U.S. patent application number 12/589641 was filed with the patent office on 2010-06-17 for detection method for human pappilomavirus (hpv) and its application in cervical cancer.
Invention is credited to Shuling Cheng.
Application Number | 20100151444 12/589641 |
Document ID | / |
Family ID | 38049389 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151444 |
Kind Code |
A1 |
Cheng; Shuling |
June 17, 2010 |
Detection method for human pappilomavirus (HPV) and its application
in cervical cancer
Abstract
Embodiments of the invention provide methods, assays, and kits
for detecting HPV infection and HPV associated epithelial cell
abnormalities, most notably those associated with pre-malignant and
malignant epithelial cell lesions. Detection of HPV DNAs, genomes,
and/or oncoproteins by nucleic acid hybridization assays and
immunological assays can be used in early clinical screening for
HPV infection and diagnosis for cervical cancer. The polypeptides,
recombinant proteins, antibodies, nucleic acids, and various
detection methods thereof are particularly useful for diagnosing
carcinomas of the uterine cervix and those at risk of developing
cervical cancer.
Inventors: |
Cheng; Shuling; (Fremont,
CA) |
Correspondence
Address: |
Shu-Ling Cheng
47853 Warm Springs Blvd.
Fremont
CA
94539
US
|
Family ID: |
38049389 |
Appl. No.: |
12/589641 |
Filed: |
October 26, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11559366 |
Nov 13, 2006 |
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12589641 |
|
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60737152 |
Nov 15, 2005 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12N 2710/20051
20130101; C12N 2710/20022 20130101; C12Q 1/708 20130101; C07K
14/005 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A papillomavirus detection composition comprising a recombinant
papillomavirus protein having native folding and being recognized
by one or more papillomavirus antibodies present from a clinical
sample.
2. The papillomavirus detection composition of claim 1, wherein the
clinical sample is selected from the group consisting of cervical
cells, cervical tissues, cervical swabs, body fluids, serum, blood,
tumors, and combination thereof.
3. The papillomavirus detection composition of claim 1, wherein the
recombinant papillomavirus protein is selected from the group
consisting of recombinant HPV-16 E6 proteins, recombinant HPV-16 E7
proteins, recombinant HPV-18 E6 proteins, recombinant HPV-18 E7
proteins, recombinant HPV-16 L1 proteins, recombinant HPV-18 L1
proteins, and combinations thereof.
4. The papillomavirus detection composition of claim 1, wherein the
recombinant papillomavirus protein is purified as a soluble
protein.
5. The papillomavirus detection composition of claim 1, wherein the
recognition of the recombinant papillomavirus protein to the one or
more papillomavirus antibodies from the clinical sample is
performed by conducting one or more immunological assays on the
clinical sample.
6. The papillomavirus detection composition of claim 5, wherein the
one or more immunological assays are selected from the group
consisting of ELISA assays, antigen assays for papillomavirus
proteins, antibody assays for antibodies against papillomavirus
proteins, assays for papillomavirus immunocomplexes, protein chip
assays, radioimmunoprecipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays.
7. A method of detecting papillomavirus infection from a clinical
sample of a human subject, comprising: obtaining a clinical sample
from the human subject; providing a papillomavirus detection
composition of claim 1; conducting one or more immunological assays
on the clinical sample; and detecting the presence of one or more
papillomavirus antibodies in the human subject using the
recombinant papillomavirus protein.
8. The method of claim 7, wherein the clinical sample is selected
from the group consisting of cervical cells, cervical tissues,
cervical swabs, body fluids, serum, blood, tumors, and combination
thereof.
9. The method of claim 7, wherein the one or more immunological
assays are selected from the group consisting of ELISA assays,
antigen assays for papillomavirus proteins, antibody assays for
antibodies against papillomavirus proteins, assays for
papillomavirus immunocomplexes, protein chip assays,
radioimmunopercipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays.
10. The method of claim 7, wherein the recombinant papillomavirus
protein is selected from the group consisting of recombinant HPV-16
E6 proteins, recombinant HPV-16 E7 proteins, recombinant HPV-18 E6
proteins, recombinant HPV-18 E7 proteins, recombinant HPV-16 L1
proteins, recombinant HPV-18 L1 proteins, and combinations
thereof.
11. A method of detecting papillomavirus infection from a clinical
sample of a human subject, comprising: obtaining a clinical sample
from the human subject; providing a papillomavirus detection
composition of claim 1; conducting one or more immunological assays
on the clinical sample; and detecting the presence of one or more
papillomavirus viral proteins in the clinical sample of the human
subject using anti-HPV antibody and the papillomavirus composition
of claim 1.
12. The method of claim 11, wherein the clinical sample is selected
from the group consisting of cervical cells, cervical tissues,
cervical swabs, body fluids, serum, blood, tumors, and combination
thereof.
13. The method of claim 11, wherein the one or more immunological
assays are selected from the group consisting of ELISA assays,
antigen assays for papillomavirus proteins, antibody assays for
antibodies against papillomavirus proteins, assays for
papillomavirus immunocomplexes, protein chip assays,
radioimmunopercipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays.
14. The method of claim 11, wherein the recombinant papillomavirus
protein is selected from the group consisting of recombinant HPV-16
E6 proteins, recombinant HPV-16 E7 proteins, recombinant HPV-18 E6
proteins, recombinant HPV-18 E7 proteins, recombinant HPV-16 L1
proteins, recombinant HPV-18 L1 proteins, and combinations
thereof.
15. Use of a papillomavirus detection composition of claim 1, for
the detection of a disease consisting of HPV infection, cervical
cancer, and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/559,366, filed Nov. 13, 2006, which claims
benefit of U.S. provisional patent application Ser. No. 60/737,152,
filed Nov. 15, 2005. Each of the aforementioned related patent
applications is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Cancer of the uterine cervix or cervical cancer is the
second most common cancer in women worldwide. Although screening
programs to identify precursor lesions of cervical cancer have
contributed to a reduction in mortality and morbidity, 500,000 new
cases of invasive cervical cancer are diagnosed worldwide and
230,000 women die of cervical cancer annually. Early detection and
diagnosis is critical for survival of cervical cancer.
[0003] Infection by human papillomaviruses (HPV) at specific
epithelium cells to induce epithelial proliferations plays an
important role for cervical carcinogenesis. About 99 percent of
confirmed cervical cancer cases are found to be associated with HPV
infection with biopsy-confirmed squamous intraepithelial lesions
(SIL) or cervical intraepithelial neoplasia (CIN). The incidence of
HPV infection, primarily transmitted through sexual contact, is
highest among young women and about 20 millions of sexually active
men and women worldwide are currently infected. Approximately 1% of
the population has genital warts and 4% of women have cervical
precancerous lesions, such as low grade of squamous intraepithelial
lesion (LGSIL) or high grade of squamous intraepithelial lesion
(HSIL). The presence of these lesions, preferentially observed in
women aged 35-40 yrs, are at high risk of progression toward
invasive cancer.
[0004] There are more than one hundred genetic types of human
papillomaviruses identified so far and only a relative few types of
HPV, such as HPV-16, -18, -31, -33, -35, -45, -51, -52 and -56,
etc., involve high risk of progression from HPV infected genital
tissue lesions to invasive cervical cancer. Infection with the vast
majority of HPV types, such as HPV-6 and -11, etc., are transient
with no permanent changes in genital tissues and are at low risk
for developing into invasive cervical cancer. However, the
development of cervical cancer is a multiple step process that
cannot be explained simply by infection with specific types of HPV.
Persistent infections with HPVs in high risk group are essential
but not exclusively required for the initiation of cervical
carcinogenesis. It is found that younger age group women are often
infected with HPV; however, clinical information reveals that most
latent or asymptomatic infections with high risk HPV types as well
as early dysplastic lesions (CIN 1) are usually self-limited and
regress spontaneously. There is a high level of correlation between
long term persistent infections with only few high-risk HPV types
and the induction of advanced CIN 2/CIN 3 lesions and/or the
progression to invasive cancer.
[0005] One additional event that appears to play a role in tumor
progression is the integration of HPV DNA genome into host genome,
which frequently disrupts the open reading frame for an early viral
gene, E2, resulting in over-expression of two important viral E6
and E7 oncoproteins and transformation of the host cells. Since
almost all cervical cancer cases harbor high risk-HPV genomes,
screening with HPV infection is important, especially long term
infection with high risk HPV types. Other factors and mutational or
secondary genetic events may also be important in the progression
and pathogenesis of invasive cervical cancers, including
recombination, integration of viral genes to host cell chromosomes,
chromosomal rearrangements, loss of constitutional heterozygosity,
and proto-oncogene activation.
[0006] In the past, screening for cervical cancer is based on
conventional cytology by papanicolaou (Pap) smear and suspicious
smears are followed up with colposcopy, and/or histological biopsy.
The use of cytological screening lead to a remarkable reduction in
the mortality of cervical cancer. However, due to subjective test
criteria, drawbacks of pap smear tests include difficulty in
obtaining samples, poor inter- and intra-observer agreement, a high
rate of false negatives (up to 20%) and false positive, the
requirements for specialized labs staffed with highly trained
personnel, and inability to identify a large proportion of
HPV-infected persons. More reproducible assays are needed to
improve the current screening method and avoid unnecessary medical
intervention and psychological distress for the affected women.
Nucleic acid methods, such as "DNA Hybrid Capture"; have been
developed, but are not ideal primarily due to their high cost,
assay operation procedures, and the requirements for facility,
equipment, and highly trained personnel. What is needed is a low
cost, simple, sensitive and specific assay that can be performed on
routine practice of a clinical lab or doctor office.
[0007] Attempts to detect the presence of HPV related antibodies in
a human subject by ELISA (enzyme linked immunoabsorbant assays)
generally lead to extremely low assay sensitivity and thus can not
be developed into a commercially suitable diagnostic test. Most of
these ELISA assays target a single viral protein or short peptide
fragments, which were not able to interact well or bind strongly
and specifically to antibodies from the human subject. The assay
specificity and sensitivity are so low such that even using samples
from patients confirmed with HPV associated invasive cervical
cancer, only 53% of the patient samples were found positive for HPV
infection. Thus, there is no successful ELISA assay available as a
diagnostic tool for clinical samples.
[0008] Therefore, there is a need to develop methods and assays for
early detection of HPV infection and assisting in diagnosis of
cervical cancer.
SUMMARY OF THE INVENTION
[0009] Embodiments of the invention generally relate to various
methods, detection assays, kits, polypeptides, recombinant
proteins, antibodies, and nucleic acids useful for detecting
general HPV infection as well as high risk HPV infection. In one
embodiment, a method of screening a human subject of papillomavirus
infection includes obtaining a clinical sample from the human
subject, obtaining a first recombinant protein encoded by an early
gene of a papillomavirus, and obtaining a second recombinant
protein encoded by a late gene of the papillomavirus. The method
further includes conducting one or more immunological assays on the
clinical sample from the human subject, detecting the presence of
an antibody to the first recombinant protein in the human subject
using the first recombinant protein, and detecting the presence of
an antibody to the second recombinant protein in the human subject
using the second recombinant protein.
[0010] In another embodiment, a method of screening a human subject
infected with a human papillomavirus includes obtaining a clinical
sample from the human subject, conducting a nucleic acid
hybridization assay on the clinical sample, detecting the presence
of a papillomavirus genome in the clinical sample from the human
subject, and conducting one or more immunological assays on the
clinical sample. The one or more immunological assays can be
performed independently or concurrently of the nucleic acid
hybridization assay using the same or different clinical sample but
from the same human subject. The one or more immunological assays
are performed to detect the presence of an antibody to an early
papillomavirus viral protein or the presence of the early
papillomavirus viral protein in the clinical sample using a first
recombinant protein of the early papillomavirus viral protein.
Further, the presence of an antibody to a late papillomavirus viral
protein or the presence of the papillomavirus late viral protein in
the clinical sample is detected using a second recombinant protein
of the late papillomavirus viral protein in the same or different
immunological assays as the immunological assay performed using the
first recombinant protein independently or concurrently.
[0011] In still another embodiment, a method of screening a human
subject of high risk human papillomavirus infection is provided.
The method includes obtaining a clinical sample from the human
subject, obtaining a first recombinant protein purified from a
first protein expression system with a first expression vector
having a portion of nucleic acid sequence corresponding to the full
length nucleic acid sequence of an early papillomavirus gene, and
obtaining a second recombinant protein purified from a second
protein expression system with a second expression vector having a
portion of nucleic acid sequence corresponding to the full length
nucleic acid sequence of a late papillomavirus gene. Further, one
or more immunological assays are conducted on the clinical sample
to detect the presence of an antibody to a viral oncoprotein or the
presence of the viral oncoprotein in the clinical sample using the
first recombinant protein, and the presence of an antibody to a
viral capsid protein or the presence of the viral capsid protein in
the clinical sample using the second recombinant protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] So that the manner in which the above recited features of
the present invention can be understood in detail, a more
particular description of the invention, briefly summarized above,
may be had by reference to embodiments, some of which are
illustrated in the appended drawings. It is to be noted, however,
that the appended drawings illustrate only typical embodiments of
this invention and are therefore not to be considered limiting of
its scope, for the invention may admit to other equally effective
embodiments.
[0013] FIG. 1 illustrates an example of a method according to one
or more embodiments of the invention.
[0014] FIG. 2A demonstrates one embodiment of an exemplary purified
recombinant protein encoded by an E6 early gene as visualized by
SDS-PAGE by staining with commassie blue.
[0015] FIG. 2B demonstrates detection of a purified recombinant
protein, HPV-16 E6 recombinant protein, by Western blot analyses
according to one or more embodiments of the invention.
[0016] FIG. 2C demonstrates the result of gel filtration column
chromatography of the purified recombinant E6 protein,
demonstrating that the purified recombinant proteins HPV-16-E6 is a
monomeric soluble protein. The purified recombinant E6 protein is
eluted later than BSA.
[0017] FIG. 3 is a SDS-PAGE gel, showing one exemplary purified
recombinant HPV-16-E7 proteins according to one or more embodiments
of the invention.
[0018] FIG. 4 demonstrates SDS-PAGE of three exemplary purified HPV
early gene recombinant proteins by commassie blue staining
according to one or more embodiments of the invention. P1:
HPV-58-E6-MBP fusion protein; P3: MBP protein; P2: HPV-16-E7-His
fusion protein; CP: HPV-16-E6-His fusion protein.
[0019] FIG. 5 demonstrates detection of three exemplary purified
HPV early gene recombinant proteins with different antibody by
Western blotting. Lane 1: HPV-16-E7-His with MAb2-1 (anti-HPV-16-E7
antibody); Lane 2: HPV-16-E7-His with MAb2-2 (anti-HPV16E7
antibody); Lane 3: HPV-16-E7-His with MAb2-3 (anti-HPV-16-E7
antibody).
[0020] FIG. 6 demonstrates detection of an exemplary purified HPV
late gene L1 recombinant protein by Western blotting using anti-his
tag antibody and chemilluminescent substrates (visualized by the
reaction of NBT and BCIP substrates) according to one or more
embodiments of the invention.
[0021] FIG. 7 is a graph showing the results of an exemplary
immunological ELISA assay for detecting a monoclonal antibody
against E6 oncoprotein on microtiter plates using a purified
recombinant protein, recombinant HPV-16 E6 protein, coated on the
bottom of the microtiter plates.
[0022] FIG. 8 is a graph showing the results of an ELISA assay for
detecting a monoclonal antibody against E6 oncoprotein on
microtiter plates using another exemplary purified recombinant
protein, a recombinant HPV-58 E6 protein, coated on the bottom of
the microtiter plates.
[0023] FIG. 9 is an exemplary graph showing the results of an ELISA
assay for detecting various monoclonal antibodies (MAb1-1, MAb1-2,
and MAb1-4) against E6 oncoprotein on microtiter plates using
purified recombinant proteins, recombinant HPV-16 E6 proteins,
coated on the bottom of the microtiter plates.
[0024] FIG. 10 is another exemplary graph showing the results of an
immunological ELISA assay for detecting a monoclonal antibody
(MAb2-1) and a polyclonal antibody (PAb2-1) against E7 oncoprotein
on microtiter plates using purified recombinant proteins,
recombinant HPV-16-E7 fusion proteins, coated on the bottom of the
microtiter plates.
[0025] FIG. 11 is another exemplary graph showing the results of an
immunological ELISA assay sensitivity for detecting monoclonal
antibody (MAb) against E7 oncoprotein using purified recombinant
proteins, recombinant HPV-16-E7 fusion proteins, coated on the
bottom of the microtiter plates.
[0026] FIG. 12 is a graph showing the results of another exemplary
immunological sandwiched ELISA assay for detecting purified
recombinant proteins, recombinant HPV-16-E7 fusion proteins, using
a combination of two monoclonal-antibodies (MAb2-1, MAb 2-3) and a
combination of polyclonal- and monoclonal-antibody (PAb2-1, MAb2-1)
against E7 oncoprotein as a pair of capture and detection
antibodies for capturing the recombinant proteins and being able to
be detected by a detection system.
[0027] FIG. 13 demonstrates the results of an exemplary
immunological protein chip assay for detecting a monoclonal
antibody against E6 oncoprotein using purified recombinant proteins
coated on a protein chip. P1 indicates recombinant HPV-58 E6-MBP
fusion protein. P3 indicates MBP protein as a control. P2 indicates
recombinant HPV-16 E7 his tag protein. P4 indicates recombinant
HPV-16 E6 his tag protein.
[0028] FIG. 14 illustrates a comparison of the results from E6
antibody test with E7 antibody test using cervical samples from
human subjects according to one or more embodiments of the
invention.
[0029] FIG. 15 illustrates a comparison of the results from
antibody tests with antigen test using cervical samples from human
subjects according to one or more embodiments of the invention.
[0030] FIG. 16 illustrates the results from E6 Ab tests, L1 Ab
tests, and PCR tests using cervical samples from human subjects
according to one or more embodiments of the invention for 173
subjects with normal pap smear scores.
[0031] FIG. 17 illustrates the results of the E6 Ab tests, L1 Ab
tests, and PCR L1 tests using cervical samples from human subjects
according to one or more embodiments of the invention for 20
subjects with abnormal pap smear scores.
DETAILED DESCRIPTION
[0032] Embodiments of the invention provide various methods,
detection assays, and kits, polypeptides, recombinant proteins,
antibodies, and nucleic acids useful for detecting general HPV
infection as well as infection with high risk HPV types. For
example, detection of HPV DNAs, genomes, early viral proteins, late
viral proteins, oncoproteins, and/or capsid proteins by nucleic
acid hybridization assays and immunological assays as described
herein can be used in early clinical screening for HPV infection.
It can also be used in diagnosing HPV-associated carcinomas of the
uterine cervix, as well as those cases associated with epithelial
cell abnormalities induced by HPV infection, pre-malignant and
malignant HPV-associated epithelial cell lesions, and those at risk
of developing HPV-associated cervical carcinoma and adenocarcinoma.
The methods as described herein can be used independently or as an
adjunct screening tool to convention cytological papanicolaou smear
tests or histological tests and the results thereof can be compared
from follow-up patient management.
[0033] FIG. 1 illustrates a method 100 of screening a human subject
of papillomavirus infection. At step 110, a clinical sample from a
human subject is obtained. The clinical sample may include, but are
not limited to, genital swabs, general fluid, cervical cells,
cervical tissues, cervical swabs, body fluids, serum, blood, urine,
lesion sites, and tumors, among others. The clinical sample may be
obtained by various methods known in the art. For example, genital
swabs from clinical hospitals can be provided, together with pap
smear scores, cytological results, and demographic history of the
clinical samples from human subjects, either normal subjects or
patients.
[0034] At step 120, one or more recombinant proteins encoded by an
early gene and/or a late gene of a papillomavirus are obtained. The
human papillomavirus may be, for example, high risk HPV types, low
risk HPV types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39,
HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6,
HPV-11, HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56,
etc. High risk human papillomaviruses include, but not limited to,
HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51,
HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, among others. Low risk
human papillomaviruses include, but not limited to, HPV-6, HPV-11,
HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, among
others.
[0035] Papillomaviruses are DNA viruses with a DNA genome, a
non-enveloped viron, and an icosahedral capsid. The
double-stranded, circular HPV DNA genome contains one coding region
for late genes, one coding region for early genes, and a non-coding
upstream regulatory region with binding sites for the various
transcription factors controlling expression of early and late
genes. Two separate open reading frames in the late gene coding
region encode viral capsid proteins L1 and L2. Capsid protein L1 is
the major capsid protein that is highly conserved among different
HPV types. Eight open reading frames in the early gene coding
region, encode eight viral early proteins, designated E1, E2, E3,
E4, E5, E6, E7, and E8. Early proteins E6 and E7 are oncoproteins
critical for host cell immortalization and transformation as well
as for long term viral replication and survival.
[0036] Infection by high risk HPVs requires two early viral
proteins, E6 and E7, which are oncoproteins because they transform
cells in vitro and their presence is needed to maintain malignancy.
Inhibition of E6 and E7 expression in precancerous or cancer
cervical tissue blocks invasive cancer progression. Inside host
tissues, E6 and E7 oncoproteins work by negatively blocking the
activities of endogenous host cellular regulatory proteins, p53 and
retinoblastoma (Rb) tumor suppression proteins, respectively, to
cause inhibition of apoptosis and deregulation of cell cycle,
leading to development of cervical cancers. E6 oncoprotein binds to
p53, a cellular factor that protects against DNA damage and
regulates apoptosis, to induce degradation of p53. By reducing the
levels of p53 protein, E6 oncoprotein prevents tumor cell death. E7
oncoprotein binds to Rb to induce degradation of Rb, disrupt normal
cell cycle, and cause cellular proliferation. The E7 oncoprotein
further destabilizes cell control through its interaction with the
cyclin-dependent kinase inhibitor protein, p21. HPV E6 and E7
oncoproteins are found to be continuously produced in transformed
genital tissues. These interactions set the stage for controlling
host cell proliferation and differentiation (i.e., transformation),
a first step in the conversion of normal cells to pre-neoplastic
cells and ultimately to the full expression of cancer
malignancy.
[0037] One additional event that appears to play a role in tumor
progression is integration of HPV DNA into host genome, which
frequently disrupts the open reading frame for E2, resulting in
over-expression of the E6 and E7 oncoproteins and possibly causing
instability of host genome. Additional cofactors and mutational
events may be important in the pathogenesis of invasive cervical
cancers and may include chromosomal rearrangements, loss of
constitutional heterozygosity, and proto-oncogene activation.
[0038] Both HPV-16 and HPV-18 are shown to immortalize human
keratinocytes in culture and are by far the most common high risk
HPV types that induce invasive cervical cancer. Infection by HPV-16
type alone is associated with over 50% of cervical cancer cases,
mostly resulting in squamous cell carcinoma. HPV-18 infection is
more likely to induce adenocarciomas. Some studies have indicated
that adenocarcinomas in cervical tissues produce more aggressive
forms of cancer with a less favorable outcome than cancers
resulting from squamous cell carcinomas. This suggests that
individuals with HPV-18 infection may have a much poorer prognosis
than those with any other form of HPV infection.
[0039] To test the hypothesis that E6 and E7 play an active role in
the maintenance of the malignant phenotype and may be ideal targets
for anti-gene therapy, studies showed antiproliferative effects of
phosphorothioate oligodeoxynucleotides (oligos) targeting HPV-16 E6
and E7 in cervical cancer cell lines and primary tumor explants.
These specific antiproliferative effects suggest that HPV-16 E6 and
E7 sequences play an active role in the malignant growth properties
of cervical cancer cells and may be ideal targets for anti-gene
therapy. Expression of two viral oncogenes, E6 and E7, in
epithelial stem cells is required to initiate and maintain cervical
carcinogenesis and results in significant over-expression of the
cellular p16INK4a protein.
[0040] Variants of HPV-16 have also been found to produce
differences in the aggressiveness of the forms of cervical cancer
they induce. For example, Asian-American HPV-16 variants are more
oncogenic than European HPV-16 variants. It has also been shown
that Asian-American and African HPV-16 variants are more likely to
produce invasive cervical cancer than European HPV-16 variants. The
more aggressive nature of some of these variants may be related to
variations in the amino acid sequences of the oncoproteins produced
by the viruses. E6 protein from Asian-American HPV-16 variants are
shown to be stronger in transforming keratinocytes and in
suppressing p53 expression than E6 protein from European HPV-16
variants and these E6 proteins differ only in several amino acids
in their sequences. Thus, in diagnosing high risk patients for
invasive cervical cancer progression, it is important to identify
not only the specific HPV type infecting the patient, but also the
variant type of the infecting HPV.
[0041] In one embodiment, the early gene that can be used herein
may include papillomavirus E6 genes, papillomavirus E7 genes, among
others. In another embodiment, the late gene that can be used
herein may include papillomavirus L1 genes, papillomavirus L2
genes, among others.
[0042] One aspect of the invention provides recombinant proteins,
such as a recombinant hybrid protein containing a full length
sequence of HPV oncogenic proteins, e.g., full-length E6, E7,
and/or L1 polypeptide sequence, which have been found very
difficult to obtain and purify due to undesirable aggregation
during protein purification, protein instability, low levels of
expression, low immunogenic responses of purified proteins. For
example, many early E6 oncoproteins contain many cysteine amino
acids and thus the correct topography of the E6 oncoproteins
requires formation of many disulfide bonds properly. In addition,
it was known that certain immunological assays using small peptides
of early E6 and E7 proteins results in extremely low assay
specificity and sensitivity and thus unsuitable as commercialized
diagnostic tools.
[0043] The one or more recombinant proteins as described herein can
be expressed in various suitable systems, such as bacterial
expression systems, viral expression systems, yeast expression
systems, mammalian expression systems, e.g., in E. coli, yeast,
baculovirus, and/or mammalian cell cultures, generally known in the
field. Although the polypeptides could be obtained by other means,
embodiments of the invention provide one or more recombinant
proteins mostly in (or close to) their native forms, which may be a
much desirable conformation for binding with antibodies from
tissues of human subjects with HPV infection in an immunological
assay.
[0044] At step 130, one or more immunological assays are conducted
on the clinical sample from the human subject. The one or more
immunological assays as developed herein lend themselves to the
high quality and properly purified recombinant proteins encoded by
HPV early and late genes, resulting in immunological assays with
very high sensitivity and specificity for screening HPV
infection.
[0045] The one or more immunological assays include, but are not
limited to, ELISA (enzyme linked immunoabsorbant assays), antigen
assays for papillomavirus proteins, antibody assays for antibodies
against papillomavirus proteins, assays for papillomavirus
immunocomplexes, protein chip assays, radioimmunopercipitation
assays, rapid membrane immunochromatographic assays, rapid stick
immunochromatographic assays, among others. The one or more
immunological assays may be non-invasive with minimal or no
additional instrument required. The basic techniques for conducting
the immunological assays can be found in "Antibodies: A Laboratory
Manual", Harlow and Lane, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. 1989; "Molecular Cloning", A Laboratory Manual,
eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory
Press, 1989. and others books and manuals known in the art.
[0046] For example, the one or more immunological-based assays may
include antibody-based assay having purified papillomarivus
proteins coated on a surface, such as bottom surfaces of a
microtiter plate, a membrane, and/or a chip. The surfaces that are
not coated can be blocked with non-binding proteins. Then, a sample
to be tested, such as a sample (samples from human subjects) likely
with antibodies against HPV virus or HPV-associated proteins can
bind to the surface by binding to the coated purified
papillomarivus proteins. The bound antibody-purified papillomarivus
protein complex can be detected by a secondary antibody and a
number of commercially available detection systems using
colormetric, chemilumenescent, or flourescent substrate. One
example of secondary antibody is a horse radish
peroxidase-conjugated secondary antibody, such as an antibody
against-human immunoglobins (specific for IgG, IgA, etc.). The
final results can be read by a microplate reader or visualized by
eye if colormetric substrates are used.
[0047] As another example, an antigen assay involves coating of a
primary antibody, such as a capture antibody having an affinity for
binding to an antigen of interest, on a surface, such as bottom
surfaces of a microtiter plate, a membrane, a chip, etc. The
antigen of interest may be, for example, a papillomarivus protein,
an oncoprotein, a capsid protein, which may be encoded by a HPV
viral gene, e.g., an early gene or a late gene, etc. After blocking
unbound portions on the surface, the clinical sample to be analyzed
can be applied to bind with the capture antibody to form an
immunocomplex, which can be detected by another primary antibody or
a detection antibody by binding to the antigen of interest. Hence,
the two primary antibodies or the pair of the capture antibody and
the detection antibody interact with the antigen of interest, much
like a sandwich. The capture antibody can be the same or different
antibody as the detection antibody as long as the two antibodies
can specifically bind to the antigen of interest, e.g., a HPV viral
protein, a HPV oncoprotein, a capsid protein, among others.
[0048] Next, the sandwiched bound antibody-antigen complex can be
detected by a secondary antibody, which have an affinity for the
detection antibody and facilitate measurement by a standard
immunological complex detection system using colormetric,
chemilumenescent, flourescent and many different kinds of
substrates. The final readouts or visualizations can be performed
by an instrument with appropriate light absorbance readers or
directly visualized by eye and compared the results to a control
sample. Positive results indicate binding of the antigen of the
interest to the primary antibodies, the capture antibody, and the
detection antibody, and thus the presence of the antigen of
interest in the clinical sample. In the contrary, negative results
indicate no binding of the antigen of the interest to the primary
antibodies and thus the absence of the antigen of interest in the
clinical sample.
[0049] The one or more immunological assays can be used to detect
at least three kinds of target proteins of interest, including, but
not limited to, antigen, antibody, and antigen/antibody
immunocomplex (also referred hereafter as antigen tests, antibody
tests, and antigen/antibody immunocomplex tests, respectively),
among others.
[0050] The formats of the one or more immunological assays may be a
microplate format (e.g., 32 wells, 48 wells or 96 wells), a
vertical or lateral membrane-based rapid test, a protein chip with
multiple spot or multiplexed. The principles of the assays are the
same as described above except detection systems vary depending on
the substrate chosen for analyzing the results in different
readouts or forms by an instrument specific designed for the
assays. In addition, the procedures, conditions, binding
specificity, developed in one type of immunological assay in one
format can be adapted into a different format of the same or a
different immunological assay, and/or a different immunological
assay in the same or a different form a.
[0051] For example, in a protein chip assay, the surface for
proteins to be coated/bound to may be, for example, a
surface-chemistry treated glass or membrane, which can be
covalently or non-covalently bind or coat with capture agents or
proteins thereto. A spotting machine with fine pins dipped with
capture agents, such as the recombinant proteins, antigens,
antibodies, or other proteins, in suitable buffers is generally
used to facilitate binding of such proteins or antibodies to the
treated surface. Like other surfaces described in the microtiter
plate format, the spotted and thus captured proteins or antibodies
bind strongly to the surface-chemistry treated surface of a protein
chip and remain on the treated surface to allow the interaction and
specific binding of the captures proteins with target proteins,
antibodies, or antigens, even after several washings of removing
non-specific binding, to be detected with a detection system having
a secondary antibody conjugated with Cy3 or Cy5. The detection of
specific interaction is obtained and measured by the intensities of
the spotted/dipped images via a microarray scanner.
[0052] More than one protein can be initially spotted and thus
bound on the treated surface to specifically capture target
proteins, antibodies, or antigens, and thus, it is possible to
interact with and bind to multiple proteins or targets
(multiplexed) in a single sample and binding of at least one of the
multiple proteins or targets on the surface of the protein chip can
be detected, performing one assay which is essentially many assays
combined together. Thus, the protein chip assays, as compared to
other assay formats, advantageously provides higher sensitivity and
multiplex format with only very minimal amount of samples required,
such as less than 50 .mu.l or less than 10 .mu.l. Such feature of
being able to detect multiple binding activities with minimal
sample requirement make it feasible to conduct many assays in one
for certain disease tissues where the amount of accessible samples
are very limited.
[0053] As another example, for a rapid immunological test, the
surface for an antibody or a protein to be coated thereon can be
membrane-based, and the binding capacity of the rapid immunological
test differs, depending on the types of target proteins,
antibodies, or antigens and background non-specific protein
contained in the samples with the target protein, antibodies, or
antigens. At least two formats of the rapid immunological test can
be used, a vertical the rapid immunological test and a lateral the
rapid immunological test.
[0054] The vertical the rapid immunological test is conducted in a
device having a membrane as a capturing/binding surface for coating
or spotting a capture agent thereon. The device further contains a
pad underneath the membrane to allow the samples and assay reagent
to flow through the membrane. Any target proteins, antibodies, or
antigens that contained in the samples and specifically interact
and bind to the capture agent will not flow through and will be
captured and retain on the surface of the membrane, even after
several washings to remove non-specific binding. A secondary
antibody conjugate with HRP or others can be applied on the surface
for detecting any protein-antibody complexes retained on the
surface and being visualized by colormetric substrates.
[0055] The lateral rapid immunological test is a one-step test
using a membrane strip with the capture proteins or antibodies
already applied/coated to designated positions on the surface
thereof. The only step the test requires is to combine obtained
samples having the target proteins or antibodies with a detecting
antibody conjugated with collateral gold particles and directly
apply the combined mixtures to the membrane strip for the sample
fluid to laterally flow through the membrane strip until the
designated positions of the surface of the membrane strip. The
capture-target-detecting protein-antibody immuno-complexes can be
formed and retained on the designated positions where the capture
proteins or antibodies are coated. Positive results can be
visualized at these designated positions and no washing or
separation is required, thus called one-step. The whole procedure
for the test takes only minutes, for example, less than 15 minutes,
and thus the test is also referred to as an one-step rapid
test.
[0056] At step 140, the presence of antibodies and/or antigens
against the one or more recombinant proteins in the human subject
can be detected. Binding of any antibodies in the biological sample
to the one or more recombinant proteins, binding of any
HPV-associated proteins/antigens in a biological sample to
antibodies obtained herein using the one or more recombinant
proteins, and/or binding of any immunocomplexes of HPV-associated
proteins in the sample to the one or more recombinant proteins and
antibodies as obtained herein, indicates the presence of HPV
infection protein in the sample.
[0057] The one or more immunological assays using the purified
recombinants proteins derived from HPV early and/or late genes and
antibodies as obtained herein serve as reliable indicators whether
HPV infection has occurred. In addition, HPV associated malignancy
or pre-malignant cell transformation can be assayed. One of the
most useful aspects of the invention is in diagnosing cervical
carcinoma, both squamous cell and adenocarcinoma as well as any
epithelial cell abnormality associated with oncogenic HPV infection
including koilocytosis; hyperkerotosis; precancerous conditions
encompasssing intraepithelial neoplasias or intraepithelial lesion;
high-grade dysplasias; and invasive or malignant cancers.
[0058] The E6 and E7 oncoproteins encoded by early E6 and E7 genes
are constitutively expressed in tumor cells, and silencing these
genes yields reversion of the malignant phenotype. Thus, the early
E6 and E7 gene products seem tumor-specific antigens, and possible
targets or probes for screening these proteins/antigens or their
antibodies thereof in immunological screening assay. These
oncoproteins can also be targets for developing vaccines for
immunotherapy to control HPV induced tumors.
[0059] For example, antibodies to the E6 and/or E7 oncoproteins
have been found in those with HPV associated neoplasms. The E6 and
E7 oncoproteins appear to be natural targets for antibody
production due to their consistent expression in cervical cancer
cells. It has been found that IgG and IgA against HPV-16 E6 and E7
oncoproteins are strongly disease associated. Antibodies against
the E6 and E7 oncoproteins are at high levels in sera from cervical
cancer patients as compared against non-cancer controls. Moreover,
such antibodies can be detectable by immunological means even when
present in lesser amounts.
[0060] Antibody tests and antigen tests for detecting antibodies
against proteins encoded by early genes (e.g., E6 and E7) and late
genes (e.g., L1) are performed. As an example, for detecting the
presence of E6, E7, or L1 antibodies in human subjects, the
concentrations of recombinant proteins, E6, E7, L1, respectively,
needed to detect an anti-E6 antibody are optimized in a microtiter
plate immunological assay format. Optimal reaction times, assay
sensitivity and variability and conditions needed to semi-quantify
the levels of E6, E7, or L1 antibodies are found and assay
sensitivity and specificity can be calculated. In one embodiment,
the sensitivity of the one or more immunological antibody test
assays as described herein is in the range of micrograms, such as
in the range of nanograms, or even picograms, etc. The specificity
of the one or more immunological antibody test assays as described
herein is in the range of about 50% or higher, such as about 70% or
higher, about 85% or higher, about 90% or higher, about 95% or
higher, or about 99% or higher.
[0061] As another example, for detecting the presence of E6, E7, or
L1 antigens in human subjects, polyclonal and monoclonal antibodies
against E6, E7, or L1 using the recombinant E6, E7, or L1 proteins
are generated and the formation of immunocomplexes due to the
binding between them are validated. Optimal reaction times, assay
sensitivity and variability and conditions needed to semi-quantify
the levels of E6, E7, or L1 antibodies, respectively, are found and
the sensitivity and specificity for the assays can calculated. In
one embodiment, the sensitivity of the one or more immunological
antigen assays as described herein is between the range of
micrograms, such as in the range of nanograms, or even picograms,
etc. The specificity of the one or more immunological antigen test
assays as described herein is in the range of aout 50% or higher,
such as about 70% or higher, about 85% or higher, about 90% or
higher, about 95% or higher, or about 99% or higher.
[0062] According to one or more aspects of the invention, the
immunological assays for detection of HPV proteins, such as E6, E7,
L1, etc., or immune response thereof due to HPV infection can be
performed in high throughput ELISA screening assays, one step rapid
immunological screening assays, and multiplexed protein chip
assays, etc., and combinations thereof. Embodiments of the
invention provides one or more assays, including an antibody,
antigen, or immunocomplex assays developed to detect HPV viral
proteins encoded by early genes (e.g., E6 and E7) and late genes
(e.g., L1). In addition, the developed antibody, antigen, or
immunocomplex assays for E6, E7, L1, protein or their antibodies
thereof in one format, for example, a microplate format, can be
adapted into a one-step immunochromatographic assay for the direct
measurement of E6, E7, L1 proteins or antibodies induced by HPV
infection.
[0063] The one-step immunochromatographic assay is a simple, fast,
and easy to operate assay, which can be conveniently developed for
point-of-care use. In general, there is simply mixing of a sample
to be tested with a detection antibody as developed herein, the
mixture can be applied onto or is already fixed on a surface (e.g.,
a membrane or a glass) for a pre-determined reaction time (e.g., in
minutes, etc.) at optimized incubation temperature, such as at room
temperature. The reaction can be optimized to be short for
convenience depending on the quality of the detection antibody used
and the assay reaction conditions. Thus, a rapid immunological test
with short waiting time period can be performed and the assay
results is generally designed to be visually scored without the
need of any detection instruments.
[0064] As another example, protein chip immunological assays for
detecting HPV proteins, such as E6, E7, L1, etc., or antibodies
thereof can be performed for rapid detection of HPV infection. In
addition, protein chip immunological assays can be designed to be
multiplexed for detecting different protein or antibodies targets
as well as in high throughput. The protein chip immunological
assays as provided herein can be used for diagnosing HPV infection
and for rapid detection of certain cervical cancer biomarkers. In
general, a surface of a chip is initially covalently bound to
antibodies, proteins, or antigens, which have an affinity to bind
the target protein of interest in a sample, by using standard
surface chemistries.
[0065] For example, purified recombinant E6, E7, and L1 proteins
are shown herein to be able to attach to surfaces of a chip and
selectively detect E6, E7, and L1 antisera in solution. As such,
protein chip immunological assays are developed to provide a rapid
readout of the presence of antibodies induced by HPV infection in a
sample. Similarly, protein chip immunological assays are developed
to provide a rapid readout of the presence of viral proteins in a
sample due to HPV infection by attaching antisera or antibodies
against HPV viral proteins encoded by early genes and/or late
genes, e.g., L1, E6 and E7, etc.
[0066] As another example, to diagnose HPV infection using protein
chip immunological assays, a capture agent, can be attached
individually to various positions on the surface of one or more
protein chips. Alternatively, the capture agent can be attached to
different positions and thus in multiplexed format to detect
different HPV infection related proteins simultaneously in one
sample. The protein chip array of proteins or antibody from all HPV
types, strains, or variants can be generated and used to screen
phenotypes of HPV infection. Thus, the use of protein chip assays
can be a very powerful screening tool to enable the design of one
protein chip or test/assay suitable for executing many or all
related immunological assays for screening or diagnosing HPV
infection. The capture agent in a protein chip assay includes, but
is not limited to, recombinant HPV viral proteins encoded by HPV
early genes and late genes, recombinant E6, E7, and L1 proteins,
antisera or antibodies against HPV viral proteins, encoded by early
genes and/or late genes, e.g., L1, E6 and E7, etc. The assay
conditions for the one or more protein chips are
optimized/standardized and tested on clinical samples. The results
from the ELISA immunological assays are also checked and correlated
with the results of the protein chip immunological assays. The
protein chip immunological assays may give better sensitivity over
microplate immunological assays because of the use of laser as
light source and better instrument designed for better detection
limit. The higher assay sensitivity and better detection instrument
enable the detection of detect extremely low amounts of antigens or
antibody induce or developed in the body of those human subjects
who are in the early stage of HPV infection or disease development
to provide better prevention and disease management.
[0067] Positive results from the immunological assays confirm that
the clinical sample may contain HPV associated proteins or antigens
to indicate current HPV infection present in the clinical sample.
It is also likely to detect past HPV infection still present in the
clinical sample by detecting immune response of past or current HPV
infection and/or the presence of antibodies induced recently or in
the past due to HPV infection, etc. Further, the one or more
immunological assays provided herein are suitable for general HPV
infection as well as infection by high risk HPV types by using
recombinant proteins derived from the genes of the HPV high risk
types.
[0068] By obtaining the results of the immunological assays
performed to detect antibodies or viral proteins derived from HPV
early genes and late genes, concurring positive results further
confirm HPV infection by conveniently obtaining one sample (more
than one sample can also be used). It was found that concurring
positive results from the one or more immunological assays
performed herein correlate very well to the clinical status of the
human subject where the clinical sample is obtained from. For
example, concurring positive results are obtained and found in a
large collection of clinical samples, to correlate well to the
histological stages of cervical intraepithelial neoplasia (CIN),
the stages or degrees/grades of the biopsy-confirmed squamous
intraepithelial lesions (SIL), the stages of progression toward
invasive cancer, carcinoma, and/or adenocarcinoma, the presence of
cytological Atypical Glands of Undetermined Significance (AGUS),
the presence of Atypical Squamous Cells of Undetermined
Significance (ASCUS), etc.
[0069] Non-concurring positive results from the two assays for the
proteins derived from HPV early genes and late genes may indicate
general HPV infection, such as infection of different HPV types as
well as cross reactivity with either one of the proteins in the two
assays directed to early or late viral proteins. For example, it is
found that there are positive results from cross reactivity to
viral proteins derived from HPV early genes but negative results
and no cross reactivity to viral proteins derived from HPV late
genes, and vice versa.
[0070] At step 150, optionally, additional nucleic acid assays,
cytological test and/or histological tests are conducted on the
clinical sample. The nucleic acid hybridization assay conducted on
the clinical sample to detect the presence of a papillomavirus
genome in the clinical sample from the human subject may include
polymerase chain reactions, nucleic acid hybridization assays, DNA
chip assays, radioactive nucleic acid hybridization and detection
assays, and non-radioactive nucleic acid hybridization and
detection assays.
[0071] The method as described herein may also include performing a
cytological papanicolaou smear assay on the clinical sample and
comparing the results of the cytological papanicolaou smear assay
with the results of the one or more immunological assays. Since HPV
can't be cultured efficiently, and the clinical performance of
serological assays is poor, diagnosis of HPV infection is almost
entirely based on molecular tools. Nucleic acid amplification
techniques such as PCR, nucleic acid-sequence based amplification,
and advances in nucleic acid-based techniques, including hybrid
capture technology (one example is a commercially available Digene
hybrid capture II test from Digene Corporation, Gaithersburg, Md.),
can be used in addition to the one or more immunological assays as
described herein as a molecular screening tool for HPV
infection.
[0072] In general, the hybrid capture II HPV DNA test is an in
vitro nucleic acid hybridization assay used for detecting high-risk
HPV types by employing RNA probes specific for thirteen high-risk
types of HPV. The hybrid capture II HPV DNA test amplified the
presence of RNA:DNA hybridized complex by coating the HPV specific
DNA probes to a microtiter plate and a detection antibody (a
monoclonal anti-DNA/RNA hybrid antibody) is used for detecting the
amplified RNA:DNA complexes, followed by the addition of
chemilumescent substrates to qualitatively detect the presence of
the DNA of the thirteen high-risk HPV types in the sample.
[0073] Thus, it requires sophisticated equipments and trained
personnel to perform the test and analyze the data using specific
microplate reader and specific software developed for the reader.
The applicability of the hybrid capture technology (Digene tests)
was limited because complex execution of techniques requires
sophisticated instrumentation and training and false positive and
false negative on general population and early HPV infected
individuals are very high, probably due to the requirements of the
presence of DNA in the test sample which can be easily degraded or
lost during sampling or sample-handling. However, the hybrid
capture tests can be used to confirm the results of the one or more
immunological assays as provided herein. Additional nucleic acid
assays, cytological test and/or histological tests are known in the
art can be used on the same clinical sample of the invention to
further concur the results of the one or more immunological
assays
[0074] The one or more immunological assays as provided herein aims
to employ user friendly procedures with simple instrument or no
additional instrument to perform in a short period of time.
Comparison of the results of the various immunological assays,
nucleic acid hybridization assays with cytological and histological
data for the human subjects as well as demographic information
serve to validate the correlation and accuracy in diagnosing HPV
infection and/or cervical cancer.
[0075] At present, there are no commercially available
immunological assays to clinically measure the presence of
HPV-associated proteins or antibodies. Embodiments of the invention
thus provide a diagnostic tool useful for diagnosis of HPV
infection and HPV related cervical cancer. In addition, the results
from the immunological assays as described herein can be used to
compare with other commercially available immunological assays
specific designed for p53 and RB. It is known that infection high
risk type HPVs, such as HPV-16 and HPV-18 may cause cervical cancer
due to the expression of E6 and E7, the viral oncoproteins that
induce cervical cell malignancy and alter/reduce the expression of
p53 and RB endogenous proteins of the host cells, leading to
cellular dysfunction and ultimately carcinoma. Thus, it is
contemplated to compare the assays results on the levels of all of
these proteins altered by HPV infection perform on clinical
samples, e.g., cervical tissues, body fluids, serum, etc., from the
same human subjects.
[0076] Changes in the expression levels of among these proteins
affected by HPV infection (e.g., E6, E7, p53, Rb, among others)
serve as a signature for high risk of contracting cervical cancer.
Elevated levels of HPV-associated viral proteins or antigens and
reduced levels of p53 and RB confirm the human subjects of not just
HPV infection but also at high risk of contracting cervical cancer.
On the contrary, unchanged levels of p53 and RB in the human
subjects but elevated levels of HPV-associated viral proteins or
antigens may indicate a general HPV infection and cervical cancer
is not yet progressed.
[0077] Accordingly, one example of a method of screening a human
subject of papillomavirus infection may include obtaining a
clinical sample from the human subject,
[0078] obtaining a first recombinant protein encoded by an early
gene of a papillomavirus, obtaining a second recombinant protein
encoded by an late gene of the papillomavirus; conducting one or
more immunological assays on the clinical sample from the human
subject, detecting the presence of an antibody to the first
recombinant protein in the human subject using the first
recombinant protein, and detecting the presence of an antibody to
the second recombinant protein in the human subject using the
second recombinant protein. The first recombinant protein may be,
for example, recombinant HPV-16 E6 proteins, recombinant HPV-16 E7
proteins, recombinant HPV-18 E6 proteins, recombinant HPV-18 E7
proteins, etc. The second recombinant protein may be, for example,
recombinant HPV-16 L1 proteins, recombinant HPV-18 L1 proteins,
among others.
[0079] Another example of a method of screening a human subject
infected with a human papillomavirus may include obtaining a
clinical sample from the human subject, conducting a nucleic acid
hybridization assay on the clinical sample, detecting the presence
of a papillomavirus genome in the clinical sample from the human
subject, conducting one or more immunological assays on the
clinical sample, detecting the presence of an antibody to an early
papillomavirus viral protein or the presence of the early
papillomavirus viral protein in the clinical sample using a first
recombinant protein of the early papillomavirus viral protein, and
detecting the presence of an antibody to a late papillomavirus
viral protein or the presence of the papillomavirus late viral
protein in the clinical sample using a second recombinant protein
of the late papillomavirus viral protein.
[0080] The early papillomavirus viral protein may include, but are
not limited to, HPV-16 E6 proteins, HPV-16 E7 proteins, HPV-18 E6
proteins, HPV-18 E7 proteins, and others. The late papillomavirus
viral protein may include, but are not limited to, HPV-16 L1
proteins, HPV-18 L1 proteins, and others. The presence of the
papillomavirus genome can be detected, for example, using a nucleic
acid probe with sequence homology to conservative DNA sequences
from a papillomavirus gene, including papillomavirus late genes, L1
genes, L2 genes, papillomavirus early genes, E2 genes, E6 genes,
and E7 genes, among others.
[0081] The one or more diagnostic immunological assays as described
therein may include taking a sample of body fluid or tissue likely
to contain antibodies against HPV associated proteins and/or HPV
antigens, reacting it with one or more recombinant proteins as
obtained and described herein, and assaying for the presence of any
antibody-antigen complexes by suitable detection systems. Positive
results confirm that the clinical sample may contain antibodies to
indicate past HPV infection and concentrated levels of the
antibodies in the present in the clinical sample. It is also likely
to detect current HPV infection, indicating strong immune response
of the human subject.
[0082] The one or more diagnostic immunological assays as described
therein may also include obtaining polyclonal antibodies,
monoclonal antibodies, and/or antiserum specific against the one or
more recombinant proteins as obtained and described herein, taking
a clinical sample likely to contain HPV associated proteins and/or
antigens, reacting it with the obtained polyclonal antibodies,
monoclonal antibodies, and/or antiserum specific for the one or
more recombinant proteins, and assaying for the presence of any
antibody-antigen complexes by suitable detection systems. Suitable
detection system may employ various colormetric, chemilumenescent,
flourescent substrates, etc., specific for a secondary antibody
used in each immunological assay.
[0083] Still, another example of a method of screening a human
subject of high risk human papillomavirus infection includes
obtaining a clinical sample from the human subject, obtaining a
first recombinant protein purified from a first protein expression
system with a first expression vector having a portion of nucleic
acid sequence corresponding to the full length nucleic acid
sequence of an early papillomavirus gene and obtaining a second
recombinant protein purified from a second protein expression
system with a second expression vector having a portion of nucleic
acid sequence corresponding to the full length nucleic acid
sequence of a late papillomavirus gene. Then, one or more
immunological assays can be conducted on the clinical sample to
detect the presence of an antibody to a viral oncoprotein or the
presence of the viral oncoprotein in the clinical sample using the
first recombinant protein and the second recombinant protein. The
first recombinant protein may be, for example, recombinant HPV-16
E6 proteins, recombinant HPV-16 E7 proteins, recombinant HPV-18 E6
proteins, and recombinant HPV-18 E7 proteins, etc. The second
recombinant protein may be, for example, recombinant HPV-16 L1
proteins, and recombinant HPV-18 L1 proteins, etc. The early
papillomavirus gene may be, for example, papillomavirus E6 genes
and papillomavirus E7 genes, etc. The late papillomavirus gene may
be, for example, papillomavirus L1 genes and papillomavirus L2
genes, etc. The early and late genes may be derived from high risk
human papillomavirus, such as HPV-16 and HPV-18, etc.
[0084] Clinically applicable vaccination programs for cervical
cancer may be available, as such, early detection to screen HPV
positive and negative infected individuals is more than ever an
urgent need to search for candidate subjects suitable of being
vaccinated. Strategies to prevent cervical cancer may thus requires
improved HPV testing/screening to cover a broad range of the
worldwide population in addition to closely follow-up those
subjects with past or present HPV infection and/or pre-cancerous
lesions.
[0085] Screening/testing for past or present HPV infection along
with a Pap smear can become the standard of care and the need is
acknowledged in clinical guidelines developed by major medical
groups including the American College of Obstetricians and
Gynecologists (ACOG), the American Cancer Society (ACS), the
Association of Reproductive Health Professionals (ARHP) and the
American Society for Colposcopy and Cervical Pathology (ASCCP).
Thus, the invention as described herein can be commercialized as a
HPV general infection assay and/or a HPV high risk type infection
assay and may play an important role as screening tests for
cervical cancer. It is proposed that cervical cancer screening
might become more efficient when it is based on combined cytology
(results of par smear test) and high risk HPV infection screening.
HPV infection screening tests may become necessary in addition to
cervical cancer screening to serve as a early quick and easy
screening, a quality control for false-negative smears, in triage
of women with equivocal smears, in follow-up of women treated for
CIN3 or cervical cancer and for the detection of cervical
adenocarcinoma.
[0086] Early diagnosis of infection with high risk HPV types is
important for successful prevention and treatment of cervical
cancer, which is one of the more deadly forms of cancer.
Importantly, it is known that infection in women for 12-15 years
with HPV is required before invasive cancer to develop. It is thus
important to be able to assay biomarkers of high risk HPV infection
as described herein to pre-screen women early, such that it will be
possible to treat HPV infection early and prevent cervical cancer
development, rather than having to rely on chemotherapy or
radiation to treat cancer malignancy. Developing the immunological
assays as described herein to detect a series of biomarkers for
general HPV infection in population as well as infection with high
risk HPVs can be used for early diagnosis and therefore prevention
of cervical cancer.
EXAMPLES
[0087] An object of the invention is to develop immune-responsive
or antibody-reactive recombinant proteins derived from early genes
and/or late genes of various HPV types and strains. It is a further
object to provide these recombinant proteins in a chemically pure
form. It is a still further object to provide simple, rapid, less
expensive and more sensitive assays/tests for diagnosing not only
HPV infection, but also most, if not all, HPV-associated
neoplasm.
I. Cloning and Production of Recombinant Proteins Encoded by HPV
Genes.
[0088] Recombinant proteins encoded by early HPV genes and late HPV
genes are obtained. Recombinant proteins can be obtained by itself
or as hybrid proteins fused transcriptionally or translational to a
portion of a full length DNA fragment for a HPV gene of interest.
The DNA sequence of the HPV gene of interest may be derived from
high risk HPV types, low risk HPV types, oncogenic HPV strains
within a HPV type, etc. An oncogenic HPV strain is an HPV strain
that is known to cause cervical cancer as determined by the
National Cancer Institute (NCI, 2001). Oncogenic HPV proteins are
early viral proteins encoded by an oncogenic HPV type or strain.
The sequences of various HPV viral genes and proteins are also
found as database entries at NCBI's Gene Bank database, as follows:
HPV16-E6: GI:9627100; HPV18-E6: GI:9626069; HPV31-E6: GI:9627109;
HPV35-E6: GI:9627127; HPV30-E6: GI:9627320; HPV39-E6: GI:9627165;
HPV45-E6: GI:9627356; HPV51-E6: GI:9627155; HPV52-E6: GI:9627370;
HPV56-E6: GI:9627383; HPV59-E6: GI:9627962; HPV58-E6: GI:9626489;
HPV33-E6: GI:9627118; HPV66-E6: GI:9628582; HPV68b-E6: GI:184383;
HPV69-E6: GI:9634605; HPV26-E6: GI:396956; HPV53-E6: GI:9627377;
HPV73: GI:1491692; HPV82: GI:9634614, HPV34 GI:396989; HPV67
GI:3228267; and HPV70 GI:1173493.
Example 1
Cloning and Production of Various Recombinant Proteins Encoded by
HPV-16, Early E6 Gene
[0089] Cloning of an exemplary oncogenic E6 early gene from an
exemplary HPV type, HPV-16, is described herein. A 474 base pair
(b.p.) DNA fragment (SEQ ID NO. 1) containing the 157 amino acid
coding region (SEQ ID NO. 2) of the HPV-16 E6 gene was obtained by
polymerase chain reaction (PCR) amplification. Primers were used
for cloning, for example, a pair of forward and reverse primers, 5'
cgcGGATCCcaccaaaagagaactgcaatgtttc 3' (SEQ ID NO. 3) and 5'
cccAAGCTTttacagctgggtttctctacgtg 3' (SEQ ID NO. 4), respectively.
The DNA sequence of the isolated DNA fragment was confirmed by
comparing with the sequence from Gene Bank database. All cloning
procedures are carried out according to the protocols described in
"Molecular Cloning", A Laboratory Manual, eds. Sambrook, Fritsch
and Maniatis, Cold Spring Harbor Laboratory Press, 1989. In
addition, E6 DNA fragments from different strains of HPV-16 can
also be cloned from different clinical samples or sources.
[0090] The obtained 474 base pair (b.p.) DNA fragment was
sub-cloned into a histidine tag expression vector, pQE30, in order
to express a his-tagged recombinant HPV-16 E6 protein. The
resulting plasmid DNA is designated, pQE30/HPV16-E6 for the
expression of His-tagged-HPV16-E6 recombinant protein. The DNA
sequence and the amino acid sequences of the resulting his-tagged
recombinant HPV-16 E6 protein are shown as SEQ ID NO. 5 (a 510 base
pair (b.p.) DNA fragment) and SEQ ID NO. 6 (a 169 amino acid fusion
protein), respectively.
[0091] Other expression vectors which are used as recombinant
protein overexpression systems with histidine tag (e.g., His.sub.6,
His.sub.8, etc.), glutathione-S-transferas (GST) fusion,
maltose-binding-protein (MBP), among others, can also be used. In
addition, the obtained HPV-16 E6 DNA fragment can be sub-cloned
into other expression systems, including maltose-binding-protein
and glutathione-S-transferase-E6 fusion protein expression systems.
Various expression systems can also be used to express E6
recombinant proteins from various HPV types and strains. For
example, E6 recombinant protein from HPV-58 was obtained and
designated as HPV-16-MBP-E6.
[0092] His tagged-HPV16-E6 and MBP-HPV-E6 recombinant proteins were
expressed in E. coli BL21(DE3) using IPTG driven induction. After
two hour induction of protein expression at 37.degree. C., GST-E6
or MBP-E6 recombinant proteins using standard protocols recommended
by the suppliers (Amersham and New England Biolabs, respectively)
were obtained and purified to a final concentration of about 1
mg/L. Longer induction time and re-flow though on protein
purification column were found to generate higher protein yield,
resulting in highly concentrated purified recombinant proteins at a
yield of about 2-10 mg/L). The purity of the recombinant GST-E6
proteins was estimated to be >90% based on PAGE analysis.
Recombinant E6 fusion proteins was used to detect the presence of
E6 antibody on clinical samples and was also be used as immunogens
for production of polyclonal antiserum and monoclonal
antibodies.
[0093] FIGS. 2A and 2B demonstrate the expression of full-length
HPV-16 E6 recombinant protein induced by IPTG analyzed by SDS-PAGE
and western blot, respectively, using anti-E6 monoclonal antibody
(MAb1-1). The molecular weight of the resulting His-tagged-HPV16-E6
recombinant protein is about 20.5 KD. The western blot was
performed on a PVDF membrane using an anti-E6 monoclonal antibody,
which is a mouse antibody, followed by a secondary antibody, an
alkaline peroxidase (AP)-goat-anti-mouse IgG1, and visualized by
the reaction of NBT and BCIP substrate mixture. The results showed
that a single major protein band and thus pure recombinant E6
protein was purified. The purity of the recombinant E6 proteins was
estimated to be about 90% or more based on PAGE analysis.
[0094] The purified recombinant E6 proteins as shown in FIG. 3A
were used in one or more immunological assays, for example, to be
used as a detecting antibody in antibody assays, etc. The purified
recombinant E6 proteins were also used to as immunogens for
generating antiserum, polyclonal antibody, and monoclonal
antibodies specific against HPV-16 E6 protein.
[0095] FIG. 2C demonstrates the result of gel filtration column
chromatography of the purified recombinant E6 protein,
demonstrating that the purified recombinant protein HPV-16-E6 is a
monomeric soluble protein with molecular size about 20.5 kDa. The
purified recombinant E6 protein is eluted later than BSA.
Example 2
Cloning and Production of Recombinant Proteins Encoded by HPV-16
Early E7 Gene
[0096] Cloning of an exemplary oncogenic E7 early gene from an
exemplary HPV type, HPV-16, is described herein. A 294 base pair
(b.p.) DNA fragment (SEQ ID NO. 7) containing the 99 amino acid
coding region (SEQ ID NO. 8) of the HPV-16 E7 gene was obtained by
polymerase chain reaction (PCR) amplification. Primers were used
for cloning, for example, a pair of forward and reverse primers, 5'
cgcGGATCCcatggagatacacctacattgc 3' (SEQ ID NO. 9) and 5'
ccgGAATTCttatggtttctgagaacagatgg 3' (SEQ ID NO. 10), respectively.
The DNA sequence of the isolated DNA fragment was confirmed by
comparing with the sequence from Gene Bank database. In addition,
E7 DNA fragments from different strains of HPV-16 can also be
cloned from different clinical samples or sources.
[0097] The obtained 294 base pair (b.p.) DNA fragment was
sub-cloned into a GST expression vector in order to express a
recombinant HPV-16 E7 GST fusion protein. The DNA sequence and the
amino acid sequences of the resulting recombinant HPV-16 E7 GST
protein are shown as SEQ ID NO. 11 (a 972 base pair (b.p.) DNA
fragment) and SEQ ID NO. 6 (a 323 amino acid fusion protein),
respectively. The molecular weight of the resulting recombinant
HPV-16 E7 GST protein is about 37.2 KD. The recombinant HPV-16 E7
GST proteins were obtained and purified to a final concentration of
about 1 mg/L. Other expression systems can also be used to express
E7 recombinant proteins from various HPV types and strains.
Recombinant E7 fusion proteins or recombinant E7 baculovirus
proteins were used to detect the presence of E7 antibody on
clinical samples and were also be used as immunogens for production
of polyclonal antiserum and monoclonal antibodies.
[0098] FIG. 3 is a SDS-PAGE gel, showing one exemplary purified
recombinant HPV-16-E7 proteins. As shown in FIG. 3, the HPV-16-E7
recombinant proteins is purified to homogeneity as a major single
band with a molecular weight of 37.2 KDa as indicated by an
arrow.
Example 3
Cloning and Production of Recombinant Proteins Encoded by HPV-16
Late L1 Gene
[0099] Cloning of an exemplary late gene from an exemplary HPV
type, HPV-16, is described herein. A 1596 base pair (b.p.) DNA
fragment (SEQ ID NO. 13) containing the 531 amino acid coding
region (SEQ ID NO. 14) of the HPV-16 L1 gene was obtained by
polymerase chain reaction (PCR) amplification. Primers were used
for PCR cloning, for example, a pair of forward and reverse
primers, 5' ccgCTCGAGatgcaggtgacttttatttacatcc 3' (SEQ ID NO. 15)
and 5' cccAAGCTTttacagcttacgttttttgcgttta 3' (SEQ ID NO. 16),
respectively. The DNA sequence of the isolated DNA fragment was
confirmed by comparing with the sequence from Gene Bank database.
In addition, L1 DNA fragments from different strains of HPV-16 can
also be cloned from different clinical samples or sources.
[0100] The obtained 1596 base pair (b.p.) DNA fragment was
sub-cloned into a baculovirus expression system in order to express
a recombinant HPV-16 L1 protein. The DNA sequence and the amino
acid sequences of the resulting recombinant HPV-16 L1 protein are
shown as SEQ ID NO. 17 (a 1716 base pair (b.p.) DNA fragment) and
SEQ ID NO. 18 (a 571 amino acid protein), respectively. The
molecular weight of the resulting recombinant HPV-16 L1 protein is
about 64.2 KD. The recombinant HPV-16 L1 proteins were obtained and
purified to a final concentration of about 1 mg/L. Other expression
systems can also be used to express L1 recombinant proteins from
various HPV types and strains.
[0101] In general, recombinant proteins from various high risk HPV
types and low risk HPV types or strains can be obtained by cloning
of early and late genes by polymerase chain reaction (PCR)
amplification using a pair of forward and reverse primers using
procedures as described herein and in various recombinant protein
expression systems. For example, a recombinant N-terminal fragment
of HPV-16 L1 protein was also obtained by expression in His-tagged
expression system. The amino acid sequence of the resulting
recombinant HPV-16 L1 N-His protein is shown as SEQ ID NO. 19 and
the molecular weight of the HPV-16 L1 N-terminal recombinant
protein is about 34 KD. C-terminal fragments can also be obtained,
Recombinant L1 proteins and/or recombinant L1 partial proteins were
used to detect the presence of L1 antibody on clinical samples and
were also be used as immunogens for production of polyclonal
antiserum and monoclonal antibodies.
[0102] FIG. 4 demonstrates SDS-PAGE of three exemplary purified HPV
recombinant proteins by commassie blue staining according to one or
more embodiments of the invention. Recombinant fusion proteins were
obtained for different HPV types, such as different high risk HPV
types, e.g., HPV-16, HPV-18, HPV-58, etc. P1 indicates a purified
recombinant HPV-58-E6-MBP fusion protein as compared to P3 for a
MBP protein alone. P2 indicates a purified recombinant
HPV-16-E7-His fusion protein and CP indicates a purified
recombinant HPV-16-E6-His fusion protein.
II. Sample Collection
[0103] Biological samples to be analyzed using the methods of the
invention may be obtained from any mammal, e.g., a human or a
non-human animal model of HPV. In many embodiments, the biological
sample is a clinical sample obtained from a living subject. In some
embodiments, the subject from whom the sample is obtained is
apparently healthy, where the analyses and/or assays are performed
as a part of routine screening. In other embodiments, the subject
is one who is susceptible to HPV, (e.g., as determined by family
history; exposure to certain environmental factors; etc.). In other
embodiments, the subject has symptoms of HPV (e.g., cervical warts,
or the like). In other embodiments, the subject has been
provisionally diagnosed as having HPV (e.g. as determined by other
tests based on, e.g., pap smears, hybrids capture, PCR tests,
etc.).
[0104] The biological sample may be derived from any cells,
tissues, organs or group of cells of the subject. In some
embodiments a cervical scrape, biopsy, or lavage is obtained from a
subject. In other embodiments, the sample is a blood or urine
sample. In some embodiments, the biological sample is processed,
e.g., to remove certain components that may interfere with an assay
or method of the invention, using methods that are standard in the
art. In some embodiments, the biological sample is processed to
enrich for proteins, e.g., by salt precipitation, and the like. In
certain embodiments, the sample is processed in the presence
proteasome inhibitor to inhibit degradation of antibodies,
proteins, or antigens and the like.
[0105] Samples as used herein include to a material or mixture of
materials, typically, although not necessarily, in fluid form,
i.e., aqueous, containing one or more components of interest and
may include any of the biological samples, clinical samples, etc.
Samples may be derived from a variety of biological sample, liquid,
or solid, such as tissue or fluid isolated from an individual,
including but not limited to, for example, plasma, serum, spinal
fluid, semen, lymph fluid, the external sections of the skin,
respiratory, intestinal, and genitourinary tracts, tears, saliva,
milk, blood cells, tumors, organs, and also samples of in vitro
cell culture constituents (including but not limited to conditioned
medium resulting from the growth of cells in cell culture medium,
putatively virally infected cells, recombinant cells, and cell
components).
Example 4
Clinical Sample Collection Procedure
[0106] All samples were taken from female patients during their
scheduled visits for gynecological examinations. After inserting a
speculum to a human subject, a brush or a cotton swab was inserted
in the endocervix and rotated to obtain endocervical cells. The
brush or swab was then removed out to smear on a slide (pap smear).
The brush or swab was then placed into about 1 ml of specimen
dilution buffer (PBS+1% BSA) and vigorously shaken to remove bound
material (mucus and cells). The diluted specimens are stored at a
-20.degree. or -80.degree. C. freezer.
[0107] Venous blood was obtained by usual phlebotomy methods, with
a 21- or 22-gauge double-pointed needle into a agar barrier tube
for a total of 7-9 ml from each subject. The blood was allowed
about 15 minutes at room temperature for clot formation and was
centrifuged for 15 minutes. Serum was aspirated away from blood
cells, using a disposable pipette, dispensed into Eppendorf tubes
as aliquots, and stored at a -20.degree. or -80.degree. C. freezer.
As a negative control with no HPV infection, serum can be obtained
from virgin females.
III. Nucleic Acid hybridization Assays for Screening of HPV
Infection
[0108] Nucleic acid hybridization assays can be used in addition to
the immunological assays provided herein.
Example 5
Isolation of DNA from samples
[0109] To prepare DNA for nucleic acid hybridization assays, the
sample of a cell suspension was vigorously shaken, and 120 .mu.l
was treated by adding 40 .mu.l of proteinase K (200 .mu.g/ml) in 3%
Triton X-100 for 1 hour at 37.degree. C. The proteinase was
inactivated by incubation at 95.degree. C. for 10 minutes.
Subsequently, 10 .mu.l of the solution was used in a reaction.
Example 6
Detecting the Presence of HPV Genome Using PCR Analyses
[0110] Primers are designed to detect the presences of HPV early
genes and late genes for various HPV types. For example, DNA
forward and reverse primers (SEQ ID NO. 20 and SEQ ID NO. 21;
5'-GCNCARGGHCAYAAYAATGG-3' and 5'-GTDGTATCHACMHCAGTAACAAA-3',
respectively. N: A+T+C+G; R: A+G; H: A+C+T; Y: T+C; D: T+A+G; M:
A+C) can be used to detect various L1 genes from different HPV
types due to the conserved sequence homology of these genes. The
primers used herein are located in the L1 open reading frame. The
presence of L1 gene can be used to detect various HPV-16 types due
to sequence homology of various HPV L1 proteins.
[0111] About 20 pmols of forward and reverse primers and about 1
.mu.l of isolated DNA, 10 mmol/L, from a test sample were mixed to
a final reaction volume of about 10 .mu.l, containing Tris-HCl, pH
9.0, 50 mmol/L KCl, 2.5 mmol/L MgCl.sub.2, 0.1% Triton X-100, 0.01%
gelatin, 200 mmol/L of each deoxynucleoside triphosphate, and 0.25
U of SuperTaq (Sphaero Q, Cambridge, UK). PCR reaction conditions
were as follows: preheating for about 1 minute at about 94.degree.
C., followed by 40 cycles of about 1 minute at about 94.degree. C.,
about 1 minute at about 45.degree. C., and about 1 minute at about
72.degree. C., and a final extension of about 5 minutes at about
72.degree. C. The PCR products were analyzed by 3% agarose gel
electrophoresis. A synthetic positive control (65 mer, SEQ ID NO.
22) for the presence of L1 gene was also synthesized and used in
PCR nucleic acid assays.
[0112] As another example, designed DNA forward primers (SEQ ID NO.
23 and SEQ ID NO. 24) and reverse primers (SEQ ID NO. 25) can be
used to detect the presence of HPV-16 by analyzing the presence of
E6-containing DNA fragments, including a 181 b.p. E6 DNA fragment
and a 286 b.p. E6 DNA fragment (SEQ ID NO. 26 and SEQ ID NO. 27,
respectively). Additional DNA forward and reverse primers were also
designed to detect E7 genes.
Example 7
Comparison with HPV DNA Hybrid Capture Assays
[0113] Hybrid Capture II HPV DNA Test from Digene Corporation were
also performed on obtained clinical samples as a comparison. The
hybrid capture II DNA test is approved by the U.S. Food and Drug
Administration to test for oncogenic HPV DNA, as reflexive
follow-up of an ASCUS (Atypical Squamous Cells of Undetermined
Significance) or other abnormal Pap results. The test was run
according to the manufacturer's protocol using the microtiter plate
based format and probes for "high carcinogenic risk" HPV types at
certified clinical laboratories. Samples readings of 1 fold or more
than the positive control (1 pg/mL HPV DNA or 5000 HPV genome
copies per test) were considered to contain DNA from a number of
high risk HPV types.
[0114] The hybrid capture test involves a molecular hybridization
that uses nonradioactive probes with amplification of the detection
of the hybrid ones for chemoluminescence. The material for analysis
is denatured and reacts with specific probes forming hybrid RNA/DNA
that are captured by antibodies that cover the walls of the tube.
Specific antibodies against RNA/DNA conjugated with alkaline
phosphatase are reacted with the immobilized RNA/DNA hybrids. By
forming a stable substrate complex for alkaline phosphatase, the
RNA/DNA hybrids are capture by antibody and detected by
chemoluminescence via spectrometry.
IV. Immunological Assays for Screening of HPV Infection
[0115] One of the initial reactions of a human subject to HPV
infection is thought to be the generation of antibodies against the
E6 and E7 oncoproteins. Presently, no immunological diagnostic
assay is commercially available to detect this immune response.
Because the amino acid sequences of various E6 and E7 proteins
(see, Table 1) are different with various degree of amino acid
sequence homology among different HPV types, host antibodies
produced in response to the same early oncoprotein from one HPV
type will be very different from another HPV type. For example,
antibodies induced in a host against oncoproteins (e.g., E6, E7,
etc.) from an oncogenic HPV type or strain (e.g., HPV-16, HPV18,
etc.) can be significant different from antibodies induced by other
proteins associated with other HPV types.
TABLE-US-00001 TABLE 1 Amino acid sequence homology of L1, E6 and
E7 for different HPV Types L1 E6 E7 HPV 16 v. HPV 18 63% 53% 42%
HPV 16 v. HPV 31 81% 65% 73% HPV 16 v. HPV 33 79% 62% 60% HPV 18 v.
HPV 31 64% 51% 38% HPV 18 v. HPV 33 65% 46% 44% HPV 31 v. HPV 33
78% 57% 59% HPV 16 v. HPV 6A 68% 35% 56% HPV 16 v. HPV 11 68% 34%
55% HPV 6A v. HPV 11 92% 81% 83%
[0116] It is proposed and tested herein that, detection of
antibodies or antigens to oncoproteins encoded by early genes, such
as E6 or E7 protein from HPV high risk types in serum, body fluid,
or cervical tissues could be an indication whether the human
subject is at high risk for cervical cancer development. In
addition, detection of antibodies or antigens to viral proteins
encoded by late genes, such as capsid proteins L1, L2 from HPV high
risk types can be used together or independently in the same or
different immunological diagnostic assays to further confirm the
risk of the subject to develop cervical cancer.
[0117] Accordingly, embodiments of the invention also provide
immunological assay systems to detect the presence of any
HPV-associated proteins, oncoproteins, and/or capsid proteins
directly in one or more immunological assays that can be performed
concurrently or separately on an obtained clinical sample.
Example 8
Western Blot Analyses for Detection of Purified Recombinant HPV
Proteins Encoded by Early Genes
[0118] FIG. 5 demonstrates the detection of three exemplary
purified recombinant proteins encoded by HPV early genes using
Western blot analysis. Lane 1 indicates the detection of
recombinant proteins, HPV-16-E7-His, and thus binding of the
recombinant proteins to a monoclonal antibody, anti-HPV-16-E7
antibody (MAb2-1). Lane 2 indicates the detection of recombinant
proteins, HPV-16-E7-His, and thus binding of the recombinant
proteins to a monoclonal antibody, anti-HPV16E7 antibody (MAb2-2).
Lane 3 indicates the detection of recombinant proteins,
HPV-16-E7-His, and thus binding of the recombinant proteins to a
monoclonal antibody, anti-HPV-16-E7 antibody (MAb2-3).
Example 8
Western Blot Analyses for Detection of HPV Associated Antigens
and/or Proteins Encoded by Late Genes
[0119] FIG. 6 demonstrates the detection of an exemplary purified
recombinant protein encoded by HPV late genes using Western Blot
analysis. Lane 1 is loaded with cell lysate from SF9 cells infected
with HPV16 L1 recombinant baculovirus for 72 hrs. Lane 2 is loaded
with cell lysate from SF9 cells infected with baculovirus without
HPV16 L1 gene for 72 hrs. The single major band visualized by
Western blotting using anti-his tag antibody and after the reaction
of NBT and BCIP substrates indicates the full-length of recombinant
L1 protein at a molecular weight of 64.2 kDa.
Example 9
Non-Radioactive Immunological Assays
[0120] Cell suspensions from collected samples were centrifuged,
and the supernatant was used to run in the assays by diluting the
supernatant 1:10 in specimen dilution buffer. Assays from this
invention are non-invasive with little or without instruments. In
general, embodiments of the invention provides in vitro enzyme
immunological assays which are non-radioactive intended for direct
detection of HPV associated proteins and/or antibodies as an
indication of HPV infection. The assays as described herein are
suitable as adjunct tests in addition to clinical physical
examination, Pap smear tests, biopsies and other clinical
tests.
[0121] An immunological assay can, for example, be performed in a
variety of different ways. Detection of the antibodies that have
bound to specific antigens can, for example, be achieved with
various antibodies to antibodies (anti-antibodies) or other
compounds with affinity for antibodies, such as protein A or
protein G. These reagents can be labelled in many different ways,
for example radioactively (radioimmunoassay), with fluorescein
(fluoro-immunoassay), or enzymatically (enzyme-linked immunoassay,
ELISA or EIA). A special case of enzymatic immunoassay is when the
antigen-antibody complexes are detected on clinical samples or
tissue sections. Such a procedure is instead referred to as
immunostaining or immuno-histocytochemistry, although the
underlying principle is the similar as for ELISA. In addition, the
formats of the immunological assays can vary and may include a
format in microplate (various number of wells), simple rapid tests,
protein chips, and others.
[0122] In an alternate embodiment, the purified recombinant
proteins can be used in similar immunological assays to detect the
presence of antibodies raised against HPV immunotherapy in the
serum, or other bodily fluid or tissues, such as those human
subjects undergoing anti-HPV vaccine treatment. The detection of
the positive results from the samples of a subject undergoing
vaccine treatment using the assays as described herein and the
purified recombinant proteins is beneficial, for example, can be
used to titrate semi-quantitative the serum sample of the subject
treated with an anti-HPV vaccine and adjust the dosage of the
vaccines.
[0123] There are at least three types, but not limited to, of
immunological assays depending the target proteins to be detected,
including antigen tests, antibody tests, and antigen/antibody
immunocomplex tests. For example, a method is provided to detect
the presence of antibodies, immunoglobulins, etc., against HPV
proteins, such as E6, E7, and/or L1, etc., in a sample from a human
subject. The purified recombinant proteins as described herein can
be used to detect antibodies against HPV E6, E7, and/or L1
proteins. The method generally includes contacting the purified
recombinant proteins with the sample and detecting any binding of
the purified recombinant proteins to the sample, wherein binding of
the purified recombinant proteins to the sample indicates the
presence of HPV-induced or HPV-associated antibodies in the sample
and thus possible HPV infection for the subject in the past or
current.
[0124] The antibodies present in the sample may be antibodies
against E6, E7, L1, and other proteins from the same HPV types and
strains as the purified recombinant proteins. Alternatively, cross
reactivity of the binding of any antibodies in the sample to the
purified recombinant proteins may result in the detection of the
presence of antibodies from different HPV types or strains and thus
indicate HPV infection of different types or strains. However, such
cross reactivity need to be confirmed since sequence homology of
some of the HPV proteins from different HPV types are low as shown
in Table 1.
[0125] Similarly, method are provided to detect the presence of
antigens, HPV-associated proteins, HPV-induced proteins, such as
E6, E7, L1, L2, p53, and/or Rb, etc., in a sample from a human
subject. The purified recombinant proteins as described herein can
be used, for example, in a sandwiched assay to detect these target
proteins or antigens. Alternatively, monoclonal antibody,
polyclonal antibodies, and antiserum against the purified
recombinant proteins can also be obtained and purified to be used
in antigen tests, antibody tests, and antigen/antibody
immunocomplex tests to indicate possible HPV infection in the past
or current by the same or different types or strains for the
subject.
Example 10
Immunological ELISA Antibody Test for Detection of HPV Associated
Antibodies
[0126] In general, ELISA is performed according to standard
procedures. For example, coating of purified recombinant proteins
is performed in about 50 .mu.l volume in a 96 well format at
4.degree. C. overnight before blocking the bottoms of a microtiter
plate with about 200 .mu.l of a blocking buffer at room temperature
for 2 hours. A sample that contains antiserum, monoclonal
antibodies, polyclonal antibodies, and other targeted HPV-induced
antibodies can be added to react with the recombinant proteins in
about 50 .mu.l volume at room temperature before washing with a
wash buffer several times. A secondary antibody, anti-human or
anti-mouse, etc., that can generically bind to the targeted
antibody was used to react with antibodies or immunocomplex bound
to the bottom of the microtiter plates. Substrate development is
performed and read out in a microtiter plate reader to measure OD
absorbance at 450 nm wavelength, OD.sub.450. In a horseradish
peroxidase immunoassay detection system, about 50 .mu.l of 3, 3',
5, 5' tetramethylbenzidine, a substrate for horseradish peroxidase
can be added to each well of the microtiter plates and the reaction
mixture is incubated at room temperature for about 5-30 minutes or
until a visually obvious green-blue color developed before stopping
the reaction mixture by adding about 50 .mu.l of 1.5 M
H.sub.2SO.sub.4 into each well.
[0127] An ELISA assay (see, e.g., Harlow and Lane (Antibodies: A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)) generally includes preparing
proteins/antigens/antibodies, coating the well of a 96 well
multiwell plate (microtiter plate) with the
antigens/proteins/antibodies, adding proteins of interest or
antibodies of interest or conjugated to a detectable compound such
as an enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) to the wells and incubating for a period of time, and
detecting the presence of the antigens/proteins/antibodies. In some
cases, the antibody of interest does not have to be conjugated to a
detectable compound; instead, a second antibody (which recognizes
the antibody of interest) conjugated to a detectable compound may
be added to the well. Also, instead of coating the well with the
antigen, the antibody may be coated to the well. In this case, a
second antibody conjugated to a detectable compound may be added
following the addition of the antigen of interest to the coated
well. One of skills in the art would be knowledgeable as to the
parameters that can be modified to increase the signal detected as
well as other variations of ELISAs known in the art.
[0128] An ELISA procedure can also be carried out in a variety of
formats. Methods for enhancement of ELISA sensitivity using several
layers of anti-antibodies, avidin-biotin complexes and
enzyme-anti-enzyme antibody complexes are well known in the art.
The solid support or surface for fixation of antigen is usually
plastic, as described here, but a variety of other solid supports
such as latex or agarose have been described. It is also not
necessary for the antigen to be directly fixed onto the solid
support/phase. There is for example a commonly used ELISA format
that fixes the specific antigen to the solid support via a
solid-phase-fixed antibody to the antigen, so-called catching
antibody ELISA or sandwich ELISA. A special case of immunoassay
which involves a blotting (transfer) of antigen to a solid support
in sheet format is termed immunoblotting. Typically, the solid
support is nitrocellulose or nylon membranes/sheets, but other
supports have been described. Various binding, mixing, incubating,
coating, or blotting interactions are involved in an ELISA assay.
Prior to an ELISA assay, the antigens or antibodies can be
separated according to their sizes by gel electrophoresis or
similar methods. Detection of antibodies bound to the specific
antigen on the sheet can be carried out in similar ways as for
other immunoassays.
[0129] The 96 well format is a high throughput screening format
useful to optimize assay procedures and conditions. Other format
with different number of wells can also be used. Positive controls
and negative controls were also performed on, for example, serum
samples from donor subjects that are positive for HPV infection and
virgin subjects without HPV infection. The immunological assays
were found to result in high sensitivity, for example, in detecting
E6, E7 and L1 antibodies. Initial titration curves were performed
and ELISA assays conditions were optimized.
[0130] FIG. 7 is a graph showing the results of an exemplary
immunological ELISA assay for detecting various monoclonal
antibodies against E6 protein on microtiter plates using a purified
recombinant protein, recombinant HPV-16 E6 protein, coated on the
bottom of the microtiter plates. The concentrations of the
recombinant HPV-16 E6 proteins used to coat the bottom of the
microtiter plates were about 750 ng, 500 ng, 250 ng, 125 ng, 62.5
ng, 31.25 ng per well, respectively, demonstrating the E6
recombinant proteins interact with different monoclonal anti-E6
antibodies with different binding affinities. The results indicate
that the purified E6 recombinant protein is capable of specific
binding to antibodies against E6 protein, thus suitable for using
as a capture agent in an antibody test to detect antibodies from
clinical samples of subjects and assay for HPV infection.
[0131] As shown in FIG. 7, a dose dependent binding assay for the
HPV-16 E6 recombinant protein showed a highest affinity with one
antibody, MAb1-1, among the three antibodies tested. The variation
in binding affinity could come from different source of immunogens
that the three antibodies are originally raised against to. (The
purified recombinant proteins as obtained are contemplated to be
better immunogens than peptides or other purified HPV proteins).
The variation in binding affinity may also indicate that different
clinical samples or different subjects of HPV infection may give
rise to different immune responses, thus may react differently with
the purified recombinant proteins as described herein. Since the E6
recombinant protein binds two out of three antibodies in FIG. 7,
the E6 recombinant protein described herein should contain at least
two or more active epitopes on the surface of its conformational
form for such binding to be detected. The results in FIG. 7
indicate the purified E6 recombinant protein can be used to capture
target antibodies from HPV infected subjects.
[0132] FIG. 8 is a graph showing the results of an ELISA assay for
detecting a monoclonal antibody against E6 oncoprotein on
microtiter plates using another exemplary purified recombinant
protein, a recombinant HPV-58 E6 protein, coated on the bottom of
the microtiter plates. The concentrations of the purified
recombinant HPV-58 E6 proteins used to coat the bottom of the
microtiter plates were about 8 .mu.g/ml, 4 .mu.g/ml, 2 .mu.g/ml per
well, respectively, demonstrating specific binding of the E6
recombinant proteins to a monoclonal anti-E6 antibody, MAb1-1, as
compared to MBP control protein which showed no binding. The
linearity of the titration curve indicates assay sensitivity may be
from micrograms to nanograms range, or even picograms or lower
range. The results in FIG. 8 confirm that the purified recombinant
HPV-58 E6 proteins is active in binding to anti-E6 antibodies, thus
suitable for using as a capture agent to detect antibodies from
samples from HPV infected subjects.
[0133] FIG. 9 is an exemplary graph showing the results of an ELISA
assay for detecting various monoclonal antibodies (MAb1-1, MAb1-2,
and MAb1-4) against E6 oncoprotein on microtiter plates using
purified recombinant proteins, recombinant HPV-16 E6 proteins,
coated on the bottom of the microtiter plates. The concentrations
of the recombinant HPV-16 E6 proteins used to coat the bottom of
the microtiter plates were about 8 .mu.g/ml, 4 .mu.g/ml, 2
.mu.g/ml, 1 .mu.g/ml, and 0.5 .mu.g/ml per well, respectively,
demonstrating the E6 recombinant proteins interact with different
monoclonal anti-E6 antibodies with different binding affinities.
The results in FIG. 9 indicate that the purified E6 recombinant
protein is capable of specific binding to antibodies against E6
protein, thus suitable for using as a capture agent in an antibody
test to detect antibodies from clinical samples of subjects and
assay for HPV infection. The linearity of the titration curves
indicates assay sensitivity may be micrograms to nanograms range,
or even picograms or lower level.
[0134] FIG. 10 is another exemplary graph showing the results of an
immunological ELISA assay for detecting a monoclonal antibody
(MAb2-1) and a polyclonal antibody (PAb2-1) against E7 oncoprotein
on microtiter plates using purified recombinant proteins,
recombinant HPV-16-E7 fusion proteins, coated on the bottom of the
microtiter plates. The concentrations of the purified recombinant
HPV-16-E7 proteins used to coat the bottom of the microtiter plates
were about 8 .mu.g/ml, 4 .mu.g/ml, 2 .mu.g/ml, 1 .mu.g/ml, and 0.5
.mu.g/ml per well, demonstrating the E7 recombinant proteins
interact with different monoclonal anti-E7 antibodies with
different binding affinities.
[0135] FIG. 11 is another exemplary graph showing the results of an
immunological ELISA assay for detecting monoclonal antibody
(MAb2-1) against E7 oncoprotein using purified recombinant
proteins, recombinant HPV-16-E7 fusion proteins, coated on the
bottom of the microtiter plates. The concentrations of the purified
recombinant HPV-16-E7 proteins used to coat the bottom of the
microtiter plates were about 4 .mu.g/ml, 1 .mu.g/ml, 0.25 .mu.g/ml,
0.125 .mu.g/ml, 62.5 ng/ml, and 31.25 ng/ml per well,
respectively.
TABLE-US-00002 TABLE 2 Subject# OD.sub.450 in E6 Ab test OD.sub.450
in E7 Ab test 12 0.41 0.30 1 0.40 0.37 20 0.94 0.60 3 1.40 1.34 57
1.38 1.12 40 1.56 1.47 Mab1-1/Pab2-1 0.35 0.48 buffer only 0.10
0.09
[0136] The results in FIGS. 10 and 11 indicate that the purified E7
recombinant protein is capable of specific binding to antibodies
against E7 protein, thus suitable for using as a capture agent in
an antibody test to detect antibodies from clinical samples of
subjects and assay for HPV infection. The linearity of the
titration curves indicates assay sensitivity may be micrograms to
nanograms range, or even picograms or lower level. Since the
purified E7 recombinant proteins bind more than one antibody
tested, the E7 recombinant protein described herein should contain
at least two, or even more than two active epitopes on the surface
of its conformational form for such binding activity to be
detected.
[0137] Table 2 illustrates the results of various ELISA antibody
tests on clinical human subjects on selected samples. The
OD.sub.450 readings for E6 antibody tests and E7 antibody tests are
shown.
Example 11
Immunological ELISA Antigen Test for Detection of HPV Associated
Antigens or Proteins
[0138] A pair of antibodies can be used as a capture antibody and a
detection antibody in an antigen test. Such as a sandwiched ELISA
assay. Different pairs of antibodies in different combinations of
mABs/mABs, polyABs/mABs, mABs/polyABs or polyABs/polyABs were
tested as the capture and detection antibodies and the sandwiched
ELISA assay conditions were optimized. The capture and detection
antibodies are chosen in different monoclonal/polyclonal
combination for a secondary antibody to interact and bind to the
resulting immunocomplex. If the optimized capture and detection
antibodies are both polyclonal antibodies or both monoclonal
antibodies, then one of the capture and detection antibodies will
be conjugated to be used with immunological assay-derived detection
substrates, such as conjugated horse radish peroxidase, and others
used in immunological assays. In general, a sandwiched ELISA in an
antigen test is performed according to standard procedures.
[0139] The purified recombinant proteins were used to raise
anti-serum, polyclonal and monoclonal antibodies by injecting to
animal species and screening with the recombinant proteins for
specific binding. Many convenient animals species can be used to
prepare the appropriate antisera, and these antisera can be used
directly. Suitable animal species include mice, rats, rabbits,
guinea pigs, or even larger mammals, such as sheep. For
administration to such animals, the recombinant proteins are
generally administered in the presence of an adjuvant, usually
Freund's complete adjuvant, and the polyclonal sera are harvested
periodically by standard techniques.
[0140] Monoclonal antibodies may be produced using the method of
Kohler and Milstein or by more recent modifications thereof by
immortalizing spleen or other antibody-producing cells from
injected animals to obtain monoclonal antibody-producing clones.
HPV positive and negative human serum samples are useful in
screening monoclonal antibody producing hybridoma to ensure the
specificity of the monoclonal antibody clones. More than one
positive clones reactive with purified E6, E7, and L1 are obtained
and further injection of the obtained cell cultures to mice or
other animal source can be used to produce ascites for purifying
the monoclonal antibodies, such as by protein A affinity column
chromatography. The purified antibody will be used as either the
capture or detection probes in our ELISA or to be conjugated with
detection enzymes, such as (HRP, AP, etc.) for ELISA substrate
detection in an absorbent, fluorescent, or chemiluminecence
detection system.
[0141] The polyclonal and monoclonal antibodies obtained are useful
for diagnosis of HPV infection in cervical biopsies, serum or
genital swabs specimen and in assessing disease levels in human or
other subjects. In particular, diagnosis using the antibodies of
the invention permits identification of patients at high risk for
malignant transformation as well as identification of the
particular phase of CIN associated with the sample. The antibodies
can also be used in analysis of serum to detect HPV virus or to
detect the virus in metastases of infected tissue, as well as to
monitor the progression of HPV immunotherapy, anti-HPV vaccines, or
other therapeutic agents directed to control of HPV infection
and/or cervical carcinoma.
[0142] The 96-well format is a high throughput screening format
useful to optimize assay procedures and conditions. Positive
controls and negative controls were also performed on, for example,
serum samples from donor subjects that are positive for HPV
infection and virgin subjects without HPV infection. Initial
titration curves were performed and ELISA assays conditions were
optimized. The immunological assays were found to result in high
sensitivity, for example, in detecting E6, E7 and L1 proteins.
[0143] FIG. 12 is a graph showing the results of an exemplary
immunological sandwiched ELISA assay for detecting purified
recombinant proteins, recombinant HPV-16-E7 fusion proteins, using
a combination of two monoclonal antibodies (MAb2-1, MAb 2-3) and a
combination of polyclonal- and monoclonal-antibody (PAb2-1, MAb2-1)
against E7 oncoprotein as a pair of capture and detection
antibodies.
[0144] Anti-E6, anti-E7, and anti-L1 capture antibody were attached
to the bottoms of the microtiter plate for coating before the
purified recombinant proteins were added. Then a detection antibody
is used to detect the captured recombinant protein bound to the
capture antibody. Optimized capture and detection antibody
concentrations were identified. The concentration of the
recombinant proteins in the reaction resulting in linearity in the
ELISA assay for antigen detection was determined. These sandwiched
ELISA assays were repeated multiple times on the same day, as well
as on different days to determine assay reproducibility and
reliability. Specificity and sensitivity for each assay were
determined. Furthermore, the ELISA assay was shown to have
selectivity in detecting cervical cancers versus other cancers, for
example, to demonstrate non-cross reactivity with samples from
ovarian or endometrial cancers. Since it is known that HPV is found
in most if not all cervical cancer cells, but is usually not
associated with other cancers, the antigen tests as described
herein should not detect antigens associated with other cancers. To
test for this selectivity, for example, extracts from tissues of
ovarian and endometrial cancer cell lines can be tested and can
also serve as negative controls in the antigen tests.
[0145] Because most of the samples assayed herein are from genital
swabs of a subject, it is possible that the subject may have
sexually transmitted disorders and therefore infected with
organisms such as Chlamydia/Gonorrhea (bacteria) and Candida
(fungi), non-cross reactivity with antigens from other sexually
transmitted organisms or other viruses besides HPV are tested.
Human serum samples from HPV infected patients and HPV negative
subjects were also tested in the antigen tests. As an example,
antigen tests were performed to detect the presence of HPV E6, E7,
and L1 proteins in a clinical sample.
[0146] Table 3 shows OD.sub.450 data from ELISA immunoassays,
comparing the results of the antigen tests and the antibody tests
for different HPV proteins for selected samples.
TABLE-US-00003 TABLE 3 results of the ELISA immunoassays and the
comparison of antigen tests and antibody tests OD.sub.450 in E6
OD.sub.450 in E7 OD.sub.450 in E6 Subject # Ab test Ab test Ag test
sample dilution 1:10 1:50 1:10 1:50 1:10 4 0.29 0.11 0.36 0.13 0.14
34 1.40 0.86 1.50 1.22 0.45 195 1.15 0.75 0.63 0.26 0.02 196 0.32
0.12 0.58 0.19 0.37 197 0.12 0.10 0.12 0.07 0.01 103 0.65 0.26 0.48
0.15 0.17 105 0.11 0.08 0.11 0.07 0.43 107 0.25 0.09 0.36 0.14 0.06
108 1.06 0.52 1.32 0.88 109 0.84 0.30 1.01 0.45 119 0.11 0.06 0.10
0.09 MAb1-1/Pab2-1 0.34 1.53 buffer only 0.05 0.06
Example 12
One-Step Rapid Immunological Assay for Detection of HPV Associated
Antigens, Proteins, or Antibodies
[0147] The one-step rapid immunological assay as provided herein is
a non-invasive and easy to run assay, similar to the types of
over-the-counter pregnancy tests and without the need of any
particular test instrument. The one-step rapid immunological assay
can be an in vitro immunochromatographic assay for direct,
qualitative detection of common HPV antigens, specific antigens for
high risk HPV types, or HPV associated antibodies. The one-step
rapid Immunological assay can be used as an adjunct test to Pap
smear examination, as point-of-care diagnosis, and/or small clinics
or labs. The one-step rapid Immunological assay is suitable for
testing condition at room temperature to simply add an obtained
sample with or without dilution, wait for a reaction time period
for the designed reactions to occur, and score the results, for
example, visualization of the results.
[0148] The one-step rapid immunological assay may be a membrane or
stick test striped with a capture agent, e.g., purified HPV
antibodies, recombinant proteins, or HPV-associated antibodies and
proteins, etc., as described herein to capture a target agent,
e.g., HPV-associated antibodies and HPV-associated proteins, etc.,
in the clinical sample, followed by an immunoassay detection
system.
[0149] The one-step rapid immunological assay can be performed
vertically on a membrane or lateral in a strip. The lateral
flow-through or diffusion one-step rapid immunological assays may
also be referred to as immunochromatographic strip tests would take
about 5-15 minutes to obtain results and is easy to use, requiring
limited training and does not require instrumentation. The basic
principles of the assay include a solid phase nitrocellulose
membrane or strip containing the capture agent to react with a swab
sample from a Pap smear. If the patient sample contains the target
agent, then the capture agent in the nitrocellulose membrane reacts
with the target agent, and a complex is formed and migrates in the
nitrocellulose membrane through diffusion or capillary action.
[0150] At a set location in the nitrocellulose membrane or strip,
colored particles coupled with anti-human (or anti-mouse or
anti-rabbit for antigen detection) immunoglobin are disposed. If
the samples contain anti-HPV antibody, the color particle coupled
with anti-human (or anti-mouse or anti-rabbit for antigen
detection) immunoglobin antibody may react with anti-HPV antibody
and form a sandwich-type immunocomplex on the solid phase
nitrocellulose membrane or strip, resulting in a visible band. If
no target agent is present in the samples, there will be no visible
band. All tests may or may not include an internal procedural
positive and negative control lines used to validate the test
results. Appearance of reactive colored lines, therefore, indicates
a positive result, while a negative test produces only one or no
line. Therefore, the presence of the target agent in samples can be
quickly detected. The assay can be very sensitive and the
nitrocellulose membranes, strips, or other suitable membranes or
strips are generally very stable, for example, may last months if
kept dry and away from heat.
[0151] The membrane or stick can also be administered to the test
human subject during sample collection and/or combined with the
cotton swabs, independently or together, to allow the designed
immunological reactions to start and thus obtain the test results
instantly, from example, right after insertion of a speculum and
the swab into the endocervix of the test human subject. Thus, the
one-step rapid immunological assay can serve as a primary screening
test. The one-step rapid immunological assay can be performed
before additional HPV confirmatory tests, including conventional
pap smear cytological tests, the immunological assays and nucleic
acid hybridization assays as described herein, or combinations
thereof.
[0152] Similar procedures and reactions condition as used in the
antigen tests and antibody tests as described herein can be
employed for the one-step rapid immunological assay. Purified
recombinant proteins as provided herein can be used to impregnate a
nitrocellulose membrane or strip. To the other end of the strip,
colored particles coupled with anti-human immunoglobin antibody can
be disposed. Samples can be added and reaction conditions were
optimized. Negative controls may include one or positions on the
membrane or strip without the purified recombinant proteins or with
only BSA, serum proteins, or other negative control proteins. Assay
specificity and sensitivity are determined and detection of ng/ml
range of the target agent is a good range for
commercialization.
Example 13
Protein Chip Immunological Assay for Detection of HPV Associated
Antigens, Proteins, or Antibodies
[0153] Protein chip immunological assays were developed to detect
antibodies using the purified recombinant HPV proteins. In
addition, the protein chip immunological assay for detecting
HPV-associated proteins, HP-induced antigens, or HPV proteins were
performed using purified polyclonal or monoclonal antibodies or
antiserum directly or in a sandwiched-type format using a capture
antibody and a detection antibody.
[0154] For example, protein chips spotted with purified recombinant
proteins, such as HPV-16 E6, HPV-16 E7, HPV-16 L1, HPV-18 E6,
HPV-18, E7, HPV-18 L1, HPV-58 E6, etc., as well as control
proteins, such as BSA were tested. Initially, the conditions were
optimized using obtained antibodies, such as mAB 1-1, mAB 1-4, mAB
1-2, mAB 2-1, mAB 2-2, mAB 2-3, polyAB 1-1, poly AB 2-1, etc. and
varied concentrations of the purified recombinant proteins spotted
on the protein chips to maximize binding. A secondary antibody
coupled to, for example, Cys5, can be added to the surface of the
protein chips. Assay specificity and sensitivity were obtained.
Positive controls and negative controls from cell culture samples
or clinical samples are also tested. In addition, commercially
available antibody pairs can be used to detect p53 and RB in the
tested sample. Accordingly, the protein chip immunological assays
provide a simple and easy readout of HPV-related proteins (proteins
of the HPV early genes and late genes, L1, E6, and E7, as well as
HPY induced p53, RB, p16INK4a and other proteins) to understand
consequent altered expressions of cellular regulators induced by
HPV infection in the host as a signature of infection by high risk
HPV types, which is likely to lead to cervical cancer.
[0155] FIG. 13 demonstrates the results of an exemplary
immunological protein chip assay for detecting a monoclonal
antibody against E6 oncoprotein using purified recombinant proteins
coated on a protein chip. As shown in FIG. 13, purified recombinant
HPV-16-E6 protein (column P4) and HPV-58-E6 protein (column P1)
were spotted onto a glass cover slide at a protein concentration of
about 100 .mu.g/ml and a solution of test sample was also spotted
for a reaction time of about two hours. As control, purified
recombinant HPV-16-E7 protein (column P2), BSA control proteins
(column BSA), and recombinant tag-proteins for HPV-58-E6 expression
system (column P3) were also attached to the slide. The slide was
then reacted with a blocking buffer (PBS/BSA) for 30 min and then
reacted with an anti-E6 monoclonal antibody, mAB1-1 (about 5
.mu.gs/ml) for 2 hrs. After the binding reaction, the slide was
washed, then dried and reacted with anti-mouse Cy5 (about 4
.mu.gs/ml) for 2 hrs to detect bound mAB1-1. As shown in FIG. 13,
the mAB1-1 antibody selectively interacts with the purified
recombinant HPV-16-E6 proteins and the purified recombinant
HPV-58-E6 (as shown in column P4 and P1, respectively), but not BSA
proteins, the purified recombinant HPV-16-E7, nor the recombinant
tag-proteins controls (as shown in columns BSA, P3, and P2,
respectively). The results indicate that the purified recombinant
HPV proteins as provided herein can specifically retain their
binding abilities to antibodies in various solid phases and thus
feasible to further develop the protein chip technology to detect a
desirable target agent bind antibody and is feasible.
V. Comparison of Cytological Analyses with the Results from the
Immunological Analyses
[0156] A large number of clinical samples were obtained for
performing immunological assays and/or nucleic acid hybridization
assays to screen for HPV infection and the results of the
cytological and/or histological assays (such as colposcopy and
biopsy) on the obtained clinical samples were also received from
hospital collaborators.
[0157] Table 4 illustrates the interpretation of Pap Smear scores
and related cytological or histological status. Pap scores of one
(1) to three (3) are considered as normal and four (4) and above as
abnormal. ASCUS: Atypical Squamous Cells of Undetermined
Significance; unusual or atypical cells in pap smear, may be
inconsequential and significance is underdetermined. AGUS: Atypical
Glands of Undetermined Significance. LSIL: Low grade of Squamous
Intraepithelial Lesion. HSIL: High grade of Squamous
Intraepithelial Lesion. SCC: Squamous Cell Carcinoma. CIN 1:
Cervical Intraepithelial Neoplasia, mild cell abnormalities. CIN2:
Cervical Intraepithelial Neoplasia with lesions appearing more
aggressive. CIN3: Cervical Intraepithelial Neoplasia with
aggressive form of dysplasia.
TABLE-US-00004 TABLE 4 interpretation of Pap Scores Status from
Score Diagnosis status colposcopy and biopsy 4 ASCUS 5 AGUS 6 LSIL
CIN 1 no cytomorphological change of HPV infection 7 LSIL CIN 1
cytomorphological change due to HPV infection 8 HSIL CIN 2 9, 10
high probability of progressing into CIN 3 invasive cancer 11 SCC
Invasive carcinoma 12 Adenocarcinoma 14 sampling disqualified for
pap smear score too much bleeding due to inflammatory or maybe
cancer, less cells, not enough cells to view the morphology 15
AGUS, favor neoplasm 16 ASCUS, maybe HSIL 17 LSIL, not sure about
HSIL
[0158] FIG. 14 illustrates a comparison of the results from E6
antibody test with E7 antibody test. The results from the HPV-16-E6
Ab test correlates well to the results from the HPV-16-E7 Ab test
to indicate possible HPV infection. In addition, the results
correlate wells with the pap scores. Abnormal pap smear results are
also shown as positive in the E6 antibody tests and the E7 antibody
tests. Some normal pap smear samples are also shown as positive in
the E6 antibody tests and the E7 antibody tests, indicating past
HPV infection, current initial or early HPV infection.
[0159] FIG. 15 illustrates a comparison of the results from
antibody tests with antigen tests. The E6 Ab tests correlates well
to the E7 Ab tests and the E6 Ag tests. Subject 107 and subject 195
were not detected as positive in the E6 Ag test and may indicate
HPV infection in the past.
[0160] Table 5 is a summary of PAP smear results from a total of
896 subjects. Out of 896 tested clinical samples, the samples from
876 subjects were tested to be Pap normal, and the samples from 20
subjects are tested to be PAP abnormal. Thus, about 97.8% in the
sample population is tested to be pap normal and about 2.2% in the
sample population is tested to be pap abnormal, reflecting a
typical rate of pap smear screening in a general population.
TABLE-US-00005 TABLE 5 summary of pap smear results for the tested
clinical samples Pap smear Subject # % Abnormal 20 subjects 2.2%
Normal 876 subjects 97.8% Total 896 subjects 100%
[0161] In addition to antibody tests and antigen tests performed
for both HPV proteins encoded by HPV early genes and late genes,
PCR nucleic acid hybridization assays were performed. Total of 442
subjects were tested on HPV-16 E6 Ab test/assay, including 282 pap
normal, PCR L1 negative subjects and 160 pap normal, PCR L1
positive subjects. The cutoff was determined based on the OD
distribution between the groups of PCR positive versus PCR
negative. Individual with more than two fold of the OD.sub.450
value of the average OD.sub.450 value from the 282 pap normal, PCR
L1 negative subjects was set as positive, otherwise are negative as
cutoff for the OD.sub.450 value.
[0162] For example, Table 6 shows the results of the samples from
the 896 subjects tested for the presence of L1 genes using PCR
nucleic acid hybridization assays. Among the 896 obtained sample,
164 samples (18%, 164/896) were tested PCR HPV-L1 positive as an
indicator for HPV infection of screening from the general
population. Among the 896 obtained, 67 samples (7.5%, 67/896) were
tested positive on HPV-16-E6 Ab tests as an indicator for HPV
infection of screening from the general population and 58 samples
out of the 67 samples (7.5%, 58/67) were confirmed as positive by
PCR L1 tests. Among the 896 obtained, 164 samples (18%, 164/896)
were tested positive on PCR L1 tests as an indicator for HPV
infection of screening from the general population.
TABLE-US-00006 TABLE 6 results of the nucleic acid hybridization
assays Pap smear Abnormal Normal Total (No. and %) PCR positive 4
subjects 160 subjects 164 subjects (0.4%) (17.9%) (18%) PCR
negative 16 subjects 716 subjects 732 subjects (1.8%) (79.9%) (82%)
Total 20 subjects 876 subjects 896 subjects (2.2%) (97.8%)
(100%)
[0163] Table 7 shows the comparison of antibody tests specific for
early and late HPV proteins on 80 selected samples from tested
subjects with positive PCR test results.
TABLE-US-00007 TABLE 7 the results of antibody tests specific for
early and late HPV proteins on 80 selected samples HPV16 HPV E6 L1
Subject # Pap Smear PCR test Ab test Ab test 21 Normal positive
negative positive 22 Normal positive negative positive 23 Normal
positive positive positive 24 Normal positive positive positive 25
Normal positive positive positive 26 Normal positive negative
negative 27 Normal positive negative positive 28 Normal positive
positive positive 29 Normal positive positive negative 30 Normal
positive negative positive 31 Normal positive positive positive 32
Normal positive positive positive 33 Normal positive negative
negative 34 Normal positive positive positive 35 Normal positive
positive positive 36 Normal positive positive positive 37 Normal
positive positive positive 38 Normal positive positive positive 39
Normal positive negative negative 40 Normal positive positive
positive 41 Normal positive positive positive 42 Normal positive
positive positive 43 Normal positive positive positive 44 Normal
positive positive positive 45 Normal positive positive positive 46
Normal positive positive positive 47 Normal positive positive
positive 48 Normal positive positive positive 49 Normal positive
positive positive 50 Normal positive positive negative 51 Normal
positive negative negative 52 Normal positive negative negative 53
Normal positive negative negative 54 Normal positive positive
positive 55 Normal positive positive positive 56 Normal positive
positive positive 57 Normal positive positive positive 58 Normal
positive positive positive 59 Normal positive negative positive 60
Normal positive negative positive 61 Normal positive positive
positive 62 Normal positive positive positive 63 Normal positive
negative positive 64 Normal positive positive positive 65 Normal
positive positive positive 66 Normal positive negative positive 67
Normal positive negative positive 68 Normal positive positive
negative 69 Normal positive negative positive 70 Normal positive
negative negative 71 Normal positive positive positive 72 Normal
positive positive positive 73 Normal positive positive positive 74
Normal positive negative negative 75 Normal positive positive s
negative 76 Normal positive negative positive 77 Normal positive
negative negative 78 Normal positive negative negative 79 Normal
positive negative positive 80 Normal positive positive positive 81
Normal positive positive positive 82 Normal positive positive
positive 83 Normal positive positive positive 84 Normal positive
negative positive 85 Normal positive positive positive 86 Normal
positive positive positive 87 Normal positive positive positive 88
Normal positive positive positive 89 Normal positive positive
positive 90 Normal positive positive positive 91 Normal positive
negative negative 92 Normal positive negative positive 93 Normal
positive negative positive 94 Normal positive negative positive 95
Normal positive positive positive 96 Normal positive negative
positive 97 Normal positive positive positive 98 Normal positive
positive positive 99 Normal positive negative positive 100 Abnormal
positive positive positive
[0164] Table 8 shows the results of antibody tests specific early
and late HPV proteins on 94 selected samples from tested subjects
with negative PCR test results. In general, the positive results
from the HPV-16-E6 Ab test correlates well to the results from the
HPV-16-L1 Ab test to indicate/confirm possible HPV infection.
TABLE-US-00008 TABLE 8 the results of antibody tests specific for
early and late HPV proteins on 94 selected PCR negative samples
HPV-16-E6 HPV-16-L1 Subject # Pap Smear PCR test Ab test Ab test
101 Normal negative positive positive 102 Normal negative negative
positive 103 Normal negative negative positive 104 Normal negative
negative negative 105 Normal negative negative negative 106 Normal
negative negative negative 107 Normal negative negative negative
108 Normal negative positive negative 109 Normal negative negative
negative 110 Normal negative negative negative 111 Normal negative
negative negative 112 Normal negative negative negative 113 Normal
negative positive negative 114 Normal negative negative negative
115 Normal negative negative negative 116 Normal negative negative
negative 117 Normal negative negative negative 118 Normal negative
positive positive 119 Normal negative negative negative 120 Normal
negative negative negative 121 Normal negative negative negative
122 Normal negative negative positive 123 Normal negative negative
negative 124 Normal negative negative negative 125 Normal negative
negative negative 126 Normal negative negative negative 127 Normal
negative negative negative 128 Normal negative negative positive
129 Normal negative negative negative 130 Normal negative negative
negative 131 Normal negative negative positive 132 Normal negative
negative negative 133 Normal negative negative negative 134 Normal
negative negative negative 135 Normal negative negative negative
136 Normal negative positive negative 137 Normal negative positive
positive 138 Normal negative negative positive 139 Normal negative
negative negative 140 Normal negative negative negative 141 Normal
negative negative negative 142 Normal negative negative negative
143 Normal negative negative negative 144 Normal negative negative
negative 145 Normal negative negative positive 146 Normal negative
negative negative 147 Normal negative negative negative 148 Normal
negative negative negative 149 Normal negative negative negative
150 Normal negative negative negative 151 Normal negative negative
negative 152 Normal negative negative negative 153 Normal negative
negative negative 154 Normal negative negative negative 155 Normal
negative negative negative 156 Normal negative negative negative
157 Normal negative positive positive 158 Normal negative negative
negative 159 Normal negative negative positive 160 Normal negative
negative negative 161 Normal negative negative negative 162 Normal
negative negative positive 163 Normal negative negative negative
164 Normal negative positive positive 165 Normal negative negative
positive 166 Normal negative positive positive 167 Normal negative
negative negative 168 Normal negative negative negative 169 Normal
negative negative negative 170 Normal negative negative negative
171 Normal negative negative negative 172 Normal negative negative
negative 173 Normal negative positive positive 174 Normal negative
negative positive 175 Normal negative negative negative 176 Normal
negative negative negative 177 Normal negative negative negative
178 Normal negative negative negative 179 Normal negative negative
negative 180 Normal negative negative negative 181 Normal negative
negative positive 182 Normal negative negative negative 183 Normal
negative negative negative 184 Normal negative negative negative
185 Normal negative negative negative 186 Normal negative positive
negative 187 Normal negative negative negative 188 Normal negative
negative negative 189 Normal negative negative positive 190 Normal
negative positive negative 191 Normal negative positive negative
192 Normal negative positive negative 193 Normal negative negative
negative 194 Normal negative positive negative
[0165] Table 9 shows the results of antibody (Ab) tests for
selective 160 samples of Pap smear Normal and PCR positive subjects
further tested to detect the presence of anti-E6 antibodies in the
samples.
TABLE-US-00009 TABLE 9 the results of antibody tests specific for
E6 HPV proteins on 160 selected samples Pap smear Normal E6 Ab and
PCR Positive tests (Total of 160 subjects) positive 55 subjects
34.3% negative 105 subjects 65.7% Total 160 subjects 100%
[0166] Table 10 shows the results of both antigen (Ag) tests and
antibody (Ab) tests further tested on selected 160 samples of Pap
smear Normal and PCR positive subjects further tested to detect the
presence of viral E6 antigens and antibodies in the samples. Strong
correlation and between-assay agreement are observed for E6 antigen
tests and E6 antibody tests.
TABLE-US-00010 TABLE 10 the results of antibody tests and antigen
tests specific for E6 early HPV antibodies and proteins on 164
selected samples Pap smear Normal and PCR Positive (Total of 160
subjects) E6Ab positive E6Ab negative total E6Ag 29 subjects 30
subjects 59 subjects positive (18.1%) (18.9%) (37.8%) E6Ag 26
subjects 75 subjects 101 subjects negative (16.2%) (46.3%) (62.2%)
total 55 subjects 105 subjects 160 subjects (34.3%) (65.2%)
(100%)
[0167] Table 11 shows the results of antibody (Ab) tests from the
164 PCR positive subjects out of the total collected 896 subjects
further tested to detect the presence of viral E6 antibodies in
these samples.
TABLE-US-00011 TABLE 11 the results of antibody tests specific for
early E6 HPV proteins on 164 selected samples E6 Ab PCR Positive
test (Total of 164 subjects) positive 57 subjects 34.8% negative
107 subjects 65.2% Total 164 subjects 100.0%
[0168] Table 12 shows the results both antigen (Ag) tests and
antibody (Ab) tests tested on the 164 PCR positive subjects out of
the total collected 896 subjects further tested to detect the
presence of viral E6 antigens and antibodies in the samples.
TABLE-US-00012 TABLE 12 the results of antibody tests and antigen
tests specific for E6 early HPV proteins on 160 selected samples
PCR Positive (Total of 164 subjects) E6 Ab test E6 Ab test positive
negative total E6 Ag test 31 subjects 31 subjects 62 subjects
positive (18.9%) (18.9%) (37.8%) E6 Ag test 26 subjects 76 subjects
102 subjects negative (15.9%) (46.3%) (62.2%) total 57 subjects 107
subjects 164 subjects (34.8%) (65.2%) (100%)
[0169] Table 13 shows the results of antibody (Ab) tests from 80
selected samples which were tested as PCR positive subjects to
further detect the presence of antibodies specific for HPV early
proteins and late proteins in these samples. These selected 80
samples are tested on antibody tests for both E6 and L1. Strong
correlation and between-assay agreement are observed for E6
antibody tests and L1 antibody tests. Therefore, one of the two
assays can be used as a confirmatory check for the other assay and
vice versa. For example, out of the 80 samples, 47 subjects (58.8%)
are double positive for both assays and 11 subjects (13.8%) are
double negative. Among the 80 samples, 69 subjects (86.2%) are
positive from either L1 or E6 test. Out of the 80 samples, 47
samples are positive on both E6 and L1 Ab tests, demonstrating very
well correlated assay results and confirmed cases of HPV infection.
In addition, 27 out of the 47 E6 positive L1 positive samples also
were tested to be E6 antigen positive. In general, L1 Ab test is
able to detect 92% (47 out of 51) of E6 Ab positive samples (51
samples) and E6 Ab test is able to detect 72% (47 out of 65) of
L1Ab positive samples (65 samples).
TABLE-US-00013 TABLE 13 the results of antibody tests specific for
early and late HPV proteins on 80 selected samples PCR Positive
(Total of 80 subjects) E6 Ab test positive E6 Ab test negative
total L1 Ab test 47 subjects 18 subjects 65 subjects positive
(58.8%) (22.5%) (81.2%) L1 Ab test 4 subjects 11 subjects 15
subjects negative (5%) (13.8%) (18.8%) total 51 subjects 29
subjects 80 subjects (63.8%) (36.3%) (100%)
[0170] Table 14 shows the results of both E6 antigen (Ag) tests and
E6 antibody (Ab) tests further tested on these selected 80 samples
to detect the presence of both E6 antigens and antibodies in these
samples.
TABLE-US-00014 TABLE 14 the results of antibody tests and antigen
tests specific for early E6 HPV proteins on 80 selected samples PCR
Positive (Total of 80 subjects) E6 Ab test positive E6 Ab test
negative total E6 Ag test 29 subjects 14 subjects 43 subjects
positive (36.3%) (17.5%) (53.8%) E6 Ag test 22 subjects 15 subjects
37 subjects negative (27.5%) (18.8%) (46.3%) total 51 subjects 29
subjects 80 subjects (63.8%) (36.3%) (100%)
[0171] In addition, selected PCR negative samples were also tested
on antibody tests and antigens tests for E6, E7, and/or L1. Table
15 shows the results of antibody (Ab) tests on 282 samples which
were also tested as PCR negative and Pap normal. The majority of
the 282 samples (about 85%) are also negative on E6 Ab tests.
TABLE-US-00015 TABLE 15 the results of antibody tests specific for
early and late HPV proteins on 282 selected samples Pap smear
Normal and PCR Negative (only 282 subjects out of 716 subjects
analyzed) E6 Ab test positive 43 subjects 15% E6 Ab test negative
239 subjects 85% Total 282 subjects 100%
[0172] Table 16 shows the results of antibody (Ab) tests for both
viral early and late proteins on selected 94 samples out of these
282 PCR negative and Pap normal to further detect the presence of
both E6 and L1 antibodies in these samples. Out of these PCR
negative and Pap normal samples, the majority of the samples were
negative on both E6 Ab test and/or L1 Ab test. Only a low
percentage of 7 samples (7.4%) are both positive on E6 Ab test
and/or L1 Ab test. In addition, among these Pap normal, PCR
negative samples, only 28 samples (7+9+12=28/94; 29.8%) are
positive to either E6 Ab test and/or L1 Ab test. For the total
obtained 896 clinical samples, randomly selected samples were
tested on antibody tests for interaction/binding to antibodies
specific for HPV early E6 proteins and HPV late L1 proteins as well
as PCR nucleic acid hybridization assays.
[0173] Table 17 shows the results for antibody tests for E6 and L1
and PCR test on selected 173 (79+94) samples with normal pap smear
scores. FIG. 16 illustrates the results from E6 Ab tests, L1 Ab
tests, and PCR tests performed on selected 173 subjects with normal
pap smear scores. Out of these 173 pap normal samples, 46 samples
(26.6%) are positive on all three tests and 53 samples (30.6%) are
positive on both E6 Ab test and L1 Ab test, demonstrating that high
level of correlation and assay specificity and sensitivity and
indicating or even confirming HPV infection, and thus high risk for
cervical cancer development in the future if the HPV infection is
not treated, even though pap smear were tested negative
(normal).
TABLE-US-00016 TABLE 16 the results of antibody tests specific for
early and late HPV proteins on 94 selected samples Pap smear Normal
and PCR Negative (Total of 94 subjects) E6 Ab test positive E6 Ab
test negative total L1 Ab test 7 subjects 13 subjects 20 subjects
positive (7.4%) (13.8%) (21.2%) L1 Ab test 8 subjects 66 subjects
74 subjects negative (8.5%) (70.3%) (78.8%) total 15 subjects 79
subjects 94 subjects (15.9%) (84.1%) (100%)
TABLE-US-00017 TABLE 17 the results of antibody tests specific for
early and late HPV proteins on 173 (79 + 94) selected samples PCR
L1 positive PCR L1 negative (selected 79 (selected 94 subject)
subjects) HPV-16-E6 HPV-16-E6 HPV-16-L1 Ab test Ab test Ab test
positive negative positive negative positive 46 18 7 13 negative 4
11 8 66 total 50 29 15 79
[0174] Out of these 173 pap normal samples, 79 samples (45.7%) are
negative on all three tests and 108 samples (62.4%) are negative on
both E6 Ab test and L1 Ab test, demonstrating that high level of
correlation of these developed assays and assay specificity and
sensitivity. The results further confirm negative HPV infection,
and thus low risk for cervical cancer development in the future,
consistent with the negative (normal) pap smear results.
[0175] Table 18 shows some possible interpretation for comparing
the results of antibody tests specific for early and late HPV
proteins.
TABLE-US-00018 TABLE 18 Assay results and possible interpretations
HPV immunoassays interpretation E6 Ab positive, HPV infection,
HPV16 related, or at high risk of L1 Ab positive progression E6 Ab
positive, HPV infection, HPV16 related, maybe at risk of early L1
Ab negative stage of progression E6 Ab negative, HPV past
infection, HPV16 related, may not be at L1 Ab positive risk of
progression E6 Ab negative, No HPV infection L1 Ab negative
[0176] Of the 896 obtained clinical samples, 20 pap abnormal
samples were further tested on antibody tests and antigen tests and
PCR tests. Table 19 illustrates the results of PCR analyses and E6
Ab test on these 20 pap abnormal subjects.
TABLE-US-00019 TABLE 19 the results of PCR analyses 20 Pap abnormal
samples Pap smear Abnormal PCR Positive PCR Negative Total E6 Ab 3
subjects 9 subjects 12 subjects positive (15%) (45%) (60%) E6Ab 1
subjects 7 subjects 8 subjects negative (5%) (35%) (40%) Total 4
subjects 16 subjects 20 subjects (20%) (80%) (100%)
[0177] FIG. 17 illustrates the results of the E6 Ab tests, E6 Ag
tests, E7 Ab tests, and PCR L1 tests for these 20 subjects with
abnormal pap smear scores. Blank indicates test results not yet
available. The positive results from the E6 antibody tests
correlate well with the positive results of the E6 antigen tests.
All five samples tested so far on E7 Ab tests were positive and
also positive on E6 Ab tests.
[0178] Overall assay agreement between the E6 Ab test and the L1 Ab
test among PCR positive samples (80/164) is 72.5% (58/85). Positive
and negative agreements are 92% (47/51) and 38% (11/29),
respectively, with positive predictive value (PPV) of about 72.3%
(47/65) and negative predictive value (NPV) of about 73.3%
(11/15).
[0179] Overall assay agreement between the E6 Ab test and the L1 Ab
test among PCR negative samples (94/282) is 78% (73/94). Positive
and negative agreements are 47% (7/15) and 84% (66/79),
respectively, with PPV of about 35% (7/20) and NPV of about 89%
(66/74).
[0180] Overall assay agreement between the E6 Ab test and PCR L1
test among pap normal samples (442/876) is 66.5% (294/442).
Positive and negative agreement are 34% (55/160) and 85% (239/282),
respectively, with PPV of about 56% and NPV of about 70%.
[0181] Kits can be developed for performing the methods and assays
provided herein. Recombinant proteins, antiserum, and antibodies
are also provided for developing these assay kits and screening for
infection with HPV-16, 18, 31, 33, 35, 45, 52, 58, 59, 66, 68b, 69,
70, 73, 82, etc., types. The kits, the immunological assays, the
recombinant proteins, antiserum, and antibodies, etc., as provided
herein are useful for a variety of diagnostic analyses, for
example, for diagnosing infection by non-oncogenic or oncogenic HPV
types or HPV strains in an individual; for determining the
likelihood of having cervical cancer, for determining a patient's
response to treatment for HPV, for determining the severity of HPV
infection in an individual, and for monitoring the progression of
HPV in an individual, among others. The kits, the immunological
assays, the recombinant proteins, antiserum, and antibodies, etc.,
as provided herein are useful in the diagnosis of infection with an
oncogenic HPV type or a strain of HPV associated with cancers,
including cervical, ovarian, breast, anus, penis, prostate, larynx
and the buccal cavity, tonsils, nasal passage, skin, bladder, head
and neck squamous-cell, occasional periungal carcinomas, as well as
benign anogenital warts. The kits, the immunological assays, the
recombinant proteins, antiserum, and antibodies, etc., as provided
herein are useful in the diagnosis of infection with an oncogenic
or a non-oncogenic type or strain of HPV associated with
Netherton's syndrome, epidermolysis verruciformis, endometriosis,
and other disorders The kits, the immunological assays, the
recombinant proteins, antiserum, and antibodies, etc., as provided
herein are useful in the diagnosis of infection with an oncogenic
or a non-oncogenic HPV type or HPV strain in adult women, adult
men, fetuses, infants, children, and immunocompromised
individuals.
[0182] The kits as described herein can further include, if
desired, one or more of various conventional components, such as,
for example, containers with one or more buffers, detection
reagents or antibodies. Printed instructions, either as inserts or
as labels, indicating quantities of the components to be used and
guidelines for their use, can also be included in the kit. In the
present disclosure it should be understood that the specified
materials and conditions are important in practicing the invention
but that unspecified materials and conditions are not excluded so
long as they do not prevent the benefits of the invention from
being realized. Exemplary embodiments of the diagnostic methods of
the invention are described above in detail.
[0183] In a subject kit, the HPV E6, E7, and/or L1 detection
reaction may be performed using an aqueous or solid substrate,
where the kit may include reagents for use with several separation
and detection platforms such as test strips, sandwich assays, etc.
In many embodiments of the test strip kit, the test strip has bound
thereto recombinant protein or antibody specific for HPV proteins,
and captures HPV induced or HPV associated proteins or antibodies
on the solid support. The kit usually includes one or more primary
or secondary antibodies for detection, which is either directly or
indirectly detectable. The kit may also include components for
conducting western blots (e.g., pre-made gels, membranes, transfer
systems, etc.); components for carrying out ELISAs (e.g., 96-well
plates); components for carrying out immunoprecipitation (e.g.
protein A); columns, especially spin columns, for affinity or size
separation of proteins or antibodies from a sample. The kit may
also contain control samples containing oncogenic or non-oncogenic
E6 and/or E7, and/or a dilution series of oncogenic E6 and/or E7,
where the dilution series represents a range of appropriate
standards with which a user of the kit can compare their results
and estimate the level of oncogenic E6 and/or E7 in their sample.
Such a dilution series may provide an estimation of the progression
of any cancer in a patient. Fluorescence, color, or autoradiogram
development results may also be compared to standard curves of
fluorescence, color or film density provided by the kit.
[0184] Assay conditions suitable for binding are those conditions
(in terms of salt concentration, pH, detergent, protein
concentration, temperature, etc.) which allow for binding to occur,
for example, between a capture agent and a target agent, between a
primary antibody and a secondary antibody, between a recombinant
protein and a protein or antibody that can bind to the recombinant
protein, etc., in solid support or in solution. Such conditions,
particularly with respect to antibodies and their antigens, are
well known in the art (see, e.g., Harlow and Lane (Antibodies: A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)). Conditions suitable for specific binding
typically permit binding partners or pairs that have a dissociation
constant (K.sub.D) of less than about 10.sup.-6 M to bind to each
other selectively.
[0185] While the foregoing is directed to embodiments of the
present invention, other and further embodiments of the invention
may be devised without departing from the basic scope thereof, and
the scope thereof is determined by the claims that follow.
Sequence CWU 1
1
271474DNApappilomavirus 1caccaaaaga gaactgcaat gtttcaggac
ccacaggagc gacccagaaa gttaccacag 60ttatgcacag agctgcaaac aactatacat
gatataatat tagaatgtgt gtactgcaag 120caacagttac tgcgacgtga
ggtatatgac tttgcttttc gggatttatg catagtatat 180agagatggga
atccatatgc tgtatgtgat aaatgtttaa agttttattc taaaattagt
240gagtatagac attattgtta tagtttgtat ggaacaacat tagaacagca
atacaacaaa 300ccgttgtgtg atttgttaat taggtgtatt aactgtcaaa
agccactgtg tcctgaagaa 360aagcaaagac atctggacaa aaagcaaaga
ttccataata taaggggtcg gtggaccggt 420cgatgtatgt cttgttgcag
atcatcaaga acacgtagag aaacccagct gtaa 4742157PRTpappilomavirus 2His
Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg1 5 10
15Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile
20 25 30Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu
Val 35 40 45Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp
Gly Asn 50 55 60Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser
Lys Ile Ser65 70 75 80Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly
Thr Thr Leu Glu Gln 85 90 95Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu
Ile Arg Cys Ile Asn Cys 100 105 110Gln Lys Pro Leu Cys Pro Glu Glu
Lys Gln Arg His Leu Asp Lys Lys 115 120 125Gln Arg Phe His Asn Ile
Arg Gly Arg Trp Thr Gly Arg Cys Met Ser 130 135 140Cys Cys Arg Ser
Ser Arg Thr Arg Arg Glu Thr Gln Leu145 150 155334DNApappilomavirus
3cgcggatccc accaaaagag aactgcaatg tttc 34432DNApappilomavirus
4cccaagcttt tacagctggg tttctctacg tg 325510DNApappilomavirus
5atgagaggat cgcatcacca tcaccatcac ggatcccacc aaaagagaac tgcaatgttt
60caggacccac aggagcgacc cagaaagtta ccacagttat gcacagagct gcaaacaact
120atacatgata taatattaga atgtgtgtac tgcaagcaac agttactgcg
acgtgaggta 180tatgactttg cttttcggga tttatgcata gtatatagag
atgggaatcc atatgctgta 240tgtgataaat gtttaaagtt ttattctaaa
attagtgagt atagacatta ttgttatagt 300ttgtatggaa caacattaga
acagcaatac aacaaaccgt tgtgtgattt gttaattagg 360tgtattaact
gtcaaaagcc actgtgtcct gaagaaaagc aaagacatct ggacaaaaag
420caaagattcc ataatataag gggtcggtgg accggtcgat gtatgtcttg
ttgcagatca 480tcaagaacac gtagagaaac ccagctgtaa
5106169PRTpappilomavirus 6Met Arg Gly Ser His His His His His His
Gly Ser His Gln Lys Arg1 5 10 15Thr Ala Met Phe Gln Asp Pro Gln Glu
Arg Pro Arg Lys Leu Pro Gln 20 25 30Leu Cys Thr Glu Leu Gln Thr Thr
Ile His Asp Ile Ile Leu Glu Cys 35 40 45Val Tyr Cys Lys Gln Gln Leu
Leu Arg Arg Glu Val Tyr Asp Phe Ala 50 55 60Phe Arg Asp Leu Cys Ile
Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val65 70 75 80Cys Asp Lys Cys
Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His 85 90 95Tyr Cys Tyr
Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys 100 105 110Pro
Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu 115 120
125Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His
130 135 140Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys
Arg Ser145 150 155 160Ser Arg Thr Arg Arg Glu Thr Gln Leu
1657300DNAPapillomavirus sylvilagi 7gatcccatgg agatacacct
acattgcatg aatatatgtt agatttgcaa ccagagacaa 60ctgatctcta ctgttatgag
caattaaatg acagctcaga ggaggaggat gaaatagatg 120gtccagctgg
acaagcagaa ccggacagag cccattacaa tattgtaacc ttttgttgca
180agtgtgactc tacgcttcgg ttgtgcgtac aaagcacaca cgtagacatt
cgtactttgg 240aagacctgtt aatgggcaca ctaggaattg tgtgccccat
ctgttctcag aaaccataag 300897PRTpappilomavirus 8His Gly Asp Thr Pro
Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro1 5 10 15Glu Thr Thr Asp
Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu 20 25 30Glu Glu Asp
Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg 35 40 45Ala His
Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu 50 55 60Arg
Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp65 70 75
80Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys
85 90 95Pro931DNApappilomavirus 9cgcggatccc atggagatac acctacattg c
311032DNApappilomavirus 10ccggaattct tatggtttct gagaacagat gg
3211972DNApappilomavirus 11atgtccccta tactaggtta ttggaaaatt
aagggccttg tgcaacccac tcgacttctt 60ttggaatatc ttgaagaaaa atatgaagag
catttgtatg agcgcgatga aggtgataaa 120tggcgaaaca aaaagtttga
attgggtttg gagtttccca atcttcctta ttatattgat 180ggtgatgtta
aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
240atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
agcggttttg 300gatattagat acggtgtttc gagaattgca tatagtaaag
actttgaaac tctcaaagtt 360gattttctta gcaagctacc tgaaatgctg
aaaatgttcg aagatcgttt atgtcataaa 420acatatttaa atggtgatca
tgtaacccat cctgacttca tgttgtatga cgctcttgat 480gttgttttat
acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
540aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
gtatatagca 600tggcctttgc agggctggca agccacgttt ggtggtggcg
accatcctcc aaaatcggat 660ctggttccgc gtggatccca tggagataca
cctacattgc atgaatatat gttagatttg 720caaccagaga caactgatct
ctactgttat gagcaattaa atgacagctc agaggaggag 780gatgaaatag
atggtccagc tggacaagca gaaccggaca gagcccatta caatattgta
840accttttgtt gcaagtgtga ctctacgctt cggttgtgcg tacaaagcac
acacgtagac 900attcgtactt tggaagacct gttaatgggc acactaggaa
ttgtgtgccc catctgttct 960cagaaaccat aa 97212323PRTpappilomavirus
12Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1
5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His
Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe
Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly
Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala
Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly
Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys
Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys Met Phe
Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His
Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155
160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp
Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln
Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys
Ser Asp Leu Val Pro Arg 210 215 220Gly Ser His Gly Asp Thr Pro Thr
Leu His Glu Tyr Met Leu Asp Leu225 230 235 240Gln Pro Glu Thr Thr
Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser 245 250 255Ser Glu Glu
Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro 260 265 270Asp
Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser 275 280
285Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu
290 295 300Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile
Cys Ser305 310 315 320Gln Lys Pro131602DNApappilomavirus
13tcgagatgca ggtgactttt atttacatcc tagttattac atgttacgaa aacgacgtaa
60acgtttacca tatttttttt cagatgtctc tttggctgcc tagtgaggcc actgtctact
120tgcctcctgt cccagtatct aaggttgtaa gcacggatga atatgttgca
cgcacaaaca 180tatattatca tgcaggaaca tccagactac ttgcagttgg
acatccctat tttcctatta 240aaaaacctaa caataacaaa atattagttc
ctaaagtatc aggattacaa tacagggtat 300ttagaataca tttacctgac
cccaataagt ttggttttcc tgacacctca ttttataatc 360cagatacaca
gcggctggtt tgggcctgtg taggtgttga ggtaggtcgt ggtcagccat
420taggtgtggg cattagtggc catcctttat taaataaatt ggatgacaca
gaaaatgcta 480gtgcttatgc agcaaatgca ggtgtggata atagagaatg
tatatctatg gattacaaac 540aaacacaatt gtgtttaatt ggttgcaaac
cacctatagg ggaacactgg ggcaaaggat 600ccccatgtac caatgttgca
gtaaatccag gtgattgtcc accattagag ttaataaaca 660cagttattca
ggatggtgat atggttcata ctggctttgg tgctatggac tttactacat
720tacaggctaa caaaagtgaa gttccactgg atatttgtac atctatttgc
aaatatccag 780attatattaa aatggtgtca gaaccatatg gcgacagctt
atttttttat ttacgaaggg 840aacaaatgtt tgttagacat ttatttaata
gggctggtac tgttggtgaa aatgtaccag 900acgatttata cattaaaggc
tctgggtcta ctgcaaattt agccagttca aattattttc 960ctacacctag
tggttctatg gttacctctg atgcccaaat attcaataaa ccttattggt
1020tacaacgagc acagggccac aataatggca tttgttgggg taaccaacta
tttgttactg 1080ttgttgatac tacacgcagt acaaatatgt cattatgtgc
tgccatatct acttcagaaa 1140ctacatataa aaatactaac tttaaggagt
acctacgaca tggggaggaa tatgatttac 1200agtttatttt tcaactgtgc
aaaataacct taactgcaga cgttatgaca tacatacatt 1260ctatgaattc
cactattttg gaggactgga attttggtct acaacctccc ccaggaggca
1320cactagaaga tacttatagg tttgtaaccc aggcaattgc ttgtcaaaaa
catacacctc 1380cagcacctaa agaagatgat ccccttaaaa aatacacttt
ttgggaagta aatttaaagg 1440aaaagttttc tgcagaccta gatcagtttc
ctttaggacg caaattttta ctacaagcag 1500gattgaaggc caaaccaaaa
tttacattag gaaaacgaaa agctacaccc accacctcat 1560ctacctctac
aactgctaaa cgcaaaaaac gtaagctgta aa 160214531PRTpappilomavirus
14Met Gln Val Thr Phe Ile Tyr Ile Leu Val Ile Thr Cys Tyr Glu Asn1
5 10 15Asp Val Asn Val Tyr His Ile Phe Phe Gln Met Ser Leu Trp Leu
Pro 20 25 30Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys
Val Val 35 40 45Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr
His Ala Gly 50 55 60Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe
Pro Ile Lys Lys65 70 75 80Pro Asn Asn Asn Lys Ile Leu Val Pro Lys
Val Ser Gly Leu Gln Tyr 85 90 95Arg Val Phe Arg Ile His Leu Pro Asp
Pro Asn Lys Phe Gly Phe Pro 100 105 110Asp Thr Ser Phe Tyr Asn Pro
Asp Thr Gln Arg Leu Val Trp Ala Cys 115 120 125Val Gly Val Glu Val
Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser 130 135 140Gly His Pro
Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala145 150 155
160Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp
165 170 175Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro
Ile Gly 180 185 190Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val
Ala Val Asn Pro 195 200 205Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn
Thr Val Ile Gln Asp Gly 210 215 220Asp Met Val His Thr Gly Phe Gly
Ala Met Asp Phe Thr Thr Leu Gln225 230 235 240Ala Asn Lys Ser Glu
Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys 245 250 255Tyr Pro Asp
Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu 260 265 270Phe
Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn 275 280
285Arg Ala Gly Thr Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys
290 295 300Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe
Pro Thr305 310 315 320Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln
Ile Phe Asn Lys Pro 325 330 335Tyr Trp Leu Gln Arg Ala Gln Gly His
Asn Asn Gly Ile Cys Trp Gly 340 345 350Asn Gln Leu Phe Val Thr Val
Val Asp Thr Thr Arg Ser Thr Asn Met 355 360 365Ser Leu Cys Ala Ala
Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr 370 375 380Asn Phe Lys
Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe385 390 395
400Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr
405 410 415Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe
Gly Leu 420 425 430Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr
Arg Phe Val Thr 435 440 445Gln Ala Ile Ala Cys Gln Lys His Thr Pro
Pro Ala Pro Lys Glu Asp 450 455 460Asp Pro Leu Lys Lys Tyr Thr Phe
Trp Glu Val Asn Leu Lys Glu Lys465 470 475 480Phe Ser Ala Asp Leu
Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu 485 490 495Gln Ala Gly
Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys 500 505 510Ala
Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Lys 515 520
525Arg Lys Leu 5301534DNApappilomavirus 15ccgctcgaga tgcaggtgac
ttttatttac atcc 341634DNApappilomavirus 16cccaagcttt tacagcttac
gttttttgcg ttta 34171716DNApappilomavirus 17atgccgcggg gttctcatca
tcatcatcat catggtatgg ctagcatgac tggtggacag 60caaatgggtc gggatctgta
cgacgatgac gataaggatc gatggggatc cgagctcgag 120atgcaggtga
cttttattta catcctagtt attacatgtt acgaaaacga cgtaaacgtt
180taccatattt tttttcagat gtctctttgg ctgcctagtg aggccactgt
ctacttgcct 240cctgtcccag tatctaaggt tgtaagcacg gatgaatatg
ttgcacgcac aaacatatat 300tatcatgcag gaacatccag actacttgca
gttggacatc cctattttcc tattaaaaaa 360cctaacaata acaaaatatt
agttcctaaa gtatcaggat tacaatacag ggtatttaga 420atacatttac
ctgaccccaa taagtttggt tttcctgaca cctcatttta taatccagat
480acacagcggc tggtttgggc ctgtgtaggt gttgaggtag gtcgtggtca
gccattaggt 540gtgggcatta gtggccatcc tttattaaat aaattggatg
acacagaaaa tgctagtgct 600tatgcagcaa atgcaggtgt ggataataga
gaatgtatat ctatggatta caaacaaaca 660caattgtgtt taattggttg
caaaccacct ataggggaac actggggcaa aggatcccca 720tgtaccaatg
ttgcagtaaa tccaggtgat tgtccaccat tagagttaat aaacacagtt
780attcaggatg gtgatatggt tcatactggc tttggtgcta tggactttac
tacattacag 840gctaacaaaa gtgaagttcc actggatatt tgtacatcta
tttgcaaata tccagattat 900attaaaatgg tgtcagaacc atatggcgac
agcttatttt tttatttacg aagggaacaa 960atgtttgtta gacatttatt
taatagggct ggtactgttg gtgaaaatgt accagacgat 1020ttatacatta
aaggctctgg gtctactgca aatttagcca gttcaaatta ttttcctaca
1080cctagtggtt ctatggttac ctctgatgcc caaatattca ataaacctta
ttggttacaa 1140cgagcacagg gccacaataa tggcatttgt tggggtaacc
aactatttgt tactgttgtt 1200gatactacac gcagtacaaa tatgtcatta
tgtgctgcca tatctacttc agaaactaca 1260tataaaaata ctaactttaa
ggagtaccta cgacatgggg aggaatatga tttacagttt 1320atttttcaac
tgtgcaaaat aaccttaact gcagacgtta tgacatacat acattctatg
1380aattccacta ttttggagga ctggaatttt ggtctacaac ctcccccagg
aggcacacta 1440gaagatactt ataggtttgt aacccaggca attgcttgtc
aaaaacatac acctccagca 1500cctaaagaag atgatcccct taaaaaatac
actttttggg aagtaaattt aaaggaaaag 1560ttttctgcag acctagatca
gtttccttta ggacgcaaat ttttactaca agcaggattg 1620aaggccaaac
caaaatttac attaggaaaa cgaaaagcta cacccaccac ctcatctacc
1680tctacaactg ctaaacgcaa aaaacgtaag ctgtaa
171618571PRTpappilomavirus 18Met Pro Arg Gly Ser His His His His
His His Gly Met Ala Ser Met1 5 10 15Thr Gly Gly Gln Gln Met Gly Arg
Asp Leu Tyr Asp Asp Asp Asp Lys 20 25 30Asp Arg Trp Gly Ser Glu Leu
Glu Met Gln Val Thr Phe Ile Tyr Ile 35 40 45Leu Val Ile Thr Cys Tyr
Glu Asn Asp Val Asn Val Tyr His Ile Phe 50 55 60Phe Gln Met Ser Leu
Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro65 70 75 80Pro Val Pro
Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg 85 90 95Thr Asn
Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly 100 105
110His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val
115 120 125Pro Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His
Leu Pro 130 135 140Asp Pro Asn Lys Phe
Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp145 150 155 160Thr Gln
Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly 165 170
175Gln Pro Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu
180 185 190Asp Asp Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly
Val Asp 195 200 205Asn Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr
Gln Leu Cys Leu 210 215 220Ile Gly Cys Lys Pro Pro Ile Gly Glu His
Trp Gly Lys Gly Ser Pro225 230 235 240Cys Thr Asn Val Ala Val Asn
Pro Gly Asp Cys Pro Pro Leu Glu Leu 245 250 255Ile Asn Thr Val Ile
Gln Asp Gly Asp Met Val His Thr Gly Phe Gly 260 265 270Ala Met Asp
Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu 275 280 285Asp
Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val 290 295
300Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu
Gln305 310 315 320Met Phe Val Arg His Leu Phe Asn Arg Ala Gly Thr
Val Gly Glu Asn 325 330 335Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser
Gly Ser Thr Ala Asn Leu 340 345 350Ala Ser Ser Asn Tyr Phe Pro Thr
Pro Ser Gly Ser Met Val Thr Ser 355 360 365Asp Ala Gln Ile Phe Asn
Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly 370 375 380His Asn Asn Gly
Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val385 390 395 400Asp
Thr Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr 405 410
415Ser Glu Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His
420 425 430Gly Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys
Ile Thr 435 440 445Leu Thr Ala Asp Val Met Thr Tyr Ile His Ser Met
Asn Ser Thr Ile 450 455 460Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro
Pro Pro Gly Gly Thr Leu465 470 475 480Glu Asp Thr Tyr Arg Phe Val
Thr Gln Ala Ile Ala Cys Gln Lys His 485 490 495Thr Pro Pro Ala Pro
Lys Glu Asp Asp Pro Leu Lys Lys Tyr Thr Phe 500 505 510Trp Glu Val
Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe 515 520 525Pro
Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro 530 535
540Lys Phe Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser
Thr545 550 555 560Ser Thr Thr Ala Lys Arg Lys Lys Arg Lys Leu 565
57019322PRTpappilomavirus 19Met Ala Ser Met Thr Gly Gly Gln Gln Met
Gly Arg Gly Ser Gln Val1 5 10 15Thr Phe Ile Tyr Ile Leu Val Ile Thr
Cys Tyr Glu Asn Asp Val Asn 20 25 30Val Tyr His Ile Phe Phe Gln Met
Ser Leu Trp Leu Pro Ser Glu Ala 35 40 45Thr Val Tyr Leu Pro Pro Val
Pro Val Ser Lys Val Val Ser Thr Asp 50 55 60Glu Tyr Val Ala Arg Thr
Asn Ile Tyr Tyr His Ala Gly Thr Ser Arg65 70 75 80Leu Leu Ala Val
Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn 85 90 95Asn Lys Ile
Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val Phe 100 105 110Arg
Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser 115 120
125Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly Val
130 135 140Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly
His Pro145 150 155 160Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala
Ser Ala Tyr Ala Ala 165 170 175Asn Ala Gly Val Asp Asn Arg Glu Cys
Ile Ser Met Asp Tyr Lys Gln 180 185 190Thr Gln Leu Cys Leu Ile Gly
Cys Lys Pro Pro Ile Gly Glu His Trp 195 200 205Gly Lys Gly Ser Pro
Cys Thr Asn Val Ala Val Asn Pro Gly Asp Cys 210 215 220Pro Pro Leu
Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val225 230 235
240His Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln Ala Asn Lys
245 250 255Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr
Pro Asp 260 265 270Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser
Leu Phe Phe Tyr 275 280 285Leu Arg Arg Glu Gln Met Phe Val Arg His
Leu Phe Asn Arg Ala Gly 290 295 300Thr Val Gly Glu Asn Val Pro Asp
Asp Leu Val Glu His His His His305 310 315 320His
His2020DNApappilomavirusmisc_feature(3)..(3)n is a, c, g, or t
20gcncargghc ayaayaatgg 202123DNApappilomavirus 21gtdgtatcha
cmhcagtaac aaa 232264DNApappilomavirus 22cvcaggghca yaayaatggc
atttgttggg gtaaccaact atttgttact gttgtdgaya 60cyac
642320DNApappilomavirus 23gttactgcga cgtgaggtat
202420DNApappilomavirus 24gtttcaggac ccacaggagc
202520DNApappilomavirus 25caacggtttg ttgtattgct
2026181DNApappilomavirus 26gttactgcga cgtgaggtat atgactttgc
ttttcgggat ttatgcatag tatatagaga 60tgggaatcca tatgctgtat gtgataaatg
tttaaagttt tattctaaaa ttagtgagta 120tagacattat tgttatagtt
tgtatggaac aacattagaa cagcaataca acaaaccgtt 180g
18127286DNApappilomavirus 27gtttcaggac ccacaggagc gacccagaaa
gttaccacag ttatgcacag agctgcaaac 60aactatacat gatataatat tagaatgtgt
gtactgcaag caacagttac tgcgacgtga 120ggtatatgac tttgcttttc
gggatttatg catagtatat agagatggga atccatatgc 180tgtatgtgat
aaatgtttaa agttttattc taaaattagt gagtatagac attattgtta
240tagtttgtat ggaacaacat tagaacagca atacaacaaa ccgttg 286
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