by SDS-PAGE and Western blot analysis of elution
fraction samples using antisera against papillomavirus L1 capsid
proteins. Those fractions containing HPV-16 L1 VLPs were
pooled.
Example 14
ALTERNATIVE AFFINITY CHROMATOGRAPHY OF HPV-16 VLP CATION EXCHANGE
CHROMATOGRAPHY ELUATES
[0231] Pooled eluates from cation exchange chromatography that
contain intracellular and extracellular recombinant HPV-16 L1 VLPs
made according to Example 12 were dialyzed against 100 volumes of
affinity loading buffer (20 mM sodium phosphate (pH 5.7), 2 mM
EGTA, and 300 mM sodium chloride) for 8-16 hrs. The dialyzed
material was loaded onto a column containing Matrex Cellufine
Sulfate (Amersham Biosciences) at a flow rate of 1 ml/min. The
ratio of packed Matrex Cellufine Sulfate to protein was 1 ml of
resin per 0.5 grams of protein. Bound proteins including
recombinant HPV-16 L1 VLPs were eluted as 1 ml fractions with
UV-monitoring at 214 nm from the column resin at a flow rate of 1
ml/min using a linear salt gradient from 300 mM to 2 M. Fractions
containing recombinant HPV-16 L1 VLPs eluted in salt fractions from
400 mM to 600 mM, as determined by SDS-PAGE and Western blot
analyses of elution fraction samples using antisera against
papillomavirus L1 capsid proteins. Those fractions containing
HPV-16 L1 VLPs were pooled.
Example 15
ANION DISPLACEMENT CHROMATOGRAPHY OF HPV-16 VLP AFFINITY
CHROMATOGRAPHY ELUATES
[0232] Pooled eluates from affinity chromatography that contain
recombinant HPV-16 L1 VLPs made according to Examples 13 or 14 were
dialyzed against 100 volumes of anion loading buffer (0.24 M
Tris-HCl (pH 8.0)) for 8-16 hrs. The dialyzed material was loaded
onto a column containing Q Sepharose FF (Amersham Biosciences), a
strong anion exchange chromatography resin, at a flow rate of 0.5
ml/min. The ratio of packed Q Sepharose to protein was 1 ml of
resin per 0.1 gram of protein. Bound proteins including recombinant
papillomavirus VLPs were displaced as 1 ml fractions with
UV-monitoring at 214 nm from the column resin at a flow rate of 0.5
ml/min using a linear gradient from 0 to 5 mg/ml dextran sulfate
(5000 MW). Fractions containing recombinant HPV-16 L1 VLPs eluted
in dextran sulfate fractions from 4 mg/ml to 5 mg/ml, as determined
by SDS-PAGE and Western blot analyses of elution fraction samples
using antisera against papillomavirus L1 capsid proteins. Those
fractions containing HPV-16 L1 VLPs were pooled. Pooled eluates
from anion exchange chromatography that contain recombinant
papillomavirus VLPs were dialyzed against 100-150 volumes of final
bulk storage buffer (5 mM Na.sub.2HPO.sub.4.7H.sub.2O, 5 mM
KH.sub.2PO.sub.4, and 500 mM NaCl (pH 6.8)) for 8-16 hrs.
Example 16
LINEAR SUCROSE GRADIENT PURIFICATION AS AN ALTERNATIVE TO L1 VLP
CHROMATOGRAPHIC VLP PURIFICATION
[0233] Intracellular and extracellular HPV VLPs were also purified
from concentrated crude cell lysates and media supernatants made
according to Examples 9 or 10 by ultracentrifugation on linear
sucrose gradients. Concentrates (5-10 g) containing HPV L1 VLPs
were loaded at a flow rate of 100-250 ml per minute onto 0-65%
linear sucrose gradient prepared in phosphate-buffered saline
solution (5 mM potassium phosphate (monobasic), 5 mM sodium
phosphate (dibasic), 154 mM sodium chloride (pH 7.2)) in a RK-2
vertical rotor (1.6 L) accelerating at 35,000 rpm in a RK
continuous flow ultracentrifuge (Schiaparelli Instruments,
Amsterdam, The Netherlands). The gradient was resolved by
centrifugation at 35,000 rpm and 15-25.degree. C. for one to two
hours residence and one hour coasting to a complete stop without
braking. Gradient material from the sucrose gradient was passed
through a UV.sub.218nm monitor and collected as 50 ml aliquots in a
fraction collector. Samples from each fraction were subjected to
SDS-PAGE and Western blot analyses to find HPV-16 L1 VLPs. Results
from these analyses indicated that recombinant HPV-16 L1 VLPs
sedimented into two bands corresponding to 43-53% sucrose and
30-40% sucrose. The baculoviruses sedimented as one band
corresponding to 30-35% sucrose. Fractions 6-9 containing
recombinant HPV-16 L1 VLPs comprised of HPV-16 L1 protein species
with molecular weights of 55 and 60 kD were pooled. Fractions
10-14, which contained recombinant HPV-16 L1 proteins with
molecular weights of 50 and 55 kD and proteolytic breakdown
products of L1 proteins, were not pooled and used as product due to
the proteolysis. The pooled L1 VLP fractions were diluted 6 fold
with the PBS solution, and subjected to a second round of
ultracentrifugation on linear sucrose gradients, except the second
gradient was 0-50% sucrose (PBS) and was run for a total of two
hours (one hour residence and one hour coasting). Sucrose gradient
fractions from 0-50% linear sucrose gradients were examined for
HPV-16 L1 VLPs by SDS-PAGE, SEC HPLC, Western blot, and ELISA
analyses. Fractions containing intact HPV VLPs displaying
conformational epitopes were pooled.
[0234] Pooled sucrose gradient fractions containing extracellular
recombinant HPV-16 L1 VLPs were diafiltered by ultrafiltration
against eight volumes of final VLP storage buffer containing 5 mM
Na.sub.2HPO.sub.4.7H.sub.2O, 5 mM KH.sub.2PO.sub.4, and 500 mM NaCl
(pH 6.8) using an Amicon M-12 Proflux Tangential Flow
Ultrafiltration System equipped with a hollow fiber ultrafiltration
cartridge (A/G Technologies Model UFP-500-C-SSA) at an initial flow
rate of 0.8 L/min and an inlet/outlet pressure of 8 psi. Triton
X-100, a nonionic detergent and surfactant, was added at a final
concentration of 0.1% to the bulk HPV VLP product, which was
incubated for two hours at ambient temperature to inactivate
residual baculoviruses. Alternatively, the bulk HPV VLP product was
irradiated with at least three rounds of ultraviolet (UV) light at
254 nm to inactivate residual baculoviruses. The treated material
was filtered aseptically through a 0.22 .mu.m membrane into
silanized borosilicate glass containers.
Example 17
SUCROSE STEP GRADIENT PURIFICATION OF HPV-16 L1 VLPS AS AN
ALTERNATIVE TO L1 VLP CHROMATOGRAPHIC VLP PURIFICATION
[0235] In yet another alternate embodiment of the present
invention, diafiltered concentrates made according to Examples 9
and containing HPV-16 L1 VLPs were purified by rate-zonal
ultracentrifugation on discontinuous sucrose step gradients.
Diafiltrates containing recombinant HPV-16 L1 VLPs were loaded onto
approximately 25% sucrose cushions (prepared in PBS solution) in a
swinging bucket rotor (Sorval Model AH 628) accelerating at 35,000
rpm and 2-8.degree. C. in an ultracentrifuge (Sorval Model OTB-65B)
for three hours. The pellets at the bottom of the sucrose cushion
were collected, while the sucrose cushion and load material were
discarded. The sucrose cushion pellets were solubilized in PBS
solution at approximately 1 g of pellet per ml of buffer 1 and
loaded onto sucrose step gradients containing six steps comprising
approximately 25, 30, 35, 40, 45, 50 and 65% sucrose. The sucrose
step gradients were resolved by ultracentrifugation in a swinging
bucket rotor at 35,000 and 2-8.degree. C. for 1 hour until
recombinant HPV VLPs separate from baculovirus particles.
[0236] Gradient material from the first round of sucrose step
gradients were monitored by ultraviolet light during collection in
a fraction collector. Gradient fractions were analyzed by SDS-PAGE
and Western blot analysis using antisera against HPV-16 L1 capsid
proteins. Peak fractions containing HPV VLPs or their component
proteins were pooled, diluted multifold with buffer solution, and
were subjected to a second round of ultracentrifugation on sucrose
step sucrose gradients. Gradient material from the second round of
sucrose step gradients were monitored by ultraviolet light during
collection in a fraction collector. Gradient fractions from the
second round of ultracentrifugation were analyzed by SDS-PAGE and
Western blot analysis using antisera against papillomavirus L1
capsid proteins. Peak fractions containing HPV-16 proteins were
pooled. The purified recombinant HPV-16 L1 VLPs in the pooled
fractions were formulated as recombinant HPV-16 L1 VLP
products.
Example 18
SUCROSE STEP GRADIENT PURIFICATION OF HPV-16 CHIMERIC L1 VLPS
[0237] In yet another alternate embodiment of the present
invention, diafiltered concentrates made according to Example 10
and containing HPV-16 chimeric VLPs were purified by rate-zonal
ultracentrifugation on discontinuous sucrose step gradients based
primarily on mass rather density of recombinant HPV-16 chimeric
VLPs in sucrose.
[0238] Diafiltrates containing recombinant HPV-16 chimeric VLPs
were loaded onto approximately 25% sucrose cushions in a swinging
bucket rotor accelerating at 35,000 rpm in an ultracentrifuge
(Sorval) for three hours. The pellets at the bottom of the sucrose
cushion were collected, while the sucrose cushion and load material
were discarded. The sucrose cushion pellets were solubilized in PBS
buffer and loaded onto sucrose step gradients containing six steps
comprising approximately 25, 30, 35, 40, 45, 50 and 65%
sucrose.
[0239] The sucrose step gradients were resolved by
ultracentrifugation in a swinging bucket rotor at 35,000 for 1 hour
until recombinant HPV VLPs were separated from baculovirus
particles. Gradient material from the first round of sucrose step
gradients was monitored by ultraviolet light during collection in a
fraction collector. Gradient fractions were analyzed by SDS-PAGE
and Western blot analysis using antisera against HPV-16 L1 capsid
proteins and L2, E7, and E2 fusion proteins. Peak fractions
containing HPV VLPs or their component proteins were pooled,
diluted multifold with buffer solution, and were subjected to a
second round of ultracentrifugation on sucrose step sucrose
gradients.
[0240] Gradient material from the second round of sucrose step
gradients were monitored by ultraviolet light during collection in
a fraction collector. Gradient fractions from the second round of
ultracentrifugation were analyzed by SDS-PAGE and Western blot
analysis using antisera against papillomavirus L1 capsid proteins
and/or L2 fusion proteins. Peak fractions containing HPV-16
proteins were pooled. The purified recombinant HPV-16 chimeric VLPs
in the pooled fractions were formulated as recombinant HPV-16
chimeric VLP products.
Example 19
FORMULATION OF FINAL BULK HPV-16 L1 VLP PRODUCTS
[0241] Residual baculovirus present in recombinant HPV-16 L1 VLPs
made according to Examples 15, 16, or 17 was inactivated by
treatment of crude, intermediate, and final bulk products with the
non-ionic detergent and surfactant, Triton X-100. Inactivation of
virus in HPV-16 L1 VLP products was provided by addition of Triton
X-100 at a final concentration 0.1% for 2 hours at 15-25.degree. C.
Following treatment, HPV VLP products were diafiltered against
4.times.1000 volumes of high salt buffer containing (5 mM
Na.sub.2HPO.sub.4.7H.sub.2O, 5 mM KH.sub.2PO.sub.4, and 500 mM NaCl
(pH 6.8)) and 2-8.degree. C. for 12 hours. Diafiltrates containing
treated recombinant HPV-16 L1 VLPs were filtered aseptically
through a 0.22 .mu.m membrane at 15-25.degree. C. Filtered HPV-16
L1 VLP final bulk products were dispensed into ten liter containers
including 316 L stainless steel tanks, silanized borosilicate glass
bottles, and polyethylene plastic bioprocess bags and stored at
2-8.degree. C. for <six (6) months or at <-70.degree. C. for
<2 years.
Example 20
ANALYSIS OF INTACT HPV VLPS
[0242] ELISA testing of solutions containing recombinant HPV-16 L1
VLPs (lot 1274), sucrose, and/or clarified supernatants from
bHPV-16 L1 infected Sf-9S insect cells mock treated or treated two
hours at room temperature with Triton X-100 (0.1% final
concentration) was performed. Mock and treated solutions containing
recombinant HPV-16 L1 VLPs were dialyzed against four changes of
1000 volumes of phosphate buffer containing 0.5 M sodium chloride.
Various amounts (250, 500, and 1000 ng) of dialysates were bound to
96-well plates and reacted with antibodies from the murine
hybridoma cell line H16.V5 that bind to HPV-16 L1 protein
conformational epitopes that elicit neutralizing antibodies to
determine the effect of Triton X-100 on these epitopes. The results
as shown in FIG. 11 demonstrated that Triton had little or no
effect on recombinant HPV-16 L1 VLPs with or without sucrose, but
five fold decrease in binding activity was noted with 1000 ng of
VLPs mixed with supernatants as compared to that of mock treated
and Triton-treated VLPs or VLPs in sucrose. These results indicated
that Triton treatment of recombinant VLPs, which effectively
afforded as much as 4 to 7 log reduction in baculovirus titers, did
not irreversibly destroy conformational L1 epitopes of recombinant
HPV-16 L1 VLPs in buffer or buffer with sucrose. However, VLPs
amidst infected extracellular materials did not retain the proper
epitope conformation after Triton treatment. The conclusion: the
best time to add Triton in the purification of recombinant HPV VLPs
was after downstream processing but prior to terminal filtration of
final bulk products.
[0243] In one set of analyses as depicted in FIG. 12, samples (5
.mu.g of phosphate buffered saline) from different lots (1207,
1244, 1265, and 1268) of recombinant HPV-16 L1 VLPs were injected
to an analytical size exclusion column and resolved according to
their mass and shape. The pre-poured column used for analytical
size exclusion chromatographic analysis was a 15 cm TSK 6000PWXL
stainless steel column (Tosohaus). The fractionation range of this
column was more than 1,000,000 to 20,000 daltons. The volume of
test sample injected into the column was 50 .mu.l. A Waters 6000
HPLC system using Millennium computer software provided the
automated mechanics and programs necessary for sample injection,
buffer transfer, column development, UV monitoring, fraction
collection, and protein data management.
[0244] As the result of the analysis, control blue dextran beads
having a molecular weight of 2,000,000 daltons was resolved as a
single peak at 10-12 minutes. The expected resolution of VLPs was
at 15-16 minutes and monomeric proteins at 24-26 minutes.
Pentameric HPV-16 L1 structures were expected to resolve at 19-20
minutes when present. Analysis of a final container vaccine vial
production lot 1207 stored at -70.degree. C. for more than two
years indicated that the recombinant HPV-16 L1 VLPs resolved as two
peaks (FIG. 12). The major peak was at 15 min. and a minor peak was
at 25 min. Upon integration of the area beneath each peak, these
data indicated that at least 95% of the L1 protein in this
production remained as VLPs after more than two years frozen at
-70.degree. C. Results of samples from bulk lots 1244, 11265, and
1268 of recombinant HPV-16 L1 VLP showed in FIG. 12 a single major
peak at 15 min. Following integration of the peak areas, >95% of
this vaccine product remained as VLPs more than two years after
production. As recombinant HPV-16 L1 VLPs dissociate, pentamers at
20 min and monomers at 25 min. appeared (data not shown).
Interestingly, bovine papillomavirus (BPV) VLPs, a relative of
HPV-16, behaved similarly in this assay. Hawaii virus (HV) VLPs,
calicivirus self-assembled macromolecules, exhibited >99% intact
VLPs.
[0245] An electron micrograph of HPV VLPs was obtained by negative
staining of 10 .mu.l aliquots of recombinant HPV-16 L1 VLPs (1
mg/ml in phosphate buffered saline) with uranyl acetate and
observing at high magnification (36,000.times.) on a transmission
electron microscope (Phillips). These results as depicted in FIG.
13 demonstrated intact and discrete virus-like particles with a
size of 40 to 55 nm and a morphology similar to papillomavirus
virions. These electron micrographs provided another example of
confirmatory evidence of high quality HPV-16 L1. Further, the SEC
HPLC assay was shown again to be an effective measurement tool for
quantitation of VLPs.
Example 21
PRODUCTION OF ANTISERA TO HOST CONTAMINANT PROTEINS
[0246] Antisera to insect cell and wild type baculovirus proteins
were produced to detect contaminating host proteins in
baculovirus-derived recombinant protein products. For antiserum
against insect cell proteins, 800 ml suspension shaker cultures of
Sf-9S and High Five insect cells were grown to cell density of
2.times.10.sup.6 cell per ml. The cultured cells were harvested by
centrifugation at 500.times.g and 2-8.degree. C. for five minutes.
The cell pellets were resuspended in 10 ml of phosphate-buffered
saline solution (5 mM sodium phosphate, dibasic, 5 mM potassium
phosphate, monobasic, 154 mM sodium chloride (pH 7.2)). The
resuspended cells were disrupted by sonication with two (2) pulses
at 200-300 watts and 2-8.degree. C. with a Branson Model 250
sonifier equipped with a 1/8" probe to produce cell lysates. The
sonicated cell lysates were clarified by centrifugation at
12,000.times.g and 2-8.degree. C. for 60 minutes to remove large
cellular debris and membranes. The clarified supernatants were
retained for protein quantitation by the BCA method, and the
pellets were discarded.
[0247] Aliquots of the clarified cell lysates were formulated into
immunogens by emulsification of equal volumes of antigen and
complete Freund's adjuvant (DIFCO) at an antigen concentration of
200 .mu.g/ml. The immunogens were administered intramuscularly
(primary) as two doses (50 .mu.g/dose) into the hindquarters of New
Zealand rabbits. At four weeks post-immunization, a second round of
immunization (booster) occurred as before except that incomplete
Freund's adjuvant was used. At eight weeks post-immunization, sera
were isolated from blood withdrawn from immunized animals.
[0248] The antibody titers of the immunized sera were determined by
Western blot analysis using nitrocellulose membranes containing
proteins from Sf-9S and High Five insect cells, as well as control
protein samples from baculovirus- and E. coli-derived recombinant
protein products. Results using these antisera from Sf-9S insect
cell proteins demonstrated that the antibodies specific for this
cell line were present, as positive binding was observed in lanes
of blots containing Sf-9S and Sf-9S infected cell proteins but not
in lanes containing purified recombinant proteins such as HPV-16 L1
proteins (FIG. 10B).
[0249] Similarly, antisera were produced in rabbits against
antigens comprised of wild-type baculovirus proteins expressed in
Sf-9S and High Five insect cells infected at an MOI of 3 pfu/cell
with AcMNPV wild-type baculovirus for three days to produce
infected cell lysates. These antisera were utilized to demonstrate
the presence of baculovirus contaminants in baculovirus-derived
recombinant protein products (FIG. 10C). Little or no reactivity
was observed in the lanes for recombinant HPV-16 L1 VLPs in Western
blots with antisera to Sf-9S cell proteins or wild type baculovirus
proteins, whereas polyclonal rabbit antisera to HPV-16 L1 proteins
were positive for the recombinant HPV-16 L1 VLP lanes (FIG.
10A).
Example 22
FORMULATION OF HPV-16 L1 VLP MONOVALENT VACCINE
[0250] Monovalent vaccines of HPV-16 VLPs were prepared by
formulation of recombinant HPV-16 L1 VLPs manufactured by the
methods in Example 18. Final bulk products of recombinant HPV-16 L1
VLPs were filtered through a 0.22 .mu.m membrane aseptically and
formulated at antigen concentrations of 20 and 100 mg/ml in
phosphate-buffered saline alone or with alum adjuvant (Alhydrogel).
The formulated products were dispensed as single dose units (0.5
cc) into sterile borosilicate glass vials (3 cc) silanized with
dimethyldichlorosilane.
[0251] Also, final bulk products of recombinant HPV-16 L1 VLPs were
formulated at antigen concentrations of 40 and 200 .mu.g/ml in
phosphate buffered saline, filtered, and dispensed as single dose
units (0.25 cc) into silanized glass vials (3 cc) for mixing just
prior to immunization with an equal volume of the adjuvant MF-50
(Chiron). The final container products were labeled, checked for
vial integrity, and stored at -20 or -70.degree. C. for final
container vials containing VLP alone and 2-8.degree. C. for final
container vials containing VLPs and alum adjuvant.
[0252] The final container products were subjected to safety and
analytical testing as required by United States federal regulations
and passed product specifications. Safety specifications included:
(1) the absence of detectable microbial contaminants, spiroplasma,
or mycoplasma, (2) endotoxins levels below 30 endotoxin units per
ml, (3) the absence of adventitious agents by in vitro and in vivo
testing, and (4) no adverse effects in adult mice and guinea pigs
as part of the general safety tests. Analytical specifications
included (1) the presence of HPV-16 L1 proteins with molecular
weights between 50 and 60 kD at a purity .gtoreq.95% as determined
by SDS-PAGE analysis coupled to scanning laser densitometry, (2)
for identity testing, positive reactivity of proteins in the
product to HPV-16 L1 antisera as determined by Western blot
analysis, (3) for potency testing, positive reactivity of product
at 100 .mu.g/ml dilution with H16.V5 antisera for conformational
epitopes as determined by ELISA testing, (4) for identity, purity,
and potency testing, at least 75% of the product was present as
intact VLPs as determined by analytical size exclusion
chromatography and negative-stain electron microscopy, and for
strength testing, protein content in the product at 20 and 100
.mu.g/ml with <5% variance as determined by BCA assay.
[0253] Upon meeting product specifications, the final container
vials were released for vaccination into healthy human volunteers
to determine the safety and immunogenicity of the final container
products as prophylactic vaccines for HPV-16 infection and
disease.
Example 23
CLINICAL INVESTIGATION OF HPV-16 VLP MONOVALENT VACCINE
[0254] The final container products as prepared by the methods of
Example 22 were used to immunize healthy human volunteers in a
double-blind, randomized clinical study at Johns Hopkins University
to determine the safety and immunogenicity of the present invention
as a monovalent vaccine to prevent HPV-16 infection and disease
(Harro et al., 2001). The study design encompassed two dosage
regimens (10 and 50 .mu.g VLP antigens) and four study arms
including placebo, HPV-16 L1 VLPs alone, HPV-16 L1 VLPs+alum
adjuvant, and HPV-16 L1 VLPs+MF-59 adjuvant. Female volunteers (72)
who were sero-negative for HIV-1, had <four lifetime sexual
partners, were not pregnant, and had normal cervical cytology and
medical history, received three doses of vaccine or placebo
intramuscularly in the deltoid muscle area at 0, 1, and 4 months.
Serum samples were collected from vaccinees at one month
post-immunization and evaluated by ELISA tests for the presence of
HPV-16 antibodies. Vaccinees were followed up to one week
post-immunization for presentation of adverse clinical signs.
[0255] The results of the clinical study indicated that the vaccine
was well-tolerated as compared to placebo, as no major adverse
effects were noted in any vaccinees. All vaccines receiving active
vaccine seroconverted (4-fold antibody rise) for HPV-16. A
dose-dependent immune response was observed in serum samples at 5
months post-immunization for those vaccinees receiving VLPs alone
or VLPs+Mf-59. However, no dose dependent response was seen in
individuals receiving VLPs+alum. The antibody titers to HPV-16
neutralizing epitopes, as determined by ELISA tests of serum
samples at five months post-immunization with 50 .mu.g doses, were
1.times.10.sup.4 E.U. for VLPs alone, 1.times.10.sup.4 E.U. for
VLPs+MF-59, and 2.2.times.10.sup.3 E.U. for VLPs+alum. The
neutralizing and ELISA antibody titers were shown to correlate well
with 0.85 degree of confidence. Thus, the vaccines were shown to be
well-tolerated and immunogenic. The antibody titers were
approximately forty-fold higher than that associated with natural
HPV-16 infections. ELISA antibody titers were demonstrated to be
reliable correlates of HPV-16 neutralizing antibody titers. Lastly,
HPV-16 L1 VLP vaccines consisting of VLPs alone at 50 .mu.g per
dose may provide protective immunity to prevent HPV-16 infection
and resolve active HPV-16 infections and disease.
[0256] All references discussed herein are incorporated by
reference. One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof and, accordingly, reference should be made to the appended
claims, rather than to the foregoing specification, as indicating
the scope of the invention.
BIBLIOGRAPHY
[0257] Bosch, F. X., Manos, M. M., Munoz, N., Sherman, M., Jansen,
A. M., Peto, J., Schiffman, M. H., Moreno, V., Kurman, R., Shah, K.
V. (1995) Prevalence of human papillomavirus in cervical cancer: a
worldwide prospective. J. Nat. Cancer Inst. 87:796-802
[0258] Breitburd, F., Kirnbauer, R., Hubbert, N. L., Nonnenmacher,
B. Trin-Dinh-Desmarquet, C., Orth, G., Schiller, J. T., and Lowy,
D. R. (1993) Immunization with virus-like particles from cottontail
rabbit papillomavirus (CRPV) can protect against experimental CRPV
infection. J. Virol. 69:3959-3963
[0259] Christiansen, N. D., Hopfi, R., DiAngelo, S. L., Cladel, N.
M., Patrick, S. D., Welsh, P. A., Budgeon, L. R., Reed, C. A., and
Kreider, J. W. (1994) Assembled baculovirus-expressed human
papillomavirus type 11 capsid protein virus-like particles are
recognized by neutralizing monoclonal antibodies and induce high
titters of neutralizing antibodies. J. Gen. Virol. 75:
2271-2276.
[0260] Christensen, N. D., Reed, C. A., Cladel, N. M., Han, R., and
Kreider, H. W. (1996) Immunization with virus-like particles
induces long-term protection of rabbits against challenge with
cottontail rabbit papillomaviruses. J. Virol. 70:960-965
[0261] Cook, J. C., Joyce, J. G., George, H. A., Schultz, L. D.,
Hurni, W. M., Jansen, K. U., Hepler, R. W., Ip, C., Lowe, R. S.,
Keller, P. M., and Lehman, E. D. 1999. Purification of Virus-like
particles of recombinant human papillomavirus type 11 major capsid
protein L1 from Sacchraromyces cerevisiae. Protein Expression
Purif. 17: 477-484.
[0262] Greenstone, H. L., Nieland, J. D., de Visser, K. E. et al.
(1998). Chimeric Papilloma Virus-like Particles Elicit Antitumor
Immunity Against the E7 Oncoprotein in an HPV 16 Tumor Model. Proc.
Natl. Acad. Sci. USA 95, 1800-1805.
[0263] Hagensee, M. and Galloway, D. (1993) Growing human
papillomaviruses and virus-like particles in the laboratory.
Papillomavirus Report 4:121-124
[0264] Harro, C. D., Pang, Y.-Y. S., Roden, R. B. S., Hildesheim,
A., Wang, Z., Reynolds, M. J., Mast, T. C., Robinson, R., Murphy,
B. R., Karron, R. A., Dillner, J., Schiller, J. T., and Lowy, D. R.
(2001) Safety and immunogenicity trial in adult volunteers of a
human papillomavirus 16 virus-like particle vaccine. J. Natl. Acad.
Sci. 93:284-292.
[0265] IARC IAfRoC. Human Papillomaviruses. Vol. 64: WHO, 1995
(Cancer IAfRo, ed. IARC Monographs on the evaluation of
carcinogenic risks to humans)
[0266] Joyce, J. G., Tung, J. S., Przysiecki, C. T., et al. (1999)
The L1 major capsid protein of human papillomavirus type 11
recombinant virus-like particles interacts with heparin and
glycosaminoglycans on human keratinocytes. J. Biol. Chem.
274:5810-5822.
[0267] Kirnbauer, R., Booy, F., Cheng, N., Lowy, D. R., and
Schiller, J. T. (1992) Papillomavirus L1 major capsid protein
self-assembles into virus-like particles that are highly
immunogenic. Proc. Natl. Acad. Sci. USA 89: 12180-12184.
[0268] Kirnbauer, R., Taub, J., Greenstone, H., Roden, R., Durst,
M., Gissman, L., Lowy, D. R., and Schiller, J. T. Efficient
self-assembly of human papillomavirus type 16 L1 and L1-L2 into
virus-like particles. J. Virol. 67:6929-6936.
[0269] Kirnbauer, R., Chandrachud, L., O'Neil, B., Wagner, E.,
Grindlay, G., Armstrong, A., McGarvie, G., Schiller, J., Lowy, D.,
Campo, M., (1996) Viruslike particles of bovine papillomavirus type
4 in prophylactic and therapeutic immunization. Virology
219:37-44.
[0270] Kurman, R. J., Henson, D. E., Herbst, A. L., Noller, K. L.,
and Schiffman, M. H. (1994) Interim guidelines for management of
abnormal cervical cytology. The 1992 National Cancer Institute
Workshop. JAMA 271:1866-1869
[0271] Levin, D. B. and Whittome, B. (2000) Codon usage in
nucleopolyhedroviruses. J. Gen. Virol. 81:2313-2325.
[0272] Luckow, V. A., Lee, S. C., Barry, G. F., and Olins, P. O.
1993. Efficient generation of infectious recombinant baculoviruses
by site-specific transposon-mediated insertion of foreign genes
into a baculovirus genome propagated in Escherichia coli. J. Virol.
67: 4566-4579.
[0273] O'Neil, P. F., and Balkovic, E. S. (1993) Virus harvesting
and affinity-based chromatography. A method for virus concentration
and purification. Bio/Technology 11: 173-178.
[0274] Parkin, D. M., Pisani P., and Ferlay, J. (1990) Estimates of
worldwide incidence from 25 cancers. Intl. J. Cancer 80:827-841
[0275] Pisani, P., Parkin, D. M., Bray, F., and Ferlay, J. (1990)
Estimates of the worldwide mortality from 25 cancers. Intl. J.
Cancer 83:18-29
[0276] Richardson, C. D. (Ed.) (1995) Methods in Molecular Biology
39, "Baculovirus Expression Protocols" Humana Press, Inc. (New
York)
[0277] Robinson, R. A., Burgess, W. H., Emerson, S. U., Leibowitz,
R. S., Sosnovtseva, S. A., Tsarev, S., and Purcell, R. H. (1998).
Structural characterization of recombinant hepatitis E virus ORF2
proteins in baculovirus-infected insect cells. Protein Expression
and Purification 12: 75-84.
[0278] Rose, R. C., Bonnez, W., Reichman, R. C., and Garcea, R. L.
(1993). Expression of human papillomavirus type 11 L1 protein in
insect cells: in vivo and in vitro assembly of viruslike particles.
J. Virol. 67:1936-44
[0279] Schiller, J. and Lowy, D. 1996. Papillomavirus-like
particles and HPV vaccine development. Seminars in Cancer Biol.
7:373-382
[0280] Shah, K. V. and Howley, P. M. Papillomaviruses. (1996) In:
Fields, B., Knipe, D. M., and Howley, P. M. Eds. Virology.
Philadelphia: Lippincott-Raven, pp. 2077-2109
[0281] Summers, M. D. and Smith, G. E. 1987. A manual of methods
for baculovirus vectors and insect cell culture procedures. Texas
Agric. Exp. Station Bull. 1855: 1-57.
[0282] Suzich, J. A., Ghim, S., Palmer-Hill, F. J., White, W. I.,
Tamura, J. K., Bell, J., Newsome, J. A., Jenson, A. B., and
Schlegel, R. (1995) Systemic immunization with papillomavirus L1
protein completely prevents the development of viral mucosal
papillomas. Proc. Natl. Acad. Sci. USA 92:11553-11557
[0283] Walboomers, J. M., Jacobs, M. C., Manosm M. M., Bosch, F.
X., Kummer, J. A., Shah, K. V., et al. (1999) Human papillomavirus
is a necessary cause of invasive cervical cancer worldwide. J.
Pathol. 189:12-19
[0284] Zhou, J., Stenzel, D. J., Sun, X.-Y., and Fraser, I. H.
(1993) Synthesis and assembly of infectious bovine papillomavirus
particles in vitro. J. Gen. Virol. 74: 763-768.
Sequence CWU 1
1
13 1 1515 DNA Artificial Sequence HPV-16 L1 codon optimized
sequence 1 atgtccctct ggctgccctc cgaggccacc gtctacctcc cccccgtccc
cgtctccaag 60 gtcgtctcca ccgatgaata cgtcgctcgc accaacatct
actaccatgc tggtacctcc 120 cgtctcctgg ctgtcggtca tccctacttc
cccatcaaga agcccaacaa caacaagatc 180 ctcgtcccca aggtctccgg
tctccaagtc cgtgtcttcc gtatccatct ccccgacccc 240 aacaagttcg
gtttccccga cacctccttc tacaaccccg atacccagcg cctgtactgg 300
gcctgcgtcg gtgtcgaggt cggtcgtggt cagcccctcg gtgtcggcat ctccggccac
360 cccctcctca acaagctcga cgacaccgag aacgcctccg cctacgccgc
caacgccggt 420 gtcgacaacc gtgagtgcat ctccatggac tacaagcaga
cccagctctg cctcatcggt 480 tgcaagcccc ccatcggtga gcactggggt
aagggttccc cctgcaccaa cgtcgccgtc 540 aaccccggtg actgcccccc
cctcgagctc atcaacaccg tcatccagga cggtgacatg 600 gtcgacaccg
gtttcggtgc catggacttc accaccctcc aggccaacaa gtccgaggtc 660
cccctcgaca tctgcacctc catctgcaag taccccgact acatcaagat ggtctccgag
720 ccctacggtg actccctctt cttctacctc cgccgcgagc agatgttcgt
ccgccacctc 780 ttcaaccgcg ccggtgctgt cggtgagaac gtccccgacg
acctctacat caagggttcc 840 ggttccaccg ccaacctcgc ttcctccaac
tacttcccca ccccctccgg ttccatggtc 900 acctccgacg cccagatctt
caacaagccc tactggctcc agcgcgctca gggtcacaac 960 aacggtatct
gctggggtaa ccagctcttc gtcaccgtcg tcgacaccac ccgctccacc 1020
aacatgtccc tctgcgccgc catctccacc tccgagacca cctacaagaa caccaacttc
1080 aaggagtacc tccgccacgg tgaggagtac gacctccagt tcatcttcca
gctctgcaag 1140 atcaccctca ccgccgacgt catgacctac atccactcca
tgaactccac catcctcgag 1200 gactggaact tcggtctcca gccccccccc
ggtggtaccc tcgaggacac ctaccgcttc 1260 gtcacctccc aggccatcgc
ctgccagaag cacacccccc ccgcccccaa ggaggacccc 1320 ctcaagaagt
acaccttctg ggaggtcaac ctcaaggaga agttctccgc cgacctcgac 1380
cagttccccc tcggtcgcaa gttcctcctc caggccggtc tcaaggccaa gcccaagttc
1440 accctcggta agcgcaaggc cacccccacc acctcctcca cctccaccac
cgccaagcgc 1500 aagaagcgca agctc 1515 2 1413 DNA Artificial
Sequence HPV-16 L2 codon optimized sequence 2 atgcgtcaca agcgttccgc
caagcgtacc aagcgtgcct ccgccaccca gctctacaag 60 acctgcaagc
aggccggtac ctgccccccc gacatcatcc ccaaggtcga gggtaagacc 120
atcgccgacc agatcctcca gtacggttcc atgggtgtct tcttcggtgg tctcggtatc
180 ggtaccggtt ccggtaccgg tggtcgtacc ggttacatcc ccctcggtac
ccgtcccccc 240 accgccaccg acaccctcgc ccccgtccgt ccccccctca
ccgtcgaccc cgtcggtccc 300 tccgacccct ccatcgtctc cctcgtcgag
gagacctcct tcatcgacgc cggtgccccc 360 acctccgtcc cctccatccc
ccccgacgtc tccggcttct ccatcaccac ctccaccgac 420 accacccccg
ccatcctcga catcaacaac accgtcacca ccgtcaccac ccacaacaac 480
cccaccttca ccgacccctc cgtcctccag ccccccaccc ccgccgagac cggtggtcac
540 ttcaccctct cctcctccac catctccacc cacaactacg aggagatccc
catggacacc 600 tttatcgtct ccaccaaccc caacaccgtc acctcctcca
cccccatccc cggttcccgt 660 cccgtcgccc gtctgggcct ctactcccgt
accacccagc aggtcaaggt cgtcgacccc 720 gccttcgtca ccacccccac
caagctcatc acctacgaca accccgccta cgagggtatc 780 gacgtcgaca
acaccctcta cttctcctcc aacgacaact ccatcaacat cgcccccgac 840
cccgacttcc tcgacatcgt cgccctccac cgtcccgccc tcacctcccg tcgcaccggc
900 atccgctact cccgtatcgg taacaagcag accctccgta cccgttccgg
taagtccatc 960 ggtgccaagg tccactacta ctacgacttc tccaccatcg
accccgccga ggagatcgag 1020 ctccagacca tcaccccctc cacctacacc
accacctccc acgccgcctc ccccacctcc 1080 atcaacaacg gtctctacga
catctacgcc gacgacttca tcaccgacac ctccaccacc 1140 cccgtcccct
ccgtcccctc cacctccctc tccggttaca tccccgccaa caccaccatc 1200
cccttcggtg gcgcctacaa catccccctc gtctccggtc ccgacatccc catcaacatc
1260 accgaccagg ccccctccct catccccatc gtccccggct ccccccagta
caccatcatc 1320 gccgacgccg gtgacttcta cctccacccc tcctactaca
tgctccgtaa gcgtcgtaag 1380 cgtctcccct acttcttctc cgacgtctcc tga
1413 3 1466 DNA Artificial Sequence HPV-16 L2/E7 fusion gene codon
optimized sequence 3 atatgcgaca caaacgttct gcaaaacgca caaaacgtgc
atcggccacc caactttata 60 aaacatgcaa acaggcaggt acatgtccac
ctgacattat acctaaggtt gaaggcaaaa 120 ctattgctga tcaaatatta
caatatggaa gtatgggtgt attttttggt gggttaggaa 180 ttggaacagg
gtctggtaca ggcggacgca ctgggtatat tccattggga acaaggcctc 240
ccacagctac agatacactt gctcctgtaa gacccccttt aacggtagat cctgtgggcc
300 cttctgatcc gtctatagtt tcgttagtgg aagaaactag ttttattgat
gctggtgcac 360 caacacctgt accttccatt cccccagatg tatcaggatt
tagtattaca acttcaactg 420 ataccacacc tgctatatta gatattaata
atactgttac tactgttact acacataata 480 atcccacttt tactgaccca
tctgtattgc agcctccaac acctgcagaa actggagggc 540 attttacact
ttcatcatcc actattagta cacataatta tgaagaaatt cctatggata 600
catttattgt tagcacaaat cctaacacag taactagtag cacacccata ccggggtctc
660 gcccagtggc acgcctagga ttatatagtc gcacaacaca acaagttaaa
gttgtagacc 720 ctgcttttgt aaccactccc actaaactta ttacatatga
taatcctgca tatgaaggta 780 tagatgtgga taatacatta tattttccta
gtaatgataa tagtattaat atagctccag 840 atcctgactt tttggatata
gttgctttac ataggccagc attaacctct aggcgtactg 900 gcattagata
cagtagaatt ggtaataaac aaacactacg tactcgtagt ggaaaatcta 960
taggtgctaa ggtacattat tattatgatt taagtactat taatcctgca gaagaaatag
1020 aattgcaaac tataacacct tctacatata ctaccccttc acatgcagcc
tcacccactt 1080 ctattaataa tggattatat gatatttatg cagatgactt
tattacagat acttttacaa 1140 ccccagtacc atctataccc tctacatcct
tatcaggtta tattcctgca aatacaacaa 1200 ttccttttgg tggtgcatac
aatattcctt tagtatcagg tcctgatata cctattaata 1260 caactgacca
aactccttca ttaattccta tagttccagg gtctccacaa tatacaatta 1320
ttgctgatgg aggtgacttt tatttacatc ctagttatta catgttacga aaacgacgta
1380 aacgtttacc atattttttt tcagatgtat cgatgcatgg agatacacct
acattgcatg 1440 aatatatgtt agatttgcaa ccagag 1466 4 2805 DNA
Artificial Sequence HPV-16 L2/E7/E2 fusion gene codon optimized
sequence 4 atgcgacaca aacgttctgc aaaacgcaca aaacgtgcat cggctaccca
actttataaa 60 acatgcaaac aggcaggtac atgtccacct gacattatac
ctaaggttga aggcaaaact 120 attgctgatc aaatattaca atatggaagt
atgggtgtat tttttggtgg gttaggaatt 180 ggaacagggt cgggtacagg
cggacgcact gggtatattc cattgggaac aaggcctccc 240 acagctacag
atacacttgc tcctgtaaga ccccctttaa cagtagatcc tgtgggccct 300
tctgatcctt ctatagtttc tttagtggaa gaaactagtt ttattgatgc tggtgcacca
360 acatctgtac cttccattcc cccagatgta tcaggattta gtattactac
ttcaactgat 420 accacacctg ctatattaga tattaataat actgttacta
ctgttactac acataataat 480 cccactttca ctgacccatc tgtattgcag
cctccaacac ctgcagaaac tggagggcat 540 tttacacttt catcatccac
tattagtaca cataattatg aagaaattcc tatggataca 600 tttattgtta
gcacaaaccc taacacagta actagtagca cacccatacc agggtctcgc 660
ccagtggcac gcctaggatt atatagtcgc acaacacaac aagttaaagt tgtagaccct
720 gcttttgtaa ccactcccac taaacttatt acatatgata atcctgcata
tgaaggtata 780 gatgtggata atacattata tttttctagt aatgataata
gtattaatat agctccagat 840 cctgactttt tggatatagt tgctttacat
aggccagcat taacctctag gcgtactggc 900 ataaggtaca gtagaattgg
taataaacaa acactacgta ctcgtagtgg aaaatctata 960 ggtgctaagg
tacattatta ttatgatttt agtaccattg atcctgcaga agaaatagaa 1020
ttacaaacta taacaccttc tacatatact accacttcac atgcagcctc acctacttct
1080 attaataatg gattatatga tatttatgca gatgacttta ttacagatac
ttctacaacc 1140 ccggtaccat ctgtaccctc tacatcttta tcaggttata
ttcctgcaaa tacaacaatt 1200 ccttttggtg gtgcatacaa tattccttta
gtatcaggtc ctgatatacc cattaatata 1260 actgaccaag ctccttcatt
aattcctata gttccagggt ctccacaata tacaattatt 1320 gctgatgcag
gtgactttta tttacatcct agttattaca tgttacgaaa acgacgtaaa 1380
cgtttaccat attttttttc agatgtatcg atgcatggag atacacctac attgcatgaa
1440 tatatgttag atttgcaacc agagacaact gatctctacg gctatgagca
attaaatgac 1500 agctcagagg aggaggatga aatagatggt ccagctggac
aagcagaacc ggacagagcc 1560 cattacaata ttgtaacctt ttgttgcaag
tgtgactcta cgcttcggtt gtgcgtacaa 1620 agcacacacg tagacattcg
tactttggaa gacctgttaa tgggcacact aggaattgtg 1680 tgccccatct
gttctcagaa accatcgatg gagactcttt gccaacgttt aaatgtgtgt 1740
caggacaaaa tactaacaca ttatgaaaat gatagtacag acctacgtga ccatatagac
1800 tattggaaac acatgcgcct agaatgtgct atttattaca aggccagaga
aatgggattt 1860 aaacatatta accaccaggt ggtgccaaca ctggctgtat
caaagaataa agcattacaa 1920 gcaattgaac tgcaactaac gttagaaaca
atatataact cacaatatag taatgaaaag 1980 tggacattac aagacgttag
ccttgaagtg tatttaactg caccaacagg atgtataaaa 2040 aaacatggat
atacagtgga agtgcagttt gatggagaca tatgcaatac aatgcattat 2100
acaaactgga cacatatata tatttgtgaa gaagcatcag taactgtggt agagggtcaa
2160 gttgactatt atggtttata ttatgttcat gaaggaatac gaacatattt
tgtgcagttt 2220 aaagatgatg cagaaaaata tagtaaaaat aaagtatggg
aagttcatgc gggtggtcag 2280 gtaatattat gtcctacatc tgtgtttagc
agcaacgaag tatcctctcc tgaaattatt 2340 aggcagcact tggccaacca
ctccgccgcg acccatacca aagccgtcgc cttgggcacc 2400 gaagaaacac
agacgactat ccagcgacca agatcagagc cagacaccgg aaacccctgc 2460
cacaccacta agttgttgca cagagactca gtggacagtg ctccaatcct cactgcattt
2520 aacagctcac acaaaggacg gattaactgt aatagtaaca ctacacccat
agtacattta 2580 aaagtggatg ctaatacttt aaaatgttta agatatagat
ttaaaaagca ttgtacattg 2640 tatactgcag tgtcgtctac atggcattgg
acaggacata atgtaaaaca taaaagtgca 2700 attgttacac ttacatatga
tagtgaatgg caacgtgacc aatttttgtc tcaagttaaa 2760 ataccaaaaa
ctattacagt gtctactgga tttatgtcta tatga 2805 5 1611 DNA Artificial
Sequence HPV-16 L2/E6 fusion gene codon optimized sequence 5
atgcgtcaca agcgttccgc caagcgtacc aagcgtgcct ccgccaccca gctctacaag
60 acctgcaagc aggccggtac ctgccccccc gacatcatcc ccaaggtcga
gggtaagacc 120 atcgccgacc agatcctcca gtacggttcc atgggtgtct
tcttcggtgg tctcggtatc 180 ggtaccggtt ccggtaccgg tggtcgtacc
ggttacatcc ccctcggtac ccgtcccccc 240 accgccaccg acaccctcgc
ccccgtccgt ccccccctca ccgtcgaccc cgtcggtccc 300 tccgacccct
ccatcgtctc cctcgtcgag gagacctcct tcatcgacgc cggtgccccc 360
acctccgtcc cctccatccc ccccgacgtc tccggcttct ccatcaccac ctccaccgac
420 accacccccg ccatcctcga catcaacaac accgtcacca ccgtcaccac
ccacaacaac 480 cccaccttca ccgacccctc cgtcctccag ccccccaccc
ccgccgagac cggtggtcac 540 ttcaccctct cctcctccac catctccacc
cacaactacg aggagatccc catggacacc 600 tttatcgtct ccaccaaccc
caacaccgtc acctcctcca cccccatccc cggttcccgt 660 cccgtcgccc
gtctgggcct ctactcccgt accacccagc aggtcaaggt cgtcgacccc 720
gccttcgtca ccacccccac caagctcatc acctacgaca accccgccta cgagggtatc
780 gacgtcgaca acaccctcta cttctcctcc aacgacaact ccatcaacat
cgcccccgac 840 cccgacttcc tcgacatcgt cgccctccac cgtcccgccc
tcacctcccg tcgcaccggc 900 atccgctact cccgtatcgg taacaagcag
accctccgta cccgttccgg taagtccatc 960 ggtgccaagg tccactacta
ctacgacttc tccaccatcg accccgccga ggagatcgag 1020 ctccagacca
tcaccccctc cacctacacc accacctccc acgccgcctc ccccacctcc 1080
atcaacaacg gtctctacga catctacgcc gacgacttca tcaccgacac ctccaccacc
1140 cccgtcccct ccgtcccctc cacctccctc tccggttaca tccccgccaa
caccaccatc 1200 cccttcggtg gcgcctacaa catccccctc gtctccggtc
ccgacatccc catcaacatc 1260 accgaccagg ccccctccct catccccatc
gtccccggct ccccccagta caccatcatc 1320 gccgacgccg gtgacttcta
cctccacccc tcctactaca tgctccgtaa gcgtcgtaag 1380 cgtctcccct
acttcttctc cgacgtctcc atgcaccaga agcgtaccgc catgttccag 1440
gacccccagg agcgtccccg taagctcccc cagctctgca ccgagctcca gaccaccatc
1500 cacgacatca tcctcgagtg cgtctactgc aagcagcagc tcctgcgtcg
tgaggtctac 1560 gacttcgctt tccgcgacct ctgcatcgtc taccgtgacg
gcaacccctg a 1611 6 505 PRT Artificial Sequence HPV-16 L1 protein 6
Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5
10 15 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr
Asn 20 25 30 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val
Gly His Pro 35 40 45 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys
Ile Leu Val Pro Lys 50 55 60 Val Ser Gly Leu Gln Tyr Arg Val Phe
Arg Ile His Leu Pro Asp Pro 65 70 75 80 Asn Lys Phe Gly Phe Pro Asp
Thr Ser Phe Tyr Asn Pro Asp Thr Gln 85 90 95 Arg Leu Val Trp Ala
Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro 100 105 110 Leu Gly Val
Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125 Thr
Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135
140 Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly
145 150 155 160 Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser
Pro Cys Thr 165 170 175 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro
Leu Glu Leu Ile Asn 180 185 190 Thr Val Ile Gln Asp Gly Asp Met Val
Asp Thr Gly Phe Gly Ala Met 195 200 205 Asp Phe Thr Thr Leu Gln Ala
Asn Lys Ser Glu Val Pro Leu Asp Ile 210 215 220 Cys Thr Ser Ile Cys
Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 Pro Tyr
Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe 245 250 255
Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260
265 270 Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala
Ser 275 280 285 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr
Ser Asp Ala 290 295 300 Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg
Ala Gln Gly His Asn 305 310 315 320 Asn Gly Ile Cys Trp Gly Asn Gln
Leu Phe Val Thr Val Val Asp Thr 325 330 335 Thr Arg Ser Thr Asn Met
Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu 340 345 350 Thr Thr Tyr Lys
Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365 Glu Tyr
Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr 370 375 380
Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu 385
390 395 400 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu
Glu Asp 405 410 415 Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys
Gln Lys His Thr 420 425 430 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys
Lys Tyr Thr Phe Trp Glu 435 440 445 Val Asn Leu Lys Glu Lys Phe Ser
Ala Asp Leu Asp Gln Phe Pro Leu 450 455 460 Gly Arg Lys Phe Leu Leu
Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480 Thr Leu Gly
Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495 Thr
Ala Lys Arg Lys Lys Arg Lys Leu 500 505 7 470 PRT Artificial
Sequence HPV-16 L2 protein 7 Met Arg His Lys Arg Ser Ala Lys Arg
Thr Lys Arg Ala Ser Ala Thr 1 5 10 15 Gln Leu Tyr Lys Thr Cys Lys
Gln Ala Gly Thr Cys Pro Pro Asp Ile 20 25 30 Ile Pro Lys Val Glu
Gly Lys Thr Ile Ala Asp Gln Ile Leu Gln Tyr 35 40 45 Gly Ser Met
Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser 50 55 60 Gly
Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro 65 70
75 80 Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val
Asp 85 90 95 Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val
Glu Glu Thr 100 105 110 Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val
Pro Ser Ile Pro Pro 115 120 125 Asp Val Ser Gly Phe Ser Ile Thr Thr
Ser Thr Asp Thr Thr Pro Ala 130 135 140 Ile Leu Asp Ile Asn Asn Thr
Val Thr Thr Val Thr Thr His Asn Asn 145 150 155 160 Pro Thr Phe Thr
Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala Glu 165 170 175 Thr Gly
Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser Thr His Asn 180 185 190
Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val Ser Thr Asn Pro Asn 195
200 205 Thr Val Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Val Ala
Arg 210 215 220 Leu Gly Leu Tyr Ser Arg Thr Thr Gln Gln Val Lys Val
Val Asp Pro 225 230 235 240 Ala Phe Val Thr Thr Pro Thr Lys Leu Ile
Thr Tyr Asp Asn Pro Ala 245 250 255 Tyr Glu Gly Ile Asp Val Asp Asn
Thr Leu Tyr Phe Ser Ser Asn Asp 260 265 270 Asn Ser Ile Asn Ile Ala
Pro Asp Pro Asp Phe Leu Asp Ile Val Ala 275 280 285 Leu His Arg Pro
Ala Leu Thr Ser Arg Arg Thr Gly Ile Arg Tyr Ser 290 295 300 Arg Ile
Gly Asn Lys Gln Thr Leu Arg Thr Arg Ser Gly Lys Ser Ile 305 310 315
320 Gly Ala Lys Val His Tyr Tyr Tyr Asp Phe Ser Thr Ile Asp Pro Ala
325 330 335 Glu Glu Ile Glu Leu Gln Thr Ile Thr Pro Ser Thr Tyr Thr
Thr Thr 340 345 350 Ser His Ala Ala Ser Pro Thr Ser Ile Asn Asn Gly
Leu Tyr Asp Ile 355 360 365 Tyr Ala Asp Asp Phe Ile Thr Asp Thr Ser
Thr Thr Pro Val Pro Ser 370 375 380 Val Pro Ser Thr Ser Leu Ser Gly
Tyr Ile Pro Ala Asn Thr Thr Ile 385 390 395 400 Pro Phe Gly Gly Ala
Tyr Asn Ile Pro Leu Val Ser Gly Pro Asp Ile
405 410 415 Pro Ile Asn Ile Thr Asp Gln Ala Pro Ser Leu Ile Pro Ile
Val Pro 420 425 430 Gly Ser Pro Gln Tyr Thr Ile Ile Ala Asp Ala Gly
Asp Phe Tyr Leu 435 440 445 His Pro Ser Tyr Tyr Met Leu Arg Lys Arg
Arg Lys Arg Leu Pro Tyr 450 455 460 Phe Phe Ser Asp Val Ser 465 470
8 488 PRT Artificial Sequence HPV-16 L2/E7 fusion protein 8 Met Arg
His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr 1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile 20
25 30 Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Asp Gln Ile Leu Gln
Tyr 35 40 45 Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly
Thr Gly Ser 50 55 60 Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu
Gly Thr Arg Pro Pro 65 70 75 80 Thr Ala Thr Asp Thr Leu Ala Pro Val
Arg Pro Pro Leu Thr Val Asp 85 90 95 Pro Val Gly Pro Ser Asp Pro
Ser Ile Val Ser Leu Val Glu Glu Thr 100 105 110 Ser Phe Ile Asp Ala
Gly Ala Pro Thr Pro Val Pro Ser Ile Pro Pro 115 120 125 Asp Val Ser
Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala 130 135 140 Ile
Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn 145 150
155 160 Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala
Glu 165 170 175 Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser
Thr His Asn 180 185 190 Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val
Ser Thr Asn Pro Asn 195 200 205 Thr Val Thr Ser Ser Thr Pro Ile Pro
Gly Ser Arg Pro Val Ala Arg 210 215 220 Leu Gly Leu Tyr Ser Arg Thr
Thr Gln Gln Val Lys Val Val Asp Pro 225 230 235 240 Ala Phe Val Thr
Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala 245 250 255 Tyr Glu
Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Pro Ser Asn Asp 260 265 270
Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val Ala 275
280 285 Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile Arg Tyr
Ser 290 295 300 Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg Ser Gly
Lys Ser Ile 305 310 315 320 Gly Ala Lys Val His Tyr Tyr Tyr Asp Leu
Ser Thr Ile Asn Pro Ala 325 330 335 Glu Glu Ile Glu Leu Gln Thr Ile
Thr Pro Ser Thr Tyr Thr Thr Pro 340 345 350 Ser His Ala Ala Ser Pro
Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile 355 360 365 Tyr Ala Asp Asp
Phe Ile Thr Asp Thr Phe Thr Thr Pro Val Pro Ser 370 375 380 Ile Pro
Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala Asn Thr Thr Ile 385 390 395
400 Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser Gly Pro Asp Ile
405 410 415 Pro Ile Asn Thr Thr Asp Gln Thr Pro Ser Leu Ile Pro Ile
Val Pro 420 425 430 Gly Ser Pro Gln Tyr Thr Ile Ile Ala Asp Gly Gly
Asp Phe Tyr Leu 435 440 445 His Pro Ser Tyr Tyr Met Leu Arg Lys Arg
Arg Lys Arg Leu Pro Tyr 450 455 460 Phe Phe Ser Asp Val Ser Met His
Gly Asp Thr Pro Thr Leu His Glu 465 470 475 480 Tyr Met Leu Asp Leu
Gln Pro Glu 485 9 805 PRT Artificial Sequence HPV-16 L2/E7/E2
fusion protein 9 Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg
Ala Ser Ala Thr 1 5 10 15 Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly
Thr Cys Pro Pro Asp Ile 20 25 30 Ile Pro Lys Val Glu Gly Lys Thr
Ile Ala Asp Gln Ile Leu Gln Tyr 35 40 45 Gly Ser Met Gly Val Phe
Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser 50 55 60 Gly Thr Gly Gly
Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro 65 70 75 80 Thr Ala
Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val Asp 85 90 95
Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr 100
105 110 Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro
Pro 115 120 125 Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr
Thr Pro Ala 130 135 140 Ile Leu Asp Ile Asn Asn Thr Val Thr Thr Val
Thr Thr His Asn Asn 145 150 155 160 Pro Thr Phe Thr Asp Pro Ser Val
Leu Gln Pro Pro Thr Pro Ala Glu 165 170 175 Thr Gly Gly His Phe Thr
Leu Ser Ser Ser Thr Ile Ser Thr His Asn 180 185 190 Tyr Glu Glu Ile
Pro Met Asp Thr Phe Ile Val Ser Thr Asn Pro Asn 195 200 205 Thr Val
Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Val Ala Arg 210 215 220
Leu Gly Leu Tyr Ser Arg Thr Thr Gln Gln Val Lys Val Val Asp Pro 225
230 235 240 Ala Phe Val Thr Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn
Pro Ala 245 250 255 Tyr Glu Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe
Ser Ser Asn Asp 260 265 270 Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp
Phe Leu Asp Ile Val Ala 275 280 285 Leu His Arg Pro Ala Leu Thr Ser
Arg Arg Thr Gly Ile Arg Tyr Ser 290 295 300 Arg Ile Gly Asn Lys Gln
Thr Leu Arg Thr Arg Ser Gly Lys Ser Ile 305 310 315 320 Gly Ala Lys
Val His Tyr Tyr Tyr Asp Phe Ser Thr Ile Asp Pro Ala 325 330 335 Glu
Glu Ile Glu Leu Gln Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr 340 345
350 Ser His Ala Ala Ser Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile
355 360 365 Tyr Ala Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val
Pro Ser 370 375 380 Val Pro Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala
Asn Thr Thr Ile 385 390 395 400 Pro Phe Gly Gly Ala Tyr Asn Ile Pro
Leu Val Ser Gly Pro Asp Ile 405 410 415 Pro Ile Asn Ile Thr Asp Gln
Ala Pro Ser Leu Ile Pro Ile Val Pro 420 425 430 Gly Ser Pro Gln Tyr
Thr Ile Ile Ala Asp Ala Gly Asp Phe Tyr Leu 435 440 445 His Pro Ser
Tyr Tyr Met Leu Arg Lys Arg Arg Lys Arg Leu Pro Tyr 450 455 460 Phe
Phe Ser Asp Val Ser Met His Gly Asp Thr Pro Thr Leu His Glu 465 470
475 480 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr
Glu 485 490 495 Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp
Gly Pro Ala 500 505 510 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn
Ile Val Thr Phe Cys 515 520 525 Cys Lys Cys Asp Ser Thr Leu Arg Leu
Cys Val Gln Ser Thr His Val 530 535 540 Asp Ile Arg Thr Leu Glu Asp
Leu Leu Met Gly Thr Leu Gly Ile Val 545 550 555 560 Cys Pro Ile Cys
Ser Gln Lys Pro Ser Met Glu Thr Leu Cys Gln Arg 565 570 575 Leu Asn
Val Cys Gln Asp Lys Ile Leu Thr His Tyr Glu Asn Asp Ser 580 585 590
Thr Asp Leu Arg Asp His Ile Asp Tyr Trp Lys His Met Arg Leu Glu 595
600 605 Cys Ala Ile Tyr Tyr Lys Ala Arg Glu Met Gly Phe Lys His Ile
Asn 610 615 620 His Gln Val Val Pro Thr Leu Ala Val Ser Lys Asn Lys
Ala Leu Gln 625 630 635 640 Ala Ile Glu Leu Gln Leu Thr Leu Glu Thr
Ile Tyr Asn Ser Gln Tyr 645 650 655 Ser Asn Glu Lys Trp Thr Leu Gln
Asp Val Ser Leu Glu Val Tyr Leu 660 665 670 Thr Ala Pro Thr Gly Cys
Ile Lys Lys His Gly Tyr Thr Val Glu Val 675 680 685 Gln Phe Asp Gly
Asp Ile Cys Asn Thr Met His Tyr Thr Asn Trp Thr 690 695 700 His Ile
Tyr Ile Cys Glu Glu Ala Ser Val Thr Val Val Glu Gly Gln 705 710 715
720 Val Asp Tyr Tyr Gly Leu Tyr Tyr Val His Glu Gly Ile Arg Thr Tyr
725 730 735 Phe Val Gln Phe Lys Asp Asp Ala Glu Lys Tyr Ser Lys Asn
Lys Val 740 745 750 Trp Glu Val His Ala Gly Gly Gln Val Ile Leu Cys
Pro Thr Ser Val 755 760 765 Phe Ser Ser Asn Glu Val Ser Ser Pro Glu
Ile Ile Arg Gln His Leu 770 775 780 Ala Asn His Ser Ala Ala Thr His
Thr Lys Ala Val Ala Leu Gly Thr 785 790 795 800 Glu Glu Thr Gln Thr
805 10 536 PRT Artificial Sequence HPV-16 L2/E6 fusion protein 10
Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr 1 5
10 15 Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp
Ile 20 25 30 Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Asp Gln Ile
Leu Gln Tyr 35 40 45 Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly
Ile Gly Thr Gly Ser 50 55 60 Gly Thr Gly Gly Arg Thr Gly Tyr Ile
Pro Leu Gly Thr Arg Pro Pro 65 70 75 80 Thr Ala Thr Asp Thr Leu Ala
Pro Val Arg Pro Pro Leu Thr Val Asp 85 90 95 Pro Val Gly Pro Ser
Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr 100 105 110 Ser Phe Ile
Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro 115 120 125 Asp
Val Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala 130 135
140 Ile Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn
145 150 155 160 Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr
Pro Ala Glu 165 170 175 Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr
Ile Ser Thr His Asn 180 185 190 Tyr Glu Glu Ile Pro Met Asp Thr Phe
Ile Val Ser Thr Asn Pro Asn 195 200 205 Thr Val Thr Ser Ser Thr Pro
Ile Pro Gly Ser Arg Pro Val Ala Arg 210 215 220 Leu Gly Leu Tyr Ser
Arg Thr Thr Gln Gln Val Lys Val Val Asp Pro 225 230 235 240 Ala Phe
Val Thr Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala 245 250 255
Tyr Glu Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Ser Ser Asn Asp 260
265 270 Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val
Ala 275 280 285 Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile
Arg Tyr Ser 290 295 300 Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg
Ser Gly Lys Ser Ile 305 310 315 320 Gly Ala Lys Val His Tyr Tyr Tyr
Asp Phe Ser Thr Ile Asp Pro Ala 325 330 335 Glu Glu Ile Glu Leu Gln
Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr 340 345 350 Ser His Ala Ala
Ser Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile 355 360 365 Tyr Ala
Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val Pro Ser 370 375 380
Val Pro Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala Asn Thr Thr Ile 385
390 395 400 Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser Gly Pro
Asp Ile 405 410 415 Pro Ile Asn Ile Thr Asp Gln Ala Pro Ser Leu Ile
Pro Ile Val Pro 420 425 430 Gly Ser Pro Gln Tyr Thr Ile Ile Ala Asp
Ala Gly Asp Phe Tyr Leu 435 440 445 His Pro Ser Tyr Tyr Met Leu Arg
Lys Arg Arg Lys Arg Leu Pro Tyr 450 455 460 Phe Phe Ser Asp Val Ser
Met His Gln Lys Arg Thr Ala Met Phe Gln 465 470 475 480 Asp Pro Gln
Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu 485 490 495 Gln
Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln 500 505
510 Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys
515 520 525 Ile Val Tyr Arg Asp Gly Asn Pro 530 535 11 1518 DNA
Artificial Sequence HPV-16 L1 wild type NVAX clone 11 atgtctcttt
ggctgcctag tgaggccact gtctacttgc ctcctgtccc agtatctaag 60
gttgtaagca cggatgaata tgttgcacgc acaaacatat attatcatgc aggaacatcc
120 agactacttg cagttggaca tccctatttt cctattaaaa aacctaacaa
taacaaaata 180 ttagttccta aagtatcagg attacaatac agggtattta
gaatacattt acctgacccc 240 aataagtttg gttttcctga cacctcattt
tataatccag atacacagcg gctggtttgg 300 gcctgtgtag gtgttgaggt
aggtcgtggt cagccattag gtgtgggcat tagtggccat 360 cctttattaa
ataaattgga tgacacagaa aatgctagtg cttatgcagc aaatgcaggt 420
gtggataata gagaatgtat atctatggat tacaaacaaa cacaattgtg tttaattggt
480 tgcaaaccac ctatagggga acactggggc aaaggatccc catgtaccaa
tgttgcagta 540 aatccaggtg attgtccacc attagagtta ataaacacag
ttattcagga tggtgatatg 600 gttgatactg gctttggtgc tatggacttt
actacattac aggctaacaa aagtgaagtt 660 ccactggata tttgtacatc
tatttgcaaa tatccagatt atattaaaat ggtgtcagaa 720 ccatatggcg
acagcttatt tttttattta cgaagggaac aaatgtttgt tagacattta 780
tttaataggg ctggtgctgt tggtgaaaat gtaccagacg atttatacat taaaggctct
840 gggtctactg caaatttagc cagttcaaat tattttccta cacctagtgg
ttctatggtt 900 acctctgatg cccaaatatt caataaacct tattggttac
aacgagcaca gggccacaat 960 aatggcattt gttggggtaa ccaactattt
gttactgttg ttgatactac acgcagtaca 1020 aatatgtcat tatgtgctgc
catatctact tcagaaacta catataaaaa tactaacttt 1080 aaggagtacc
tacgacatgg ggaggaatat gatttacagt ttatttttca actgtgcaaa 1140
ataaccttaa ctgcagacgt tatgacatac atacattcta tgaattccac tattttggag
1200 gactggaatt ttggtctaca acctccccca ggaggcacac tagaagatac
ttataggttt 1260 gtaacatccc aggcaattgc ttgtcaaaaa catacacctc
cagcacctaa agaagatccc 1320 cttaaaaaat acactttttg ggaagtaaat
ttaaaggaaa agttttctgc agacctagat 1380 cagtttcctt taggacgcaa
atttttacta caagcaggat tgaaggccaa accaaaattt 1440 acattaggaa
aacgaaaagc tacacccacc acctcatcta cctctacaac tgctaaacgc 1500
aaaaaacgta agctgtaa 1518 12 1518 DNA Artificial Sequence HPV-16 L1
wild type clone (GenBank K02718) 12 atgtctcttt ggctgcctag
tgaggccact gtctacttgc ctcctgtccc agtatctaag 60 gttgtaagca
cggatgaata tgttgcacgc acaaacatat attatcatgc aggaacatcc 120
agactacttg cagttggaca tccctatttt cctattaaaa aacctaacaa taacaaaata
180 ttagttccta aagtatcagg attacaatac agggtattta gaatacattt
acctgacccc 240 aataagtttg gttttcctga cacctcattt tataatccag
atacacagcg gctggtttgg 300 gcctgtgtag gtgttgaggt aggtcgtggt
cagccattag gtgtgggcat tagtggccat 360 cctttattaa ataaattgga
tgacacagaa aatgctagtg cttatgcagc aaatgcaggt 420 gtggataata
gagaatgtat atctatggat tacaaacaaa cacaattgtg tttaattggt 480
tgcaaaccac ctatagggga acactggggc aaaggatccc catgtaccaa tgttgcagta
540 aatccaggtg attgtccacc attagagtta ataaacacag ttattcagga
tggtgatatg 600 gttcatactg gctttggtgc tatggacttt actacattac
aggctaacaa aagtgaagtt 660 ccactggata tttgtacatc tatttgcaaa
tatccagatt atattaaaat ggtgtcagaa 720 ccatatggcg acagcttatt
tttttattta cgaagggaac aaatgtttgt tagacattta 780 tttaataggg
ctggtactgt tggtgaaaat gtaccagacg atttatacat taaaggctct 840
gggtctactg caaatttagc cagttcaaat tattttccta cacctagtgg ttctatggtt
900 acctctgatg cccaaatatt caataaacct tattggttac aacgagcaca
gggccacaat 960 aatggcattt gttggggtaa ccaactattt gttactgttg
ttgatactac acgcagtaca 1020 aatatgtcat tatgtgctgc catatctact
tcagaaacta catataaaaa tactaacttt 1080 aaggagtacc tacgacatgg
ggaggaatat gatttacagt ttatttttca actgtgcaaa 1140 ataaccttaa
ctgcagacgt tatgacatac atacattcta tgaattccac tattttggag 1200
gactggaatt ttggtctaca acctccccca ggaggcacac tagaagatac ttataggttt
1260 gtaacccagg caattgcttg tcaaaaacat acacctccag cacctaaaga
agatgatccc 1320 cttaaaaaat acactttttg ggaagtaaat ttaaaggaaa
agttttctgc agacctagat 1380 cagtttcctt taggacgcaa atttttacta
caagcaggat tgaaggccaa accaaaattt 1440 acattaggaa aacgaaaagc
tacacccacc acctcatcta cctctacaac tgctaaacgc 1500 aaaaaacgta
agctgtaa 1518 13 1413 DNA Artificial Sequence HPV-16 L2 wild type
NVAX clone 13 atgcgacaca
aacgttctgc aaaacgcaca aaacgtgcat cggctaccca actttataaa 60
acatgcaaac aggcaggtac atgtccacct gacattatac ctaaggttga aggcaaaact
120 attgctgatc aaatattaca atatggaagt atgggtgtat tttttggtgg
gttaggaatt 180 ggaacagggt cgggtacagg cggacgcact gggtatattc
cattgggaac aaggcctccc 240 acagctacag atacacttgc tcctgtaaga
ccccctttaa cagtagatcc tgtgggccct 300 tctgatcctt ctatagtttc
tttagtggaa gaaactagtt ttattgatgc tggtgcacca 360 acatctgtac
cttccattcc cccagatgta tcaggattta gtattactac ttcaactgat 420
accacacctg ctatattaga tattaataat actgttacta ctgttactac acataataat
480 cccactttca ctgacccatc tgtattgcag cctccaacac ctgcagaaac
tggagggcat 540 tttacacttt catcatccac tattagtaca cataattatg
aagaaattcc tatggataca 600 tttattgtta gcacaaaccc taacacagta
actagtagca cacccatacc agggtctcgc 660 ccagtggcac gcctaggatt
atatagtcgc acaacacaac aagttaaagt tgtagaccct 720 gcttttgtaa
ccactcccac taaacttatt acatatgata atcctgcata tgaaggtata 780
gatgtggata atacattata tttttctagt aatgataata gtattaatat agctccagat
840 cctgactttt tggatatagt tgctttacat aggccagcat taacctctag
gcgtactggc 900 ataaggtaca gtagaattgg taataaacaa acactacgta
ctcgtagtgg aaaatctata 960 ggtgctaagg tacattatta ttatgatttt
agtaccattg atcctgcaga agaaatagaa 1020 ttacaaacta taacaccttc
tacatatact accacttcac atgcagcctc acctacttct 1080 attaataatg
gattatatga tatttatgca gatgacttta ttacagatac ttctacaacc 1140
ccggtaccat ctgtaccctc tacatcttta tcaggttata ttcctgcaaa tacaacaatt
1200 ccttttggtg gtgcatacaa tattccttta gtatcaggtc ctgatatacc
cattaatata 1260 actgaccaag ctccttcatt aattcctata gttccagggt
ctccacaata tacaattatt 1320 gctgatgcag gtgactttta tttacatcct
agttattaca tgttacgaaa acgacgtaaa 1380 cgtttaccat attttttttc
agatgtatcc tga 1413
* * * * *