U.S. patent number 4,777,239 [Application Number 06/884,184] was granted by the patent office on 1988-10-11 for diagnostic peptides of human papilloma virus.
This patent grant is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Joel M. Palefsky, Gary K. Schoolnik.
United States Patent |
4,777,239 |
Schoolnik , et al. |
October 11, 1988 |
Diagnostic peptides of human papilloma virus
Abstract
A series of seventeen synthetic peptides which are capable of
raising antibodies specific for certain desired human papilloma
virus (HPV) are useful in diagnosis and therapy of conditions
associated with HPV infection.
Inventors: |
Schoolnik; Gary K. (Palo Alto,
CA), Palefsky; Joel M. (Redwood City, CA) |
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University (Stanford, CA)
|
Family
ID: |
25384124 |
Appl.
No.: |
06/884,184 |
Filed: |
July 10, 1986 |
Current U.S.
Class: |
530/326; 530/327;
530/328; 530/387.9; 530/389.4; 530/389.7; 530/389.8; 530/391.3;
930/220; 930/DIG.811 |
Current CPC
Class: |
C07K
14/005 (20130101); C07K 16/084 (20130101); A61K
39/00 (20130101); C12N 2710/20022 (20130101) |
Current International
Class: |
C07K
14/005 (20060101); C07K 14/025 (20060101); C07K
16/08 (20060101); A61K 39/00 (20060101); C07K
007/06 (); C07K 007/08 (); C07K 015/14 () |
Field of
Search: |
;935/32
;530/324,326,327,328,387 ;435/236 ;514/12 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
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Biol. Abstr., vol. 79(1985), 104971. .
Biol. Abstr., vol. 69(1980), 6847. .
Chem. Abstr., vol. 78(1973), 111744. .
Chem. Abstr., vol. 102, (1985), 46249. .
Chem. Abstr., vol. 103 (1985), 123891. .
Chem. Abstr., vol. 104 (1986) 49602. .
Chem. Abstr., vol. 104 (1986) 180834. .
Chem. Abstr., vol. 82 (1975) 82646. .
Chem. Abstr., vol. 83 (1975) 39938. .
Chem. Abstr., vol. 87 (1977) 180411. .
Chem. Abstr., vol. 87 (1977) 147435. .
Chem. Abstr., vol. 94 (1981), 1141. .
Chem. Abstr., vol. 95 (1981), 148419. .
Chem. Abstr., vol. 99 (1983), 33750. .
Chem. Abstr., vol. 101, (1984), 73029. .
Biol. Abstr., vol. 68 (1979) 22851..
|
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Ciotti & Murashige, Irell &
Manella
Claims
We claim:
1. A peptide having, as an antigenic region, an amino acid sequence
selected from the group consisting of
(1) Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu (representing
residues 147-158 of E6 except Ser was substituted for Cys at
position 1);
(2) Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys,
repreenting residues 9-23 of E6;
(3) Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys,
representing residues 24-37 of E6;
(4) Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys,
representing residues 45-58 of E6;
(5) Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg, representing residues
127-136;
(6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala, representing
residues 40-50 of E7;
(7) Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met, representing residues 4-12
of E7;
(8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile-Asp-Gly, representing
residues 29-40 of E7;
(9) Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser, representing
residues 75-85 of E2;
(10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro, representing residues
210-219 of E2;
(11) His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly,
representing residues 218-230 of E2;
(12) Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val, representing
residues 339-350 of E2;
(13) Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro, representing
residues 48-58 of E4;
(14) Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro,
representing amino acids 20-34 of E4;
(15) Arg-Leu-Tyr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn, representing
amino acids 476-48 of E1; and
(16) Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys,
representing amino acids 218-230 of E2;
wherein all the foregoing are derived from Type 16; and
(17) Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys,
representing amino acids 6-17 of the E6 reading frame of Type
6B.
2. A peptide selected from the group consisting of
(1) Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu;
(2)
Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys;
(3) Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys;
(4) Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys;
(5) Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg;
(6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala;
(7) Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met;
(8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile-Asp-Gly;
(9) Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser;
(10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro;
(11) His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly;
(12) Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val;
(13) Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro;
(14)
Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro;
(15) Arg-Leu-Tyr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn;
(16) Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys;
(17) Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys.
3. The peptide of claim 2 selected from the group consisting of
peptides (1) and (5) through (15) which further contains a Cys
residue at its C-terminus.
4. The peptide of claim 1 conjugated to antigenically neutral
carrier protein.
5. The peptide of claim 1 conjugated to label.
6. Antibodies immunospecific for the peptide of claim 1.
7. The antibodies of claim 6 conjugated to label.
8. A process to prepare a peptide having a single antigenic region
characteristic of a specific type of HPV infection, which process
comprises:
selecting a region of the deduced amino acid sequence encoded by an
open reading frame of a subject HPV virus DNA which has low amino
acid sequence homology with the analogous region of other HPV
types,
subjecting said deduced amino acid sequence to analysis to
determine regions of at least eight amino acid residues containing
both reverse turns and regions of hydrophilicity, and
synthesizing a peptide containing a single antigenic determinant of
said at least eight amino acid residues corresponding to a region
containing at least one reverse turn and high hydrophilicity.
9. A process to prepare a peptide having a single antigenic region
characteristic of HPV infection regardless of specific type, which
process comprises:
selecting a region of the deduced amino acid sequence encoded by an
open reading frame of a subject HPV virus DNA which has high amino
acid sequence homology with the analogous region of other HPV
types,
subjecting said deduced amino acid sequence to analysis to
determine regions of at least eight amino acid residues containing
both reverse turns and regions of hydrophilicity, and
synthesizing a peptide containing a single antigenic determinant of
said at least eight amino acid residues corresponding to a region
containing at least one reverse turn and high hydrophilicity.
10. A peptide having a single antigenic region prepared by the
process of claim 8.
11. A peptide having a single antigenic region prepared by the
process of claim 9.
12. The peptide of claim 10 conjugated to antigenically neutral
carrier protein.
13. The peptide of claim 11 conjugated to antigenically neutral
carrier protein.
14. The peptide of claim 10 conjugated to label.
15. The peptide of claim 11 conjugated to label.
16. Antibodies immunospecific for the peptide of claim 10.
17. Antibodies immunospecific for the peptide of claim 11.
18. Antibodies of claim 16 conjugated to label.
19. Antibodies of claim 17 conjugated to label.
Description
TECHNICAL FIELD
The invention relates to vaccines and diagnostics relevant to human
papilloma virus (HPV) infection. In particular, synthetic peptides
corresponding to regions of putative peptides for types of HPV
which infect the genital region raise antibodies useful in
diagnosis and in protection against infection.
BACKGROUND ART
Human papilloma virus appears to be associated with the development
of cervical carcinoma, a malignant condition which appears to be
preceded by several stages of cervical intraepithelial neoplasia
(CIN). The association of HPV infection with CIN has long been
recognized (Meiseles, A., et al, Gynecol Oncol (1981) 12:
3111-3123; Crum, C. P., et al, ibid (1983) 15: 88-94; Syrjanen, K.
J., Obstet Gynecol Surv (1984) 39: 252-265). In fact, IgG reactive
with a group-specific papilloma virus antigen was detected in 93%
of women with cervical carcinoma and 60% of those with CIN, but not
in any control subjects (Baird, P. J., Lancet (1983) ii: 17-18),
and the presence of HPV DNA in these lesions has been recognized by
several groups.
There are approximately forty different types of HPV, which are
classified by DNA sequence homology using hybridization techniques.
Samples having more than 50% homology, as judged by hybridization,
are placed into the same type designation. The various types appear
to be rather tissue specific. HPV-6, HPV-11, HPV-16, HPV-18, and
HPV-31 appear to be associated with the genital tract; others
appear to be associated with warts or epidermal dysplasias in other
tissues. However, HPV-6 and HPV-11 are associated with condyloma
type lesions, while HPV-16, HPV-18 and HPV-31 are associated with
cervical intraepithelial neoplasia, including invasive
carcinoma.
The relationship of HPV infection to the development of CIN and
cervical carcinoma is unclear, however it has been postulated that
HPV acts as an initiator in cervical carcinogenesis and that
malignant transformation depends on interaction with other factors
(Zur Hausen, H., et al, Lancet (1982) ii: 1370). The incidence of
HPV infection appears to be increasing as shown by a 700% increase
in patient visits related to genital HPV infections in both males
and females between 1966 and 1981 (Center for Disease Control:
Nonreported Sexually Transmitted Diseases (1979) MMWR 28: 61) and
the presence of HPV in more than 3% of pap smears of women under 30
(Ferenczy, A., et al, Am J Surg Pathol (1981) 5: 661-670).
The nature of HPV-16 in particular, and papilloma viruses in
general has been well studied recently. HPV-16 is a member of the
Papova virus group and contains a 7904 bp double-stranded DNA
genome (Siedorf, K., et al, Virology (1985) 145: 181-185). The
capsid is 50 nm and contains 72 capsomers (Klug, A., J. Mol Biol
(1965) 11: 403-423). There are a number of subtypes of HPV-16 which
are isolates showing greater than 50% homology (Coggin, Cancer
Research (1979) 39: 545-546), but differences in restriction in
endonuclease patterns.
The DNAs of several papilloma viruses have been sequenced,
including several HPV types, bovine papilloma virus (BPV) and
cottontail rabbit papilloma virus (CRPV). All of these display
similar patterns of nucleotide sequence with respect to open
reading frames. The open reading frames can be functionally divided
into early region (E) and late regions (L); the E region is
postulated to encode proteins needed for replication and
transformation; and L region to encode the viral capsid proteins
(Danos, O., et al, J. Invest Derm (1984) 83: 7s-11s).
The detection of HPV in cervical samples has been different because
there is no tissue culture system capable of supporting virus
harvested from the tissue to be tested through its replication
cycle (Tichman, et al, J Invest Derm (1984) 83: 25-65). There is,
however, a recently reported in vitro transformation assay
(Yasumoto, S. J. Virol (1986) 57: 572-577). Tsurokawa, U. et al
Proct Nat'l Acad Sci (USA) (1986) 83: 2200-2203. It is believed
that because of analogy with the better studied BPV and CRPV
systems, the proteins encoded by several early open reading frames,
for example, E6, E5, E7 and E2 (see FIG. 1) are important in HPV
genital infections. However, no system for utilization of peptides
associated with these regions has been suggested either as an aid
to diagnosis or in the synthesis of a vaccine.
DISCLOSURE OF THE INVENTION
The present invention provides peptides selected on the basis of
predicted secondary structure and hydrophilicity from proteins or
peptides encoded by selected open reading frames. The secondary
structure and hydrophilicity are deduced from the amino acid
sequence of these proteins according to methods disclosed by Hopp,
T., et al, Proc Natl Acad Sci (USA) (1981) 78: 3824; Levitt, M., J
Mol Biol (1976) 104: 59; and Chou, P., et al, Biochem (1974) 13:
211. The results of these deductions permit the construction of
peptides which elicit antibodies reactive with the entire protein,
as is further described below. Two general types of such antigenic
peptides are prepared. Peptide regions identified as being specific
to HPV-16 or other HPV type-specific determinants by lack of
homology with other HPV types lead to the peptides which are useful
to raise antibodies for diagnostic, protective, and therapeutic
purposes against HPV-16 or other virus type per se. Peptide regions
which are homologous among the various types of HPV of interest are
useful as broad spectrum diagnostics an vaccines, and elicit
antibodies that are broad spectrum diagnostics.
Accordingly, in one aspect, the invention is directed to peptides
of the following sequences deduced from the noted regions of
HPV-16:
(1) Ser-Arg-Ser-Ser-Arg-Thr-Arg-Arg-Glu-Thr-Gln-Leu (representing
residues 147-158 of E6 except Ser was substituted for Cys at
position 1);
(2) Phe-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys,
representing residues 9-23 of E6;
(3) Thr-Glu-Leu-Gln-Thr-Thr-Ile-His-Asp-Ile-Ile-Leu-Glu-Cys,
representing residues 24-37 of E6;
(4) Leu-Arg-Arg-Glu-Val-Tyr-Asp-Phe-Ala-Phe-Arg-Asp-Leu-Cys,
representing residues 45-58 of E6;
(5) Asp-Lys-Lys-Gln-Arg-Phe-His-Asn-Ile-Arg, representing residues
127-136;
(6) Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala, representing
residues 40-50 of E7;
(7) Asp-Thr-Pro-Thr-Leu-His-Glu-Tyr-Met, representing residues 4-12
of E7;
(8) Asn-Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu-Ile-Asp-Gly, representing
residues 29-40 of E7;
(9) Leu-Gln-Leu-Thr-Leu-Glu-Thr-Ile-Tyr-Asn-Ser, representing
residues of 75-85 of E2;
(10) Ile-Ile-Arg-Gln-His-Leu-Ala-Asn-His-Pro, representing residues
210-219 of E2;
(11) His-Pro-Ala-Ala-Thr-His-Thr-Lys-Ala-Val-Ala-Leu-Gly,
representing residues 218-230 of E2;
(12) Ser-Glu-Trp-Gln-Arg-Asp-Gln-Phe-Leu-Ser-Gln-Val, representing
residues 339-350 of E2;
(13) Asp-Gln-Asp-Gln-Ser-Gln-Thr-Pro-Glu-Thr-Pro, representing
residues 48-58 of E4;
(14) Gly-Ser-Thr-Trp-Pro-Thr-Thr-Pro-Pro-Arg-Pro-Ile-Pro-Lys-Pro,
representing amino acids 20-34 of E4;
(15) Arg-Leu-Thr-Leu-His-Glu-Asp-Glu-Asp-Lys-Glu-Asn, representing
amino acids 476-487 of E1; and
(16) Ala-Pro-Ile-Leu-Thr-Ala-Phe-Asn-Ser-Ser-His-Lys-Gly-Cys,
representing amino acids 218-230 of E2;
wherein all the foregoing are derived from Type 16; and
(17) Glu-Ser-Ala-Asn-Ala-Ser-Thr-Ser-Ala-Thr-Thr-Ile-Cys,
representing amino acids 6-17 of the E6 reading frame of Type
6B.
Each of the foregoing peptides, designated herein peptide 1,
peptide 2, etc, i.e. peptides 1-17, may, if it does not already
have this residue, be prepared with an additional C-terminal
cysteine for ease in conjugation to a neutral carrier, and in
another aspect, the invention relates to peptides containing this
additional cysteine.
In addition, the invention relates to a method for synthesizing
peptides useful in the various aspects of the invention, which
method comprises preparing an analysis of the secondary structure
and hydrophilicity of peptides encoded by the open reading frames
of the DNA corresponding to HPV viral types associated with genital
infection, and selecting regions of secondary structure
corresponding to areas on the surface capable of eliciting
antibodies reactive with the entire protein. The selected peptide
regions are then prepared synthetically using either solid phase
synthesis or other suitable techniques. The invention also relates
to peptides prepared using this method. The peptides thus prepared
can be designed as specific to a particular virus type or may be
capable of raising antibodies cross-reacting against the range of
HPV associated with genital infection depending on the use desired.
Like the specific peptides numbered 1-17 above, these may also be
prepared with a C-terminal cysteine for ease in conjugation.
In further aspects, the invention relates to the foregoing peptides
conjugated to carriers capable of conferring immunogenicity on
these peptides, to antisera raised against these peptides and the
antibodies contained in these sera, to methods of diagnosing the
presence of HPV in tissue utilizing these antisera, to methods of
detecting anti-HPV antibodies using the peptides per se, and to
kits useful in such assays.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the distribution of open reading frames in various
papilloma viruses.
FIG. 2 shows comparison of the amino acid sequences in the E2 open
reading frame for various human papilloma viruses.
FIG. 3 shows comparison of the amino acid sequences in the E6 open
reading frame for various human papilloma viruses.
FIG. 4 shows comparison of the amino acid sequences in the E7 open
reading frame for various human papilloma viruses.
FIG. 5 shows comparison of the amino acid sequences in the L1 open
reading frame for various human papilloma viruses.
FIG. 6 shows comparison of the amino acid sequences in the L2 open
reading frame for various human papilloma viruses.
FIG. 7 shows a typical analysis plot using the referenced methods
to ascertain secondary structure in hydrophilicity.
FIG. 8 shows the staining of a cervical biopsy at stage CIN2 using
immunoperoxidase staining mediated by the antibodies of the
invention:
FIG. 8A shows the section stained with immune serum preabsorbed
with the immunizing peptide. FIG. 8B shows the same section stained
with the immune serum, and FIG. 8C shows a magnification of the
stain in FIG. 8B.
FIG. 9 shows the ability of the immunoperoxidase stain mediated by
antibody of the invention to detect a single malignant cell.
MODES OF CARRYING OUT THE INVENTION
The peptides of the invention are used to raise antibodies, either
in subjects for which protection against infection by HPV is
desired, i.e. as vaccines or to heighten the immune response to an
HPV infection already present. They are also injected into
production species to obtain antisera useful in diagnosis. In lieu
of the polyclonal antisera obtained in the production subjects,
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 for injected
animals to obtain antibody-producing clones.
In any event, the polyclonal or monoclonal antibodies obtained are
useful for diagnosis of HPV infection in cervical biopsies or pap
smears 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 the virus or to detect the virus in
metastases of infected tissue, as well as to monitor the progress
of therapy with antiviral or other therapeutic agents directed to
control of the infection or carcinoma. The antibodies, if corrected
for species variations can also be used as passive therapy.
In a converse diagnosis, the peptides of the invention can be used
in similar immunoassays to detect the presence of antibodies raised
against HPV in the serum of persons suspected of harboring HPV
infections or to titrate the sera of persons treated with an
anti-HPV vaccine. The use of synthetic peptides for this purpose
has the advantage of providing a relatively low cost and
reproducible reagent for use in these tests.
Direct administration of the proteins to a host can confer either
protective immunity against HPV or, if the subject is already
infected, a boost to the subject's own immune response to permit
more effective combat of the progress of the disease. For all
applications, the peptides are administered in immunogenic form.
Since the peptides are relatively short, this necessitates
conjugation with an immunogenicity conferring carrier material.
This carrier material should ideally be antigenically
neutral--i.e., ineffective in raising antibodies against itself.
Antigenic neutrality is, of course, an ideal state as many carriers
which are actually satisfactory do contain some antigenic regions
which are capable of raising antibodies in the host. However, this
may still be acceptable if the antigenic regions are in fact
different from those of the peptide of interest, which is quite
easy to achieve, or if the antibodies raised against the carrier
portions are harmless to the subject.
The peptides of the invention are designed for their in vitro
synthesis by choosing appropriate regions of the protein encoded by
the open reading frames of the HPV types of interest. Regions are
chosen for their immunogenic capability and, depending on the use
required, for their ability to serve as type specific or broad
range vaccines and diagnostics.
Once designed, the peptides of the invention are prepared by any
convenient means, commonly by chemical peptide synthesis using
solid phase techniques. For conjunction to carrier protein, it is
convenient to synthesize these peptides with an additional cysteine
residue, for example, at the C-terminus to provide a sulfhydryl
group for convenient linkage. Depending on the nature of the
linkers used, however, other approaches to form the conjugates are
possible. The conjugated peptides are then administered to subject
animals.
In the peptides are to be administered as vaccines, they are
formulated according to conventional methods for such
administration to the subject to be protected. If they are to be
used directly, as diagnostic reagents, they are purified and
packaged for such use. If they are to be used to produce antibodies
for diagnostic purposes, convenient test animals can be used to
prepare the appropriate antisera, and these antisera used directly.
Suitable hosts include mice, rats, rabbits, guinea pigs, or even
larger mammals such as sheep. For administration to such animals,
the peptides linked to carrier are generally administered in the
presence of an adjuvant, usually Freund's complete adjuvant, and
the polyclonal sera are harvested periodically by standard
techniques.
If the antibodies are to be used for therapeutic purposes, it is
generally desirable to confer species characteristics upon them
compatible with the subject to be treated. Accordingly, it is often
desirable to prepare these antibodies in monoclonal form since
fusion with suitable partners is capable of conferring the desired
characteristics on the secreted monoclonals.
These matters are set forth in greater detail below.
Selection of Peptides
At least five HPV types are known to be associated with genital
infection, and additional types may be isolated in the future, as
it is not certain that all existing types have in fact been
detected, and it is expected that mutation will result in
appearance of previously non-existent forms. Of the five types now
known, HPV-6 and HPV-11 are associated with benign conditions,
while HPV-16, HPV-18 and HPV-31 appear to be associated with
malignant transformation. All five of these types, and presumably
those still to be found, exhibit similar organization of their DNA,
so that all contain, for example, the open reading frames
associated with the putative early (E) proteins and late (L)
proteins. Depending on the use for which the peptide is intended,
appropriate regions of the early or late putative proteins are
selected.
The early proteins, as they are thought to be associated with viral
replication and transformation, are particularly useful as portions
of immunogens administered to production animals to generate
antibodies needed for diagnosis of the progress of HPV infection.
They are also useful as vaccine components. Reagents associated
with the late proteins, as these appear to be associated with
capside proteins, are also useful as vaccines. They are also
capable of raising antibodies which are diagnostic for the presence
of free virus in the bloodstream and are themselves useful in
detecting antibodies raised against the whole virus. Therefore, the
first step in the analysis is to ascertain the stage of viral
infection which forms the subject matter for the utility.
The second decision point relates to whether the peptide is
designed for applications specific to a particular type or a broad
spectrum of genital infection-causing HPV. If detection of antigen
or antibody associated with a particular type is desired, it is
desirable to have a type-specific peptide as the basis for the
reagent or antiserum. On the other hand, if a vaccine protective
against HPV genital infections in general is desired, or if it is
satisfactory to determine the presence or absence of any HPV
genital infection, then a peptide associated with a broad spectrum
of these virions is desired.
To design species-specific vaccines, regions of the putative
proteins encoded by the open reading frames are selected which are
not homologous from strain to strain, but characteristic of the
particular type of interest. If a broad spectrum peptide is
required, reagents or homology are chosen. FIGS. 2-6 show
comparisons of amino acid sequences for various open reading frames
in several HPV types. These sequences show various regions of
homology for these proteins; for example, in the early protein
encoding region E2, the region between positions 109-119 is much
more homologous than the region between 139 and 148 of the
sequence; the region between residues 331 and 350 contains
considerable homology, whereas that between positions 241-260 does
not.
The third level of selection relates to secondary structure and
hydrophilicity. Regions are selected which are considered likely to
raise antibodies in the host which are cross-reactive against the
entire protein on the basis of the ability of this particular
subsection of the protein to mimic its own secondary structure in
the native state. Generally, if there is no additional information,
one is limited to the use of areas of conformational flexibility
such as the N-terminal and C-terminal regions because these regions
of the native protein are assumed to exhibit the same spectrum of
conformations as the synthesized peptide. A difficulty with this
approach, however, is that due to the multiplicity of
possibilities, antibody production against a desired conformation
may represent a small portion of the antibodies raised and the
process is relatively inefficient.
A superior approach is based on ascertaining regions of the protein
which are likely to have the same conformation in the intact
protein as is exhibited by the synthesized peptide. These are
believed to be regions having reversed or beta turns as the
formation of these turns is generally associated with the surface
of the protein, and is also dependent on the amino acid sequences
in close proximity with each other. Methods to ascertain the
locations of the desired beta turns are known in the art and
described in the paper of Chou and Fassman referenced above. FIG. 7
shows an illustrative plot obtained accordng to these procedures
showing the regions of alpha helixes, beta sheets and reverse turns
for the protein encoded by the E7 open reading frame of HPV-16. The
results also provide data as to regions of hydrophobicity and
hydrophilicity which are significant in the formation of these beta
turns. Thus, using FIG. 7 as an illustration, those regions
considered likely to exhibit the desired immunogenicity are
illustrated as peptides 1-5. In selecting these peptide regions,
information is obtained from the plot which shows the presence of
beta turns and associated regions of hydrophilicity. This analysis
can, of course, be done with respect to the peptides encoded in the
open reading frames of any of the desired HPV types.
When the appropriate region is chosen by this analysis, peptides of
8-15 amino acid residues which include this region are synthesized
as described below. It is believed that at least 8 amino acids are
needed to provide sufficient sequence; peptides longer than 15
amino acids could be synthesized, but at a sacrifice of
economy.
Peptide Synthesis
As used herein, "peptide", "polypeptide", and "protein" are used
interchangeably, and refer to amino acid sequences of a variety of
lengths, either in their neutral (uncharged) forms or in forms
which are salts, and either free of modifications such as
glycosylation, side chain oxidation, or phosphorylation or
containing these modifications. It is well understood in the art
that amino acid sequences contain acidic and basic groups, and that
the particular ionization state exhibited by the peptide is
dependent on the pH of the surrounding medium when the protein is
in solution, or that of the medium from which it was obtained if
the protein is in solid form. Also included in the definition are
proteins modified by additional substituents attached to the amino
acid side chains, such as glycosyl units, lipids, or inorganic ions
such as phosphates, as well as modifications relating to chemical
conversions of the chains, such as oxidation of sulfhydryl groups.
Thus, "peptide" or its equivalent terms is intended to include the
appropriate amino acid sequence referenced, subject to those of the
foregoing modifications which do not destroy its immunospecific
antigenic properties.
All of the peptides of the invention are sufficiently short that
chemical synthesis, using methods now standard in the art, is
feasible. A review of such methods is given by, for example,
Margolin, A., et al, Ann Rev Biochem (1970) 39: 481. In most of
these procedures, the C-terminal amino acid is bound to a solid
support, and reacted with the next amino acid in sequence which has
been protected at the amino group to prevent self-condensation.
After the initial coupling, the NH.sub.2 protecting group is
removed, and the coupling process repeated with the amino acid next
in order. Polypeptides of considerable chain length have been
synthesized in this way. The only requirement is that the amino
acid sequence desired to be produced is known.
Recombinant DNA methodology provides an alternative way of
synthesizing the desired peptides. The DNA coding sequence for the
desired peptide or protein is ligated into an expression vector
suitable for transforming a recipient cell, which is thus caused to
express the gene and produce the protein. The DNA coding sequences
are sufficiently short to be prepared synthetically using means
known in the art; see, e.g., Edge, M. P., et al, Nature (1981) 292:
756.
The coding sequence is placed under the control of control
sequences compatible with recombinant hosts in plasmids containing
convenient restriction sites for insertion of the desired coding
sequence. For example, for bacterial hosts typical of such plasmids
are pUC8, and pUC13 available from Messing, J., at the University
of Minnesota; (see, e.g., Messing, et al, Nucleic Acids Res (1981)
9: 309) or pBR322, available from New England Biolabs. Suitable
promoters include, for example, the .beta.-lactamase
(penicillinase) and lactose (lac) promoter systems (Chang, et al,
Nature (1977) 198: 1056 and the tryptophan (trp) promoter system
(Goeddel, D., et al, Nucleic Acids Res (1980) 8: 4057). The
resulting bacterial expression vectors are transformed into
suitable bacterial hosts using the calcium chloride method
described by Cohen, S. N., et al, Proc Natl Acad Sci (USA) (1972)
69: 2110, and the transformants selected and cultured.
Alternatively, these peptides can be produced in nonbacterial
recombinant hosts using appropriate control sequences, vectors and
transformation techniques.
Synthesis of Conjugates
Because the peptide sequences of the invention are considered too
small to be immunogenic, they are linked to carrier substances in
order to confer this property upon them. The carrier substances
should be antigenically neutral in the subject generating the
antisera.
By "substantially antigenically neutral carrier" is meant a
material to which the peptides of the invention may be attached to
render them immunogenic, but which does not itself elicit
antibodies which will be detrimental to the host, or contain
antigenic sites which interfere with the antigenic function of the
invention peptides. For example, in subjects which are not beef
eaters, such as rabbits or mice, bovine serum albumin (BSA) could
be used. For human use, however, carriers are limited to proteins
which do not raise antibodies to materials commonly and
nonpathogenically encountered by humans. For example, human serum
albumin (HSA) or tetanus toxoid protein could be used.
The conjugates can be formed in a variety of ways. For example,
there are a large number of heterobifunctional agents which
generate a disulfide link at one functional end group and a peptide
link at the other, and these have been used extensively. The most
popular of these is N-succidimidyl-3-(2-pyridyl dithio) propionate
(SPDP). This reagent creates a disulfide linkage between itself and
a cysteine residue in one protein and an amide linkage through the
amino on a lysine, or other free amino group in the other. A
variety of such disulfide/amide forming agents are known. See, for
example, Immun Rev (1982) 62: 185. Other bifunctional coupling
agents form a thioether rather than a disulfide linkage. Many of
these thioether forming agents are commercially available and
include reactive esters of 6-maleimidocaproic acid, 2 bromoacetic
acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)
cyclohexane-1-carboxylic acid and the like. The carboxyl groups can
be activated by combining them with succinimide or
1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. A particularly
preferred coupling agent for the method of this invention is
succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC)
obtained from Pierce Company, Rockford, IL.
The foregoing list is not meant to be exhaustive, and modifications
of the named compounds can clearly be used. However, if a disulfide
or thioether linkage to the peptide is to be employed, and the
peptide contains no convenient cysteine, an additional cysteine
residue at either terminus can be added when the peptide is
prepared. As only shorter peptides require conjugation to carrier,
these residues can be included conveniently during chemical
synthesis.
Vaccine Preparation
Preparation of vaccines which contain peptide sequences as active
ingredients is well understood in the art. Typically, such vaccines
are prepared as injectables, either as liquid solutions or
suspension; solid forms suitable for solution or suspension in
liquid prior to injection may also be prepared. The preparation may
also be emulsified. The active immunogenic ingredient is often
mixed with excipients which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof. In addition, if desired, the vaccine may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants which enhance
the effectiveness of the vaccine. The vaccines are conventionally
administered parenterally, by injection, for example, either
subcutaneously or intramuscularly. Additional formulations which
are suitable for other modes of administration include
suppositories and, in some cases, oral formulations.
The vaccines are administered in a manner compatible with the
dosage formulation, and in such amount as will be therapeutically
effective and immunogenic. The quantity to be administered depends
on the subject to be treated, capacity of the subject's immune
system to synthesize antibodies, and the degree of protection
desired. Precise amounts of active ingredient required to be
administered depend on the judgment of the practitioner and are
peculiar to each individual. However, suitable dosage ranges for
subcutaneous or muscular injection are of the order to 50-500 .mu.g
active ingredient per individual. Suitable regimes for initial
administration and booster shots are also variable, but are
typified by an initial administration followed in one-two week
intervals by a subsequent injection or other administration.
Preparation of Antisera
For preparation of diagnostic antisera, hyperimmune sera can be
prepared, for example, in rabbits using standard immunization
techniques comprising an initial injection and boosting. The sera
can be periodically tritrated in a solid phase assay against the
immunogenic peptide. For preparation of monoclonal panels, the
immunized animals are sacrificed and spleen cells harvested for
immortalization to obtain cells capable of producing antibodies.
The supernatants of successful immortalized cells are screened for
the production of antibodies reactive with the injected peptide. If
therapeutic uses are to be made of the monoclonals, the preparation
of antisera and immortalization may involve techniques capable of
conferring suitable species characteristics on the secreted
antibodies. Techniques to obtain antibodies with human
characteristics are summarized and disclosed in Teng, N. N. H., at
al. in Human Hybridomas and Monoclonal Antibodies (1985), Engleman,
E., et al. Ed., Plenum Press, p. 71.
Diagnostics
The prepared antisera can be used in a variety of diagnostic
assays. All of these are, of course, immunoassays and the design of
such assays is of tremendous variety. Perhaps most convenient are
solid phase supported immunoassays which employ "sandwiches" in
which at least one layer is comprised of the antibodies of the
invention and another layer is comprised of sample. In one possible
embodiment, the antisera prepared according to the method of the
invention are to coat a solid support, such as, for example, a
microtiter plate to provide a surface specifically reactive with
the HPV components of the sample to be tested. The plates are then
treated with sample, and then with an additional antibody also
reactive with the desired HPV antigen. The third layer may itself
contain a radioactive, fluorescent, or enzymic label or may be
reactive with still another layer containing such labels. The
variety of protocols for such assays is well understood in the art,
and any suitable protocol is acceptable.
With regard to the indications for which the antibodies of the
invention are useful in diagnosis, the most commonly encountered
use is clearly the diagnosis of the presence or absence of HPV-16
or other relevant HPV either in a pap smear or in cervical biopsy
or in biopsies of other tissue. The presence of HPV-16,
specifically, and also of HPV-18 or HPV-31, in a cervical biopsy or
pap smear is indicative of a high risk for malignant transformation
resulting in cervical carcinoma.
The progress of HPV infection to result in the malignant state is
believed to pass through three CIN stages, commonly labeled CIN1,
CIN2, and CIN3 before finally converting to a malignant transformed
carcinoma. The various stages may be associated with expression of
different genes of the HPV virus with resulting changes in the
level of differentiation of the infected tissue. Therefore, an
objective distinction between CIN3 and CIN2 can be made using these
antisera, since the differentiated state associated with CIN3 fails
to produce a positive response against the antisera raised against
the peptides 1-4 of the invention, while that associated with CIN2
does do so. Thus, if the morphological characteristics as to the
stage of development of the lesion are unclear, these two stages
can be distinguished by virtue of their reactivity with the
antisera.
In addition, the antisera are useful in detecting the HPV virus in
blood or serum, thus providing a basis for antigen detection in
these fluids, or in foreign tissues which would indicate the
presence of metastases from an original site. The results of
treatment of carcinoma associated with HPV infection can also be
followed by assessing the levels of species reactive with these
antisera in serum or other tissues. Thus, if the disease is
treated, for example, by administration of interferon, the effect
of this drug can be monitored by tracking the levels of HPV
antigens in serum using the antibodies in this assay.
Conversely, the peptides of the invention can be used in
immunoassays to test for the presence or absence of anti-HPV
antibodies in serum. The protocol for such assays is similar to
that associated with the use of antisera to test for the presence
or absence of antigen, except that the sample and reagent are
interchanged. These proteins are a convenient source of pure HPV
material which thereby permit assays with a minimum of cross
reaction.
Therapeutic Uses
The antibodies can also be used therapeutically so long as they are
compatible with the host to be treated. Monoclonal preparations
having the proper species characteristics are most suitable for
this application of the invention which is conducted by injecting
into a person already suffering from HPV infection sufficient
antibody preparation to combat the progress of the infection.
As noted above, the peptides themselves, in addition to being used
as vaccines in advance of infection for protection against the
disease, can also be used as an immunomodulation agent when the
subject is already infected. By supplying additional immunogenic
peptides, the antibody response mounted by the patient is
fortified, and is thus more effective in disease control.
Kits
The diagnostic antibodies or peptides of the invention can
conveniently be packaged into kits, for the conduct of the
immunoassays in which they are the essential reagent. The
components of such kits include, typically, the peptide or antibody
preparation of the invention, labeled antibody or other protein to
permit detection, and optionally, suitable supports and containers
for conducting the protocols described. In addition, such kits will
contain instructions for utilizing the peptides or antisera of the
invention in the context of the samples to be tested and the
equipment and reagents provided.
EXAMPLES
The following examples are intended to illustrate but not to limit
the invention.
EXAMPLE 1
Preparation of Peptides
Peptides 1-17, further containing a C-terminal cysteine residue are
prepared by the solid phase techniques disclosed in Chirgwin, J.
M., et al, Biochem (1979) 18: 5294 using t-Boc protected amino
acids and amino acid derivatized polystyrene resin, supplied by
Peninsula Laboratories Inc., Belmont, CA. Asp, Glu, Thr, and Ser
side chains are protected as O-benzyl esters; Arg and His side
chains are protected with tosyl groups; Cys is protected with
p-methoxybenzyl; Lys by O-chlorobenzyloxycarbonyl, and Tyr by
2,6-dichloro benzyl. The couplings are performed with 2.5-3 molar
excess of t-Boc amino acid and dicyclohexylcarbodiamide (DCC). For
couplings of Asn or Gln, a 2.5-fold molar excess of
N-hydroxytriazole is also included. Coupling is monitored with
ninhydrin and coupling is continued until 99% efficiency is
obtained.
After synthesis of the desired chain, protecting groups and resin
are cleaved simultaneously by anhydrous hydrogen fluoride in the
presence of dimethyl sulfoxide and anisol. The cleaved peptide is
extracted with ether, isolated from the resin by extraction with 5%
acetic acid, and lyophilized several times. The purity of the final
product is determined by reverse phase HPLC on Licosorb RP18 (Merck
Dormstat FRG) and by amino acid analysis.
EXAMPLE 2
Conjugation to Carrier Protein
Each of the peptides 1-7, synthesized in Example 1 is conjugated to
thyroglobin or to bovine serum albumin. In either case, 10 mg of
the carrier protein are dissolved in 3 ml PBS, pH 7.4, and mixed
with 1 ml distilled DMF containing 5 mg of the cross-linker
m-malenimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) for
thyroglobin and succinimidyl 4-(N-malenimido
methyl)cyclohexane-1-carboxylate (SMCC) for bovine serum
albumin.
In the meantime, each of the peptides 1-17 is reduced with sodium
borohydride for 15 min on ice to ensure the presence of a free
sulfhydryl. After destroying excess borohydride with HCl, the
neutralized and reduced peptide is combined with the carrier
cross-linker conjugate and stirred overnight at room temperature.
The resulting peptide carrier conjugate is isolated by gel
filtration on Sephadex G25 in 0.1M ammonium bicarbonate buffer, pH
7.5, and the molar ratio of peptides to carrier determined in the
product.
EXAMPLE 3
Preparation of Antisera
Hyperimmune sera are prepared in female New Zealand white rabbits
using the conjugates prepared in Example 2. Two rabbits are
immunized for each of the peptides 1-17 carrier conjugates. Five
hundred .mu.g of thyroglobin-MBS-peptide conjugate are emulsified
in Freund's adjuvant and injected subcutaneously and
intramuscularly at multiple cites. The rabbits are boosted at six
weeks with the same immunogen emulsified with incomplete Freund's
adjuvant, and one week later the animals are bled by cardiac
puncture. The animals are boosted one week later, bled two weeks
later, boosted a third time three weeks later and bled a third time
four weeks after the initial cardiac puncture. The antisera are
then evaluated in the microtiter solid phase binding assay using
peptide-SMCC-BSA conjugate as antigen.
EXAMPLE 4
Titration of Antisera
Wells of disposable polystyrene U microtiter plates are coated with
peptide-BSA conjugate in 0.1M sodium carbonate buffer, pH 9.6 for
12 hr at room temperature. The wells are then washed three times
with NaCl-brij. Serially diluted antisera are added in PBS-brij-BSA
and incubated at 37.degree. C. for 2 hr and washed. Approximately
20,000 cpm of .sup.125 I-protein A in PBS-BSA-brig is added to each
well and incubated at 37.degree. C. for 1 hr and washed. The wells
are assayed for radioactivity in a gamma scintillation counter,
negative controls are nonsensitized wells, wells which are not
exposed to antisera and wells exposed to preimmune sera. Each
sample is performed in triplicate and recorded as the mean.
EXAMPLE 5
Use of Antisera in Immunoassays
Four of the antisera prepared in Example 4 are used to assess
cervical swabs and biopsies for the presence of HPV virus in
correlation to various CIN stages and malignant cervical carcinoma.
Samples are obtained from patients referred to dysplasia clinics
for history of abnormal pap smears. Cervical biopsy material is
frozen and stored.
To conduct the assays, several protocols may be employed, including
immunoperoxidase staining of pap smears and cervical biopsies, dot
immunobinding of cervical smears, Western blot, and
immunoprecipitation.
For cervical swabs immunobinding on nitrocellulose paper is
conveniently employed. Cervical swabs are spotted onto a 1 cm.sup.2
section of nitrocellulose paper, pore size 0.2 .mu.m as described
by Jahn, et al, Proc Natl Acad Sci (USA) (1984) 81: 1684-1687. The
paper is air dried and fixed for 15 min in a 10% v/v acetic acid,
25% v/v isoproponyl solution, rinsed, preincubated for 5 min in
Tris-buffered saline and incubated in blocking buffer consisting of
Tris-saline-5% BSA. The paper is then incubated with antiserum to
peptides 1, 2, 3 and 4 above in Tris-saline-BSA-0.1% Triton X-100
for 2 hr, rinsed and immersed again in blocking buffer for 30 min.
The paper is then incubated in 300,000 cpm of .sup.125 I-protein A
in Tris-saline-BSA-Triton-X-100 for 1 hr at 37.degree. C., washed
and dried. The squares are cut out and assayed for radioactivity in
a gamma scintillation counter.
For biopsies, immunoperoxidase staining is most convenient. The
avidin/biotin method described by Hsu, S. M., et al. Advances in
Immunohistochemistry (1984) Medical Publishers, pp. 31-42 was
used.
The tissue sections are immersed in 3% hydrogen peroxide to block
endogenous peroxidase activity. The slides are then washed in
distilled water and PBS, and then immersed in 3% normal goat serum
(Vector Laboratories) to block nonspecific binding. The peptide
antisera are then added in dilutions ranging from 1:100 to 1:2000
and incubated overnight at 4.degree. C. The slides are rinsed in
PBS and incubated with goat anti-rabbit IgG, and then reacted with
avidin/biotin complex (ABC) reagent (Vector Laboratories) for a
minimum of 30 min. Labeling antibodies (ABC stain) is added for 30
min and the slides again rinsed in PBS. Chromogen reaction using
diaminobenzedine (DAB) solution (0.05% Tris pH 7.6, DAB, 30%
hydrogen peroxide) is then developed for 5 min, and the samples are
rinsed, counterstained with hematoxylin and cover slipped. A
positive staining is indicated by a black-brown chromogenic
precipitate of DAB.
FIG. 8 shows the results of staining of a single biopsy which is
assessed as CIN 2 by pathology. FIG. 8A shows this section stained
using rabbit antiserum prepared against peptide 1, but preadsorbed
with the peptide by incubating the serum in the presence of excess
peptide 1. As FIG. 8A shows, no DAB precipitate is detectable; the
epithelial layer is clearly distinguishable from the stroma by
apparent cell morphology with clear definition of the basement
membrane. FIG. 8B is the same section stained using the same
antisera, but unadsorbed with peptide 1. The epithelium clearly
shows the presence of the CIN2 characteristics in this tissue. The
HPV antigen is associated with this stage as demonstrated by the
brown stain apparent in the photograph. The antigen does not appear
in the stroma, since penetration of the basement membrane has not
yet occurred in CIN2. FIG. 8C is a magnification of the stained
area at the surface of the epithelium shown in FIG. 8B.
FIG. 9 shows the ability of the immunoperoxidase method of the
invention to detect the presence of a single malignant cell in the
stroma. FIG. 9 is a magnified view of stained tissue from a patient
where malignant transformation has already occurred and a
pathologic diagnosis of cervical carcinoma has already been made.
The cell at the center of the photograph is an isolated malignant
cell exhibiting the HPV antigens.
The results illustrated in FIGS. 8 and 9 were obtained in the
course of a study involving eleven patients as described above. In
these assays, controls were run to show that no immunoreaction
(brown stain) was ever observed using pre-immune serum or serum
previously incubated with the immunizing peptide. These controls
were run in all tests. Additional controls were also run with
antisera to peptide 1, using samples known to contain HPV-16,
HPV-18, HPV-6 or HPV-11. Only HPV-16 and 18 gave positive results
and higher color intensity was obtained with HPV-16; this cross
reactivity with HPV-18 shows that peptide 1 generates cross-type
reacting antisera. Normal cervical swabs were also used as controls
and no reaction was obtained using antisera obtained from any of
the four peptides tested.
However, patients having known dysplasias did show positive results
as set forth in Table 1 below. The results shown in Table 1
indicate that the test detects presence of the virus and tracks the
progress of tissue differentiation caused by the stepwise
expression of the viral genome.
TABLE 1 ______________________________________ Peptide # 1 2 3 4
______________________________________ 2 patients CIN1 HPV-16 DNA
(-) - - - - 6 patients CIN1 HPV-16 DNA (+) + + + + 4 patients CIN2
HPV-16 DNA (+) + + + + 2 patients CIN3 HPV-16 DNA (+) - - - - 1
patient SSC HPV-16 DNA (+) + - + + normal Adj CIN3 HPV-16 DNA (+) +
+ + + ______________________________________
The first two patients, listed in row 1 of Table 1, were assessed
as having CIN1 stage lesions by pathology. However, the absence of
HPV-16 virus in these patients (indicated by the (-) was determined
by Southern blot DNA hybridization performed on the smear. As
shown, these patients also tested negative for the presence of
HPV-16 using the antisera. On the other hand, the six patients also
showing CIN1 by pathology, but shown to harbor HPV-16 by Southern
blot DNA hybridization (labeled (+)), gave positive results with
the antisera. The four patients assessed as CIN2 gave clear
positive tests with the antisera for the presence of HPV.
However, two patients assessed as CIN3 were unreactive with all
sera presumably because of the differentiation state of the tissue
at this stage. The biopsy from one patient was assessed
independently, but the other CIN3 tissue was obtained adjacent to
one of the CIN2 samples in the preceding row. This confirms that
the test with antiserum makes possible an objective differentiation
between CIN2 and CIN3. This is sometimes difficult to ascertain by
visual inspection: i.e. the findings of a single pathologist will
generally be internally consistent, but there are frequently
differences between the assessments given by different
pathologists.
As shown in the next-to-bottom line of the table, a patient having
a known carcinoma was positive with respect to peptides 1, 3 and 4
but negative with respect to peptide 2, presumably indicating
alteration in genomic integration, transcription, or
post-transcription events which may occur when the cells become
malignant. The last line of the table gives the results for normal
tissue obtained from a patient who also had CIN3 tissue which had
tested negative against all antisera, but which CIN3 tissue had
been shown to contain HPV-16 DNA by Southern blot. The positive
results indicate that latent virus, and even gene expression
products remain in the normal cells when adjacent tissues are in
later stages of the infection.
* * * * *