1. Field of Invention
The present invention relates to the field of the specific peptides reactive with antibodies formed against oncoproteins of the human papillomavirus (HPV). More particularly, the invention relates to peptides isolated, purified or derived from the sequences of the early coding regions of the E2, E4, E6, and E7 oncoproteins and late coding regions (L1 capsid proteins) of HPV; Harvey Ras (H-Ras), Kirsten Ras (K-Ras), and phosphatase and tensin homologue (PTEN) tumor suppressor proteins; and Chlamydia trachomatis heat shock protein 60 (CHSP60 groEL1), and the method for use for the detection and/or diagnosis of cervical epithelia cell abnormalities, precancerous clinical conditions and cervical carcinomas associated with HPV, tumor suppressor H-Ras, K-Ras, and PTEN, and CHSP60 groEL1, mutated or deleted peptides, via an immunoassay.
2. Background of the Invention
Cervical cancer is second only to breast cancer as the most frequent female malignancy and cause of death worldwide. International Agency for Research on Cancer (IARC) has estimated that in the year 2000 there was approximately 500,000 cases of cervical cancer worldwide. In the United States there are about 130,000 new cases of invasive cervical cancer each year and >4,000 deaths. Almost 80% of new cases occur among women in developing countries, with 80-85% of them appearing at late, incurable stages making incidence equivalent to mortality. Comparable worldwide figures are approximately 10 million cases of high grade dysplasia, 30 million cases of low grade dysplasia, and 300 million cases of cervical infection without cytologic abnormalities. This disease is due to infections by certain human papillomaviruses (HPV), as are many head and neck cancers as well as penile, urethral, anal and possibly some bladder cancers. On the basis of the strength of their association with cervical cancer, genital HPVs are placed into different risk categories as follows, high risk: HPV-16, 18, 31, 45 (each found in at least 5% of invasive cancers); intermediate risk: HPV-33, 35, 39, 51, 52, 56, 58, 59, 68 (each found in between 1% and 5% of invasive cancer); low risk: HPV-6, 11 and many others (rarely found in invasive cancer, but associated with genital condyloma). Another consequence is laryngeal papillomatosis in babies born to HPV-infected women, a pathology that kills 25,000 children per year. Such dire consequences of unmonitored HPV infections are a priority issue of major medical entities with a worldwide perspective like the World Health Organization, the Program for Applied Technology in Health, and the Johns Hopkins Program for International Education in Reproductive Health (JHPIEGO).
Cervical cancer is relatively common in the People's Republic of China, but little research has been published in the West about the relationship of these cancers to HPV infection in Chinese women. Cancers overwhelmingly stem from HPV 16 and 18, but may also be associated with HPV 31, 33, 35, 45, 51, 52, 56 and 58. One study of women from the Sichuan province in China demonstrated a significantly altered risk of cervical cancer associated with HPV 16 or HPV 33 infection at 95% confidence interval. Another study found evidence of HPV infection in 57% of women with invasive cervical cancer who live in Shanxi province, where the incidence of cervical cancer is extremely high at 1,026 per 100,000. A hospital-based study of women from Shanghai, P.R.C. revealed the prevalence of HPV infection and types of HPVs found in cervical cancer patients. This study provides new information about the occurrence of infection with HPV type 52, 58, two HPV types that are relatively uncommon in cervical cancer patients in the Americas, Europe, Africa and South Asia.
Human Papillomavirus (HPV) infection has been strongly associated with development of cervical cancer neoplasia. The virus is detected in almost 100% of women with invasive cervical cancer. Also the cancer itself may increase susceptibility to HPV infection. Routing cervical cancer screening in asymptomatic women has proven successful. The presence of HPV in cytologically normal Papanicolaou (Pap) smears is associated with a significantly increased risk of developing invasive cervical cancer. In particular, infection with high risk or multiple HPV genotypes result in a strongly increased risk of developing cervical cancer.
Although HPV infections are extremely prevalent in young populations, most HPV infections spontaneously clear. Only a small minority of individuals who are exposed to HPV have cervical cancer neoplasia. Thus, microbe-or host-specific cofactors Chlamydia trachomatis (C trachomatis) must be involved in the pathogenesis. Chlamydia trachomatis infection is an independent risk factor for the development of invasive cervical sequamous cell carcinoma (SCC). Chlamydial trachomatis infection is one of the most common sexually transmitted diseases (STDs) in the United States, with between 3 and 4 million new infections each year. Females with a chlamydial infection may have vaginal redness and discomfort accompanied by a vaginal discharge. Males may have a discharge from the urethra (the opening where urine comes out) and burning upon urination. Depending on the extent of the infection, there may occasionally be a low-grade fever. People with chlamydial infections often have no symptoms at all. Although an infected person may have no symptoms, he can still spread the infection to other sexual partners. Chlamydia trachomatis causes infections that can affect the eyes, lungs, or urogenital (urinary-genital) area, depending on the age of the person infected and how the infection is transmitted. Chlamydia trachomatis infections may spread to the upper reproductive tract, including the uterus, fallopian tubes, and ovaries and may cause pelvic inflammatory disease. Scarring of the fallopian tubes after chlamydial infection may cause permanent damage to the reproductive system, resulting in infertility. Chlamydial infections also increase the risk that bacteria will cause secondary infections in the pelvic organs, genitals, or rectum. Most females with chlamydial infections will be asymptomatic (they will not have any symptoms).
In industrialized countries Chlamydia trachomatis infections are more common than gonorrhea, another STD, but many people contract both infections simultaneously. Doctors estimate that among patients with gonorrhea, approximately 25% of men and up to 50% of women also have chlamydial infections. Experts believe that 5% to 25% of all pregnant women in the United States currently have chlamydial infections, and 50% of these mothers who deliver vaginally will infect their infants with Chlamydia at birth. Almost half of the infected children will develop a chlamydial conjunctivitis, and close to 20% will develop chlamydial pneumonia.
Heat shock proteins (HSP60) are recognized as immunodominant clamydial antigens, and several immunoepidemiologic studies have found an association with immune responses to Chlamydia trachomatis. Antibody to Chlamydial Heat shock proteins (HSP60) are associated with cervical squamous cell carcinoma. Antibodies to Heat shock proteins (CHSP60) appear to reflect persistent Chlamydia infection. (Jorma Paavonen., et al: Am J Obst; Gynecol 2003; 189; 1287-92; Tarja Anttila., et al: JAMA, 285(1): (47-51), 2001.
Mutational activation of K-Ras gene is implicated in the development of premalignant cervical lesions and HPV infection may be an important step in the development of premalignant cervical lesion. (Prokopakis P. Sourvinos G., et al: Oncol Rep, 9(1): 129-33, 2002.) K-Ras gene mutations were detected in 15% of cases while HPV genome was found in 36% of HPV type related cases and HPV 18 at a higher rate than HPV 16, 71% and 29% respectively. High-risk HPV infections coexist with K-Ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri. (Stenzel A., et al: Pathol Res Pract, 9: 597-603, 2001.)
Harvey (H-Ras), Kirsten (K-Ras), and Neuroblastoma (N-Ras) genes have been localized to chromosomes 11, 12 and 1 respectively, in humans. All the ras oncogenes (H, K and N-Ras) encode for a 21-KDa (P21) protein, 189 amino acids long. Ras mutations in cervical carcinomas are low and preferentially occur at codon 12 of the K-ras gene. The point mutations at codon 12 of the K-Ras gene in premalignant and malignant cervical lesions range from 17% to 24%. This data suggests that the mutational activation of the K-Ras gene may be involved in the initial stages of cervical carcinogenesis. Although the presence of HPV is detected in over 90% of cervical carcinomas, it is insufficient to conclude that carcinogenesis has occurred. In vitro studies have demonstrated that an activated H-Ras gene can induce tumorrigenetic conversion of HPV-immortalized cervical kerationlcytes, indicating a cooperative effect between the ras and E6/E7 genes in cellular transformation. (Ioannis N. Mammas., et al: Gynecologic Oncology 92: 941-948, 2004.)
Studies have demonstrated an early immune recognition of E4 protein and mutated ras gene. Patients with condyloma and cervical intraepithelial neoplasia (CIN) also showed a higher prevalence of Ras antibodies (˜40%) than cervical cancer patients (10%). The high prevalence of antibodies against Ras and E4 proteins in premalignant lesions opens the possibility of using both antibodies as early markers for potential cervical cancer patients. The levels of anti-Ras antibodies were relatively high in sera from patients with condyloma and CIN lesions. The presence of anti-Ras antibodies in early lesions of the cervix could be used as an early marker in cervical cancer patients.
Normal human cellular H-Ras, K-Ras and N-Ras genes have the potential for activation to oncogenes by mutations occurring in codons 12, 13 and 61; such mutated, activated and transforming K-Ras genes have been identified and isolated from human tumors and cultured tumor cells. Although the expression patterns of K-Ras proto-oncogene proteins in normal human tissues are known, similar information for activated K-Ras oncogene encoded p21's and its relevance to human disease diagnosis and prognosis remains to be determined. (A. Pedroza-Saavedra., et al: Arch Virol 145: 603-623, 2000.)
Phosphatase and tensin homologue (PTEN) tumor suppressor protein has been demonstrated in the role of epigenetic and genetic changes of PTEN in the development of sequamous cell carcinoma (SCC) of the uterine cervix and their value as prognostic factor. Tumor suppressor gene, PTEN (also known as MMAC-1 and PTEN-1) located at chromosome 10q23.3 has an important role in controlling cell growth, inducing cell cycle arrest, promoting apoptosis, down regulating adhesion and suppressing cell migration. Deletion and/or mutation of the PTEN gene has been found in a variety of human cancers, including brain, prostate, breast, thyroid, lung and endometrium. More specifically, deletion and/or mutation of the PTEN gene has been found in 30-50% of endometrial cancer cases, 40% in cases where hyper-methylation of the promoter region of PTEN occurs, and 36% in a CIN-H and cervical carcinoma immunostaining study. The detection of PTEN and related specific PTEN antibodies is an early event in the development of cervical cancer carcinoma and is effective in silencing PTEN expression. Loss of PTEN function subsequent to HPV infection therefore might contribute to progression into carcinoma after deletion and/or mutation and methylation of the PTEN gene. (Tak-Hong Cheung., et al: Gynecologic Oncology, 93: 621-627, 2004; Takeo Minaguchi., et al: Cancer Letters 210: 57-62, 2004.)
The key of the molecular basis of immune responses is that immunogens are degraded, through a complex series of cellular processing events, into small peptide fragments. There are two pathways, endogenous and exogenous for degradation of proteins that share several similarities, but are also associated with fundamental differences. Small peptides of HPV oncoprotein fragments, tumor suppressor Ras and CHSP60 protein are bound by specific cellular receptors called Major Histocompatibility Complex (MHC) molecules. In the case of human cells MHC molecules are known as Human Leukocyte Antigen (HLA) molecules. The major biological function of the HLA molecules is to bind peptides. HLA class I molecules mostly bind peptides derived from the endogenous processing pathway and thus from proteins derived from inside cells. HLA class I molecules are most important for antiviral and anticancer immunity. Peptides derived from the extracellular milieu tend to bind to HLA class II molecules. These molecules are important for helper T-cell responses, which regulate antibody and cytotoxic responses.
T cells are key players in regulating a specific immune response. Activation of cytotoxic T-cells requires recognition of specific peptides bound to Major Histocompatibility Complex (MHC) class I molecules or HLA class I molecules in the case of human cells. The critical role of the human leukocyte antigen (HLA) system in presenting peptides to antigen-specific T cell receptors is that T cell responses that clear viral infections can be induced when the major histocompatibility gene products are complexed with peptides derived from HPV viral antigens and present on the surface of antigen-presenting cells. MHC-peptide complexes are potential tools for diagnosis and treatment of pathogens and cancer, as well as for the development of peptide vaccines. Only one in 100 to 200 potential binders actually binds to a certain MHC molecule, as the activation of cytotoxic T-cells requires recognition of specific peptides bound to MHC class I molecules. The complex between peptides and HLA receptors is transported to the cell surface where it can be recognized by T cells expressing a specific T-cell receptor (TCR) for that particular peptide-HLA combination. The peptides bound by HLA molecules and recognized by these specific T cells are called epitopes. A good prediction method for MHC class I binding peptides can reduce the number of candidate binders that need to be synthesized and tested. (Arkadium Chil, et al: Acta Obstet Scand, 82: 1146-1152), 2003.
Major Histocompatibility Complex (MHC) Class I, II and an oligopeptide (>90%) are 8-11 amino acids in length and encoded by a viral gene, tumor suppressor and CHSP60. Peptides are pre-dominantly generated from the hyproducts of proteasomal degration. The molecules are cell surface glycoproteins, which take an active part in host immune reactions. Here, the MHC class I molecule is folded through the concentrated chaperone (tapasin). The oncogenetic HPV E2, E4, E5, E6, E7 oncoproteins, tumor suppressor Ras proteins and CHSP60 are coded by three classes of genes. Class I and Class II gene products are directly associated with immune reactions with Class II gene products playing an indirect role.
Class I genes encode the principle subunits of MHC I glycoprotein called H2-K, H2-d, H2-l in mice and HLA-A, B, C in humans. Proteins encoded by these genes are present virtually on all nucleated cells. Class I molecules interact with the amino (N-) and carboxyl (C-) terminals of the bound peptide, leaving a bulge in the middle. These N- & C- terminal interactions together with closed peptide binding groove restrict the length of interacting peptide to 8-10 amino acids. However peptide-binding groove of Class II molecules is open at both ends and the interactions of peptide are more diffuse thereby a more variable length is allowed (generally 10-28 amino acids). The involvement of MHC class-II with HPV early proteins is in response to almost all antigens. (A. Sette, et al: Tissue Antigens, 59: 443-451, 2002; Margaret M. Madeleine, et al: JID, 186(1) 1565-1574, 2002.)
For the Chinese population based on HLA, microsatellites and other gene loci have divided the Chinese population into two groups, the Southern and the Northern. Dai and Buyi were two representative Southern Chinese population with Jing also belonging to the Southern group. The most frequent DPB1 allele was DPB1 *0501, DPB1 *1301, DPB1 *0401, and DPB1 *020102. Frequent DRB1 alleles are DRB1 *150201, 030101, DRB1 *090102. The most predominant DQB1 alleles was DQB1 *030101/0309, DQB1 *050201, and DQB1 *0201/0202. The high affinity binding prediction of peptides by HMCPred program runs as a GCI server and covers a range of different human HMC allele peptide specificity models, which include: class I (HLA-A *0101, HLA-A *0102, HLA-A *0202, HLA-A *0203, HLA-A *0206, HLA-A *0301, HLA-A *1101, HLA-A *3301, HLA-A 6801, HLA-A 6802, HLA-A 3501) and class II (HLA-A-DRB1 *0101, HLA-A-DRB1 *0401 and HLA-A-DRB1 *0701) alleles. All their alleles exist at a high frequency within human populations and have significant literature binding dates.
The first reported association between HLA class II gene and cervical cancer were DQw3, a serologic designation that has subsequently been subdivided into 3 other serologic designations: DQ7, DQ8, and DQ9. The specificities include DQB1 0301, *0303 and 0304 for DQ7, DQB1 *0302 and 0305, DQB1 0302 and 0305 for DQ8 and DQB1 0303 for DQ9. Other allele groups that have been reported with increased risk include DRB1 *11, DRB1 *15, DQB1 *06, and related haplotypes DRB1 *0401, DRB1 *0301, DRB1 * 1101, DRB1 *1501, and DQB1 *0602. In contrast to an increased risk of invasive disease associated with DRB1 *1501-DQB1 *0602 according to Hispanic American and British studies, Hildesheim et al. reported a decreased risk of HPV-16 containing high grade aquamous intraepithelial cervical lesions associated with DRB1 *1501-DQB1 *0602 based on a U.S. study of white women in Portland, Oreg. area. (Jin-Hia Lin, et al: Human Immunology 2003: 64: 830-834; Pingping Guan, et al: Nucleic Acid Research 2003 Vol. 31 No.13 3621-3624; Harpresst Singh, et al: Bioinformatics 2003: Vo. 19 No.8 1009-1014).
While the prior art provides diagnosis and early prediction of cancer or cellular abnormalities based on Papanicolaou (Pap) smear screening, these tests do not provide HPV type, sensitive, rapid, simple, and economical detection of genital condyloma, squamous cell carcinoma (SCC), squamous interaepithelial lesion (SIL), low-grade squamous interaepithelial lesion (LSIL), high-grade squamous interaepithelial lesion (HSIL), cervical intraepithelial neoplasia (CIN) and invasive cervical cancer.
There is a need in the art, therefore, for an early diagnosis and better treatment of cervical cancer utilizing diagnosis kits and specific HPV peptide vaccines. The prior approaches are primarily focused on detection of immunoassays of HPV, tumor suppressors and heat shock proteins of immunodominant clamydial antigens.
Accordingly, it is a principal object of the present invention to provide a method of detecting specific antibodies against the E2, E4, E6, E7 oncoproteins, tumor suppressor proteins and Chlamydia trachomatis heat shock protein antigens, utilizing novel protein and peptide sequences for the early detection of genital condyloma, squamous cell carcinoma (SCC), squamous interaepithelial lesion (SIL), low-grade squamous interaepithelial lesion (LSIL), high-grade squamous interaepithelial lesion (HSIL), cervical intraepithelial neoplasia (CIN) and invasive cervical cancer.
A primary object of the present invention is to provide novel antibody reactive protein sequences or peptides isolated, purified or derived from the HPV proteins, tumor suppressor proteins (H-Ras, K-Ras and PTEN), and Chlamydia trachomatis heat shock protein 60 (CHSP60 groEL1).
A further object of the present invention is to provide a simple, rapid, less expensive, specific and more sensitive test for detecting or diagnosing not only HPV infections, but also most, if not all, HPV associated neoplasms.
It is a further object of the present invention to provide these peptides in a chemically pure form.
It is still another object of the present invention to provide novel antibody reactive protein sequences or peptides isolated, purified or derived from HPV genotypes 6, 11, 16, 18 31, 33, 45, 52 and 58.
In addition, it is an object of the present invention to provide novel antibody reactive protein sequences or peptides isolated from early and late coding regions of HPV linked to other protein sequences or peptides isolated from HPV E2, E4, H6, E7 oncoproteins and L1 capsid proteins, H-Ras, K-Ras, PTEN, or CHSP60 groEL1, by adding neutral, genetically coded amino acid residues as a media linker between the peptides and epitopes.
It is a further object of the present invention to provide a method for detecting or diagnosing cancer or cellular abnormalities comprising the steps of: reacting a sample of body fluid or tissue likely to contain IgG and IgA specific antibodies to one or more protein sequences or peptides isolated from the E2, E4, E6 and E7 early coding regions and late coding regions of L1 capsid proteins of HPV, and characterized by a linkage to another protein sequence or peptide isolated from the E2, E4, E6, and E7 early coding regions of HPV; forming an antibody-peptide complex comprising at least one of the isolated protein sequences or peptides and the sample antibodies; and detecting the antibody-peptide complex.
It is a further object of the present invention to provide a method for detecting or diagnosing cancer or cellular abnormalities through detection or diagnosis of HPV associated cell abnormalities.
It is also an object of the present invention to provide a method for detecting or diagnosing cancer or cellular abnormalities through detection or diagnosis of HPV associated precancerous or premalignant conditions.
Further objects of the invention will become apparent from the following description.