Presentation on human papiloma virus and its briefly description

Human papiloma virus... And beginning threat of cervical cancer

1. HUMAN PAPILLOMAVIRUS SUBMITTED TO : Dr. YOGINDER PAL SINGH. (SENIOR SCIENTIST IN SRL MOLECULAR BIOLOGIST) REPRESENTED BY : RISHITA MITRA (MSc. BIOTECH)

2. CONTENTS INTRODUCTION DISCOVERY AND HISTORY OF HPV HUMAN PAPILLOMA VIRUS HPV - Structure of HPV - Proteins associated with HPV HPV T ypes MECHANISM OF INFECTION Host Immune Response HPV and Cervical Cancer HPV – PATHOGENISIS OF CERVICAL CANCER

3. EPIDEMIOLOGY MODES OF TRANSMISSION SYMPTOMS OF HPV HOW THE HPV IS DIAGNOSIS HPV DETECTION METHODS HPV TREATMENT CONCLUSION

4. INTRODUCTION HPV stands for human papillomavirus. There are more than 200 types of HPV. Some types produce warts plantar warts on the feet and common hand warts. About 40 types of HPV can infect the genital area the vulva, vagina, cervix, rectum, anus, penis, or scrotum.Genital HPV infections are very common. HPV is so common that nearly all sexually active men and women get it at some point in their lives. But most people who have HPV don't know it. Most HPV infections have no harmful effect at all. Some types of HPV may cause genital warts. These are called low-risk types of HPV. Some types of HPV may cause cell changes that sometimes lead to cervical cancer and certain other genital and throat cancers. These are called high-risk types.

5. Noble Prize Winner 2008 Detected HPV DNA in Cancer cells Structure of HPV Prof. Harald zur Huse n

6. Human papillomavirus (HPV) Papillomaviruses are small, non enveloped, double-stranded DNA viruses encased in a 72-sided icosahedral protein capsid. The HPV genome consists of circular, double-stranded DNA of approximately 7,900 nucleotide base pairs.

7. HPV Structure Non Enveloped Double Stranded DNA Virus Small DNA viruses with about 7900 base pairs 6 early (E) and 2 late (L) proteins L1-capsid protein **E6 and E7 are oncogenic ! Each type has less than 90% base pair homology with any other type

8. to be contd... Genome : Circular, d/s DNA ~8kbp associated with cellular histones to form a chromatin-like substance. At least 12 different HPV genomes have been sequenced, and the genetic organization of all is similar.

9. Proteins associated with HPV E1 DNA-dependent ATPase, ATP dependent helices: allow unwinding of the viral genome and act as an elongation factor for DNA replication. E2 Responsible for recognition and binding of origin of replication. E4 Late Expression: C terminal binds intermediate filament, allowing release of virus-like particles. E5 Obstruction of growth suppression mechanisms: e.g EGF receptor; activation of mitogenic signalling pathways via transcription factors: c-Jun and c-Fos. Inactivation of p21. E6 Binding p53 protein. E7 binds to pRB/p107. Gene Function L1 Major capsid protein: can form virus-like particles. L2 Minor capsid protein: possible DNA packaging protein. Structural Pr oteins

10. to be contd...(I mages of L1 proteins and L2 Proteins) L1 protein in COPV L2 Major Protein

11. Classification of Proteins E2 PROTEINS E2 binds DNA as a dimer and is sequence specific. The overall secondary structure of the dimer consists of an eight-stranded beta-barrel (magenta) and two pairs of alpha-helices (purple). Also it is a trans-acting transcriptional activator. The protein is capable of activating a conditional enhancer in the viral long control region, or lcr . E4 PROTEINS Ability to aggregate into cytoplasm and nuclear inclusion granules. Inclusions are most noticeable in cutaneous lesions. E4 expression coincides with the onset of vegetative viral DNA replication and occurs immediately after cells have left the basal layer (higher layers for mucosal types). Involvement in virus maturation or vegetative viral DNA replication possibly by sequestering inhibitory factors E4 may interfere with normal differentiation in order to create the conditions required for high level virus synthesis. In the upper layers, E4 and L1 are expressed from the same bicistronic message An important role in productive infection seems likely.

12. TRANSFORMATION IN HPV E2 binds to the early promoter and decreases expression of E6/E7; loss of E2 is thus the first stage in transformation. E6 binds to p53 via a cellular protein ("p100") and targets it for degradation via the ubiquitin pathway. E7 binds pRB and prevents phosphorylation. This would normally result in apoptosis BUT: Both E6 and E7 interact with a number of cellular proteins which influence the outcome of infection:

13. Cycle Representing How Virsus Is Transformed

14. HPV TYPES The common HPV types can be divided into three major categories based on their oncogenic potential: 1.Low risk HPV-6,11,42,43,44 2.Intermediate risk HPV-33,35,39,52,58 3.High risk HPV-16,18,31,45

15. to be contd Low risk types most commonly cause genital warts. HPV-6 is the most common type associated with genital warts. Seventy-five percent cases of cervical cancer are infected with high risk types. HPV-16 is the predominant type in pathogenesis of squamous cell cancer. HPV-18 is the predominant pathogen in adenocarcinoma of cervix.

16. MECHANISM OF INFECTION All HPV exhibit extreme specificity for infection on epithelial cells. The papillomavirus epitheliotrophy resides in the interaction of specific transmission factors with the viral regulatory region LCR. The infection normally results in hyperproliferation of the host cell and may lead to transformation and immortalization.

17. LIFE CYCLE

18. Host Immune Response Humoral : There is development of HPV specific neutralizing antibodies either of immunoglobulin IgA and IgG These antibodies prevent infection of anogenital mucosal surface. These are detected against L1 & L2 HPV capsid protein.

19. Cell Mediated Immunity : Whenever the pathogens enter the human cell-Cell Mediated immunity ( CMI) mechanism is switched on in following way. Specific CD8 +, cytotoxic T lymphocytes , CD4 and Helper T lymphocytes are activated and play important role, required to clear the infected cells. Cytotoxic T lymphocytes (CTL) recognise the early proteins in the infected cells in conjuction with host major histocompatibility complex (MHC) class I molecule. Helper T cells recognise longer peptide fragments complexed with MHC class II molecules. Both these cytotoxic T lymphocytes and Helper T lymphocytes promote specific antibody production through cytokines induction.

20. HPV and Cervical Cancer

21. CERVICAL NEO P LASIA AND HPV Infections with HPV cause approximately 10 % of the global burden of human cancers and at least 500,000 deaths annually . Infection with specific HPV (16,18) types is necessary for the development of the vast majority of cervical cancers (>99.7%) and the immediate precursor lesion (CIN 3) . The magnitude of the association between HPV and cervical cancer is higher than the association between smoking and lung cancer.

22. The four major steps in the development of cervical cancer are- (i) infection of the metaplastic epithelium of the transformation zone with one or more carcinogenic HPV types; (ii) viral persistence rather than clearance reflecting the host immune response; (iii) clonal progression of persistently infected epithelium to cervical precancer (CIN 3); and progression to (iv) clinical cancer (macro and micro invasion).

23. Model of Cervical Carcinogenesis Latent Infection ↓ Productive Viral Infection ↓ Cellular Transformation ↓ Transformation to Malignancy

24. HPV – PATHOGENISIS OF CERVICAL CANCER The HPV genome is usually maintained as a stable viral episome, independent of the host cell genome, in the nucleus of infected cells. It codes for only eight genes In some high-grade CIN lesions, and more frequently in cervical cancer, HPV genomes are covalently bonded or integrated into the host chromosomes . This integration event occurs at random within the host cell genome but is highly specific in relation to the viral genome, involving the E1 and E2 genes, with important consequences for regulation of viral gene expression . This integration of the HPV genome into the host genome is associated with invasive cancer and might serve as an important biomarker to distinguish HPV infection from precancer .

25. The late genes, L1 and L2, the sequences of which are highly conserved among all papilloma viruses, encode the common capsid proteins. These viral proteins reflect late viral gene expression and are exclusively present in well-differentiated keratinocytes . Both proteins play an important role in mediating efficient virus infectivity.

26. 1. The proteins encoded by the E6 and E7 genes of high-risk HPV types, particularly HPV 16 (clade A9) and 18 (clade A7), are directly involved in cellular transformation in the presence of an active oncogene . 2. E6 and E7 are the primary HPV oncoproteins with numerous cellular targets . 3. Both E6 and E7 proteins can immortalize primary keratinocytes from cervical epithelium and can influence transcription from viral and cellular promoters . 4. The activity of these viral oncoproteins results in genomic instability, leading to the malignant phenotype. 5. E6 proteins of high-risk HPV types bind the tumor suppressor protein p53 . 6. This induces ubiquitination and degradation of p53, removing the p53-dependent control of the host cell cycle.

27. E6 also increases telomerase activity in keratinocytes through increased transcription of the telomerase catalytic subunit gene (hTERT) through induction of c-myc . Telomerase activity is usually absent in somatic cells, leading to shortening of telomeres with successive cell divisions and to eventual cell senescence. E6 mediation of telomerase activity may predispose to long-term infection and the development of cancer. Recently E6 and E7 viral oncogenes have been shown to antagonize BRCA-mediated inhibition of the hTERT promoter .

28. The E7 gene product is a nuclear phosphoprotein that associates with the product of the retinoblastoma gene (pRb), which is a tumor suppressor gene important in the negative control of cell growth . E7 is the primary transforming protein. Degradation of p53 by E6 and the functional inactivation of pRb by E7 represent the main mechanisms whereby expression of HPV E6 and E7 oncoproteins subverts the function of the negative regulators of the cell cycle . Deregulated expression of the viral oncogenes is a predisposing factor to the development of HPV-associated cancers .

29. The products of the E2 gene are involved in transcriptional regulation of the HPV genome. The process of HPV integration into the cellular genome, which occurs in some high-grade CIN lesions and most invasive cervical cancers, disrupts the E2 gene . This results in increased levels of E6 and E7 expression, correlating with increased immortalization activity . Both E6 and E7 proteins are expressed at low levels in the process of HPV infection

30. Persistent infection critical for development of neoplastic change

31. EPIDEMOLOGY

32. PERCENTAGE SHOWING HPV AFFECTING GLOBALLY

33. MODES OF TRANSMISSION 1 . Sexual transmission- Genital HPV infection are primarily through sexual contact. Infectivity rate is approximately 65%. The risk factors for transmision are: - Number of lifetime sexual partners -younger age group -cigarette smoking -use of oral contraceptive pills 2. Extragenital skin transmission: Skin to skin or genital to skin transmission has been observed in periungal and congenital warts. 3 .Formites: Transmission through formites is arare documentation, eg. Gloves, surgical instruments. 4. Genital HPV types appear to be uncommonly transmitted in neonatal period. The only clinically expressed HPV disease that is acquired at birth is laryngeal papillomatosis. This is mainly caused by HPV-6 and 11.

34. Symptoms of HPV lesions of the skin epithelium or the mucosal linings of the anogenital area, oral cavity, and respiratory tract. 2. The most common (and benign) symptomatic presentation of an HPV infection is condylomata acuminata (genital warts). 3. Other visible HPV infections are the plantar warts and the common warts. 4. Asymptomatic/sub clinical HPV infection.

35. Clinical Manifestation of HPV The incubation period for genital warts is usually between three and six months, but it may last for years after exposure . small bumps that appear in the genital area or anus. They may be single or clusters and have a cauliflower-like appearance as they grow larger. Women: Genital warts usually start as In women, genital warts appear on the vulva, in the vagina, on the cervix, or in the anal area. In men, they appear on the foreskin, head or shaft of the penis, and in the anal area, urethra, and scrotummouth or throat of a person who has had sexual contact with an infected person

36. Genital Wart –Vulva And Perineum HOW IT IS AFFECTED Condyloma acuminata, vaginal wall

37. HOW THE HPV IS DIAGNOSIS 1. Genital warts - They can be visualised by gross inspection 2- Subclinical HPV infection is diagnosed by colposcopy . After application of 3-5 percent acetic acid , subclinical HPV has shiny- white colour with irregular borders and satellite lesions. Vaginal subclinical infection may exibit reverse punctation. 3- Squamous intraepithelial lesion(SIL) of cervix: They are detected mainly by routine cytological screening test Paps smear.

38. HPV DETECTION METHODS HPV can be detected by following methods such as Pap Smear Method Colposcopy Some other methods used for detection of HPV are Nucleic acid Hybridization Assays Signal Amplification Assays Nucleic acid Amplifications Assays

39. Pap Smear Method A Pap smear is a simple, quick, and essentially painless screening test (procedure) for cancer or precancer of the uterine Cells collected from a woman's cervix during a pelvic exam are spread on a microscope slide for examination. The cells are evaluated for abnormalities, specifically for pre- cancerous and cancerous changes. A woman may experience a small amount of spotting (light vaginal bleeding) immediately after a Pap smear, but heavy or excessive bleeding is not normal. Cervical cancer screening is recommended every 3 years for women aged 21-65.cervix.The Pap smear is analyzed according to a uniform standardized system known as the Bethesda System. An abnormal Pap smear may show precancerous changes that can be treated at an early stage, before cancer develops. A recording of the woman's menstruation status and whether and when she had abnormal Pap smears previously is essential to the reader of the current Pap smear. Up to 80% of women diagnosed with invasive cancer of the cervix have not had a Pap smear in the past 5 years.

40. How is a Pap smear done? A speculum is then inserted into the vaginal area (the birth canal). (A speculum is an instrument that allows the vagina and the cervix to be viewed and examined.) A small brush or swab is inserted into the opening of the cervix and twirled around to collect a sample of cells. A second sample is also collected on the surface of the cervix as part of the Pap smear The samples are placed in a solution from which cells are isolated and used to produce slides for laboratory evaluation.

41. Colposcopy It is the method in which a special magnifying device is used for examine the affected parts of vulva , vagina and cervix.This test is most often done when the result of a Pap test is abnormal. Why It Is Done ? It is done to look at the cervix for problem areas when a Pap test was abnormal. If an area of abnormal tissue is found, a biopsy is often done. It is done to check whether a sore or other problem (such as genital warts) found on or around the vagina and cervix. It is done to follow up on abnormal areas seen on a previous colposcopy. It can also be done to see if treatment for a problem worked. 4. It is done to look at the cervix for problem areas if an HPV test shows a high-risk type of HPV.

42. How it is Done? Colposcopy is usually done by a gynecologist .If a biopsy is done, the sample will be looked at by a pathologist. This test can be done in your doctor's office. The doctor will insert a lubricated tool called a speculum into the vagina. It gently spreads apart the vaginal walls so the doctor can see inside the vagina and the cervix. The colposcope is moved near the vagina.Vinegar (acetic acid) or iodine may be used inside the cervix to make abnormal areas easier to see. Photos and videos of vagina and cervix are captured. If a sample of tissue is needed from inside the opening of the cervix, a test called endocervical curettage (ECC) will be done. This area can't be seen by the colposcope. So a small sharp-edged tool called a curette is gently put into the area to take a sample. ECC takes less than a minute to do. It may cause mild cramping. An ECC is not done during pregnancy.

43. SOME OTHER METHODS TO DETECT HPV

44. Nucleic acid-hybridization assays Initially, techniques such as Southern blotting, in situ hybridization, and dot-blot hybridization used radio-labeled nucleic acid hybridization assays to detect HPV infection in cervical samples. Although these techniques generated high-quality information, the disadvantages of these direct-probe approaches include low sensitivity, the need for relatively large amounts of purified DNA, and time-consuming procedures . Signal-amplification assays: The Digene® HPV test using Hybrid Capture® 2 (hc2) technology, and the Cervista® HPV HR assay are the only methods that currently have FDA (Food and Drug Administration) approval for diagnostic testing. The Hybrid Capture® 2 system (hc2, Digene Corp., USA) is a non-radioactive signal-amplification method based on the hybridization of the target HPV-DNA to labeled RNA probes in solution

45. This test detects 13 HR-HPV types (-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59 and -68) or 5 LR-types (-6, -11, -42, -43, and -44). Nucleic acid-amplification methods : PapilloCheck® This assay detects and genotypes 24 HPV types in a single reaction (HPV -6, -11, -16, -18, -31, -33, -35, -39, -40, -42, -43, -44, -45, -51, -52, -53, -55, -56, -58, -59, -66, -68, -70, -73, and -82). The assay uses a multiplex PCR with fluorescent primers to amplify a 350 bp fragment of the E1 gene of HPV, comprising 28 probes, each in 5 replicate spots fixed on a DNA chip. Co-amplification of the human ADAT1 gene is used as internal control. The main advantages of the PapilloCheck® assayis HR/LR-HPV identification, and detection of multiple infections, and may be considered a reliable screening test [30].

46. B . PCR METHOD: The HPV-PCR protocols use consensus primers such as PGMY09/PGMY1 and GP5+/GP6+, which allow amplification of a large number of HPV genotypes in a single reaction. The primers target conserved regions of the HPV genome, such as the L1 capsid gene . After amplification, the HPV genotypes can be determined separately, using techniques such as restriction-fragment length polymorphism (RFLP), linear probe assays, direct sequencing, or genotype-specific primers. C. Real-time PCR: This assay is a reliable, sensitive, and specific diagnostic tool for detection and genotyping of targeted HPV genotypes in tissue specimens [44] and cellular samples. The advantages of this method are: Ability to detect viral load. The use of different fluorochromes that emit fluorescence, as the PCR reaction proceeds, the reactions can be performed in multiples and can amplify different nucleic-acid targets. Nucleic acids can be detected even in very small concentrations, using a 7-log dynamic range to extrapolate the viral load/concentration over the standard curve; and finally It is extremely reproducible, rapid, and applicable to clinical samples .

47. D. HPV Genome Sequencing: The dideoxy chain-termination technique (Sanger technique) was first described for genome sequencing more than three decades ago. Fluorescently labeled nucleotides were incorporated into Sanger sequencing, and advances have led to increasing expansion and development of high-quality, thorough sequencing . However, it has not been validated for clinical use. Similar to dideoxy sequencing methods, pyrosequencing is applicable to any source of DNA or RNA that can be amplified by PCR (blood, saliva, cell line, plasma, serum, tissue, formalin-fixed paraffin-embedded samples, and whole genome-amplified DNA).The primary advantage is simplicity: the readout sequence itself is obtained, rather than a fluorescent signal that must be converted to a sequence. Second, it is faster and less expensive: savings result from its sequence-by-synthesis process where a DNA sequence is read in real time, and it is synthesized by addition of inexpensive, unlabeled nucleotides; and finally, the method is uniquely quantitative. E. HPV-mRNA Detection: E6 and E7 are the main genes responsible for cell transformation mediated by HR-HPV, and they modulate the activities of cellular proteins that regulate the cell cycle . Thus, the presence of E6/E7 can be a specific marker for diagnosing precancerous lesions by HPV

48. Thus, the presence of E6/E7 can be a specific marker for diagnosing precancerous lesions by HPV . For this reason, the search for transcripts of E6/E7 could increase the specificity and sensitivity of the tests in screening for cervical lesions that have a greater chance of progressing, compared with a simple detection of HPV-DNA. The main techniques used to detect mRNA for E6/E7 oncogenes are two commercial assays: PreTect® Proofer and APTIMA® HPV Assay The chemistry is based on transcription-mediated amplification of full-length E6/E7 transcripts preempted by target capture. The APTIMA® HPV assay Itdetects HPV E6/E7 mRNA of the 14 HR (-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68), which provides better sensitivity than the Proofer test, which detects only 5 HR-HPV [62].

49. HPV TREATMENT The development of a vaccine for HPV could lead to a potential reduction in the incidence of cervical cancer and its precursor lesions, other associated cancers (anal, penile, vaginal, vulvar), and genital warts . Recently three separate trials have been performed to test the efficacy of various HPV vaccines. Each trial was able to show that the vaccine they were using was efficacious in preventing persistent HPV infection . V accination of young sexually active women may still provide some protection. Routine performance of Pap smears or HPV DNA testing prior to vaccination is not recommended, although such screening may be appropriate for sexually active women. Cervical cancer screening should continue for the immunized population to screen for disease caused by nonvaccine HPV types, to monitor the continued efficacy of the vaccination program (which may not be 100%), and to screen HPV infected women as the vaccine is not therapeutic.

50. TYPES OF VACCINES: HPV4 (Gardasil) contains types 16 and 18 (high risk) and types 6 and 11 (low risk) HPV2 (Cervarix) contains types 16 and 18 (high risk) Both vaccines are supplied as a liquid in a single dose vial or syringe Neither vaccine contains an antibiotic or a preservative

51. HUMAN PAPILLOMA VIRUS VACCINES HPV4 vaccine is approved for - females 9 through 26 years of age for the prevention of cervical cancers, precancers and genital warts - males 9 through 26 years of age for the prevention of genital warts HPV2 vaccine is approved for -females 10 through 25 years of age for the prevention of cervical cancers and precancers -not approved for males or for the prevention of genital warts .

52. OTHER TREATMENT METHOD Genital warts can be treated by a doctor and by different methods. Podofilox gel: A patient-applied treatment for external genital warts. Imiquimod cream: A patient-applied treatment. Chemical treatments (including trichloracetic acid and podophyllin), which must be applied by a trained health care provider to destroy warts. Genital warts can be treated by a doctor and by different methods. Cryotherapy: Uses liquid nitrogen to freeze off the warts. Laser therapy: Uses a laser beam or intense lights to destroy the warts. Electrosurgery: Uses and electric current to burn off the warts. S urgery: Can cut away the wart in one office visit . Interferon: an antiviral drug, which can be injected directly into warts.

53. CONCLUSION Cervical cancer remains as a leading cause of morbidity and mortality for women worldwide HPV integration may alter mRNA expression via deletion, amplification or genomic rearrangement which may have implication for their expression in cervical cancer. Depending on the nature of their targets, mRNA can funtion as either tumor surppressor mRNAs or onocogenic mRNAs. This findings suggest many approaches to mRNA specific personalized treatment and molecular targeted thearapy Therefore mRNA are likely to be imporatant in diagnosis and treatment of cervical cancer.