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.

1 . A method of screening a human subject of papillomavirus infection, comprising:
 obtaining a clinical sample from the human subject;   providing one or more first antibodies against one or more recombinant proteins encoded by one or more papillomavirus genes;   providing one or more second antibodies against one or more endogenous proteins of host human cells;   conducting one or more immunological assays on the clinical sample from the human subject;   detecting the presence of one or more papillomavirus proteins encoded by the one or more papillomavirus genes in the human subject using the one or more first antibodies; and   detecting the presence of the one or more endogenous proteins in the human subject using the one or more second antibodies.   
     
     
         2 . The method of  claim 1 , further comprising comparing the results of the immunological assays on the levels of the papillomavirus proteins and the endogenous proteins in the clinical sample of the human subject as altered by HPV infection. 
     
     
         3 . The method of  claim 2 , further comprising comparing on the levels of the papillomavirus proteins and the expression levels of the endogenous proteins in the human subject with the levels of these proteins in healthy human subjects as measured by the immunological assays. 
     
     
         4 . The method 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, pap smear scrapes, and combinations thereof. 
     
     
         5 . The method of  claim 1 , further comprising 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. 
     
     
         6 . The method of  claim 1 , wherein the one or more papillomavirus genes are selected from the group consisting of papillomavirus E6 genes, papillomavirus E7 genes, papillomavirus L1 genes, papillomavirus L2 genes, and combinations thereof. 
     
     
         7 . The method of  claim 6 , wherein the levels of the one or more papillomavirus proteins from the clinical sample as measured by the one or more immunological assays are elevated by comparing to the levels of these proteins in a healthy subject. 
     
     
         8 . The method of  claim 1 , wherein the one or more endogenous proteins are selected from the group consisting of p53 protein, RB protein, p16INK4a, p21, and combinations thereof. 
     
     
         9 . The method of  claim 8 , wherein the levels of the one or more endogenous proteins from the clinical sample as measured by the one or more immunological assays are reduced by comparing to the levels of these proteins in a healthy subject. 
     
     
         10 . The method of  claim 8 , wherein the levels of the one or more endogenous proteins from the clinical sample as measured by the one or more immunological assays are increased by comparing to the levels of these proteins in a healthy subject. 
     
     
         11 . The method of  claim 1 , wherein the one or more papillomavirus proteins are 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, HPV-16 L1 proteins, recombinant HPV-18 L1 proteins, and combinations thereof. 
     
     
         12 . The method of  claim 1 , further comprising:
 conducting a nucleic acid hybridization assay on the clinical sample; and   detecting the presence of a papillomavirus genome in the clinical sample from the human subject.   
     
     
         13 . The method of  claim 1 , wherein the one or more immunological assays are selected from the group consisting of ELISA (enzyme linked immunoabsorbent assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, immunostaining, immuno-histochemistry, protein chip assays, radioimmunopercipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays. 
     
     
         14 . A method of screening a human subject infected with a human papillomavirus, comprising:
 obtaining a clinical sample from the human subject;   providing one or more recombinant papillomavirus proteins encoded by one or more papillomavirus genes;   detecting the presence of altered levels of one or more papillomavirus antibodies in the clinical sample altered by HPV infection, as compared to the levels in samples from healthy subjects, by conducting one or more immunological assays on the clinical sample using the one or more recombinant papillomavirus proteins; and   detecting the expression levels of one or more endogenous proteins in the clinical sample as compared to the expression levels of the endogenous proteins in samples from healthy subjects.   
     
     
         15 . The method of  claim 14 , further comprising 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. 
     
     
         16 . The method of  claim 14 , wherein the one or more papillomavirus genes are selected from the group consisting of papillomavirus E6 genes, papillomavirus E7 genes, papillomavirus L1 genes, papillomavirus L2 genes, and combinations thereof. 
     
     
         17 . The method of  claim 14 , wherein the human papillomavirus is selected from the group consisting of 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, and combinations thereof. 
     
     
         18 . The method of  claim 14 , wherein the one or more papillomavirus genes are selected from the group consisting of papillomavirus E6 genes, papillomavirus E7 genes, papillomavirus L1 genes, papillomavirus L2 genes, and combinations thereof. 
     
     
         19 . The method of  claim 14 , wherein the one or more endogenous proteins are selected from the group consisting of p53 protein, RB protein, p161NK4a, p21, and combinations thereof. 
     
     
         20 . The method of  claim 14 , wherein the one or more papillomavirus proteins are 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, HPV-16 L1 proteins, recombinant HPV-18 L1 proteins, and combinations thereof. 
     
     
         21 . The method of  claim 14 , 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. 
     
     
         22 . The method of  claim 14 , wherein the one or more immunological assays are selected from the group consisting of ELISA (enzyme linked immunoabsorbent assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, immunostaining, immuno-histochemistry, protein chip assays, radioimmunopercipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays. 
     
     
         23 . A method of screening a human subject of infection of a human papillomavirus, comprising:
 obtaining a clinical sample from the human subject;   providing one or more recombinant papillomavirus proteins purified from protein expression systems with expression vectors having a portion of nucleic acid sequence corresponding to the full length nucleic acid sequence of one or more papillomavirus genes;   providing one or more first antibodies against the one or more recombinant proteins encoded by the one or more papillomavirus genes;   providing one or more second antibodies against one or more endogenous proteins of host human cells;   conducting one or more immunological assays on the clinical sample from the human subject;   detecting the presence of one or more papillomavirus proteins encoded by the one or more papillomavirus genes in the human subject using the one or more first antibodies; and   detecting the presence of the one or more endogenous proteins in the human subject using the one or more second antibodies.   
     
     
         24 . The method of  claim 23 , wherein the human papillomavirus is selected from the group consisting of 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, and combinations thereof. 
     
     
         25 . The method of  claim 23  wherein the one or more papillomavirus genes are selected from the group consisting of papillomavirus E6 genes, papillomavirus E7 genes, papillomavirus L1 genes, papillomavirus L2 genes, and combinations thereof, and wherein the one or more endogenous proteins are selected from the group consisting of p53 protein, RB protein, p16INK4a, p21, and combinations thereof. 
     
     
         26 . The method of  claim 23 , wherein the one or more immunological assays are selected from the group consisting of ELISA (enzyme linked immunoabsorbent assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, immunostaining, immuno-histochemistry, protein chip assays, radioimmunopercipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays.