Human Papillomaviruses: Natural History and Virology

1. Human Papillomaviruses: Natural History and Virology Elizabeth R. Unger Ph.D., M.D. Acting Chief, Papillomavirus Section Centers for Disease Control and Prevention November 29, 2001

2. Papillomaviruses • Non-enveloped dsDNA viruses • Circular genome ~8 kb • 55-nm spherical capsid coat • Widely distributed in higher vertebrates • Tight species specificity • Tropism for squamous epithelium • Associated with warts and papillomas

3. Genome Organization • Similar for all papillomaviruses • Only one strand transcribed • Open reading frames (ORFs) named in relation to bovine papillomavirus genes • “Early” genes E1-E7 (but no E3 in HPV) • “Late” genes L1 and L2, coding for major and minor capsid proteins

4. HPV Genome: Episome

5. Simple Genome • Dependent on host cell for replication, transcription and translation • Viral functions tightly linked to cellular differentiation • Poly-cistronic viral transcripts • Multiple promoters, multiple splice patterns • Promoter usage linked to differentiation

6. HPV URR • Upstream regulatory region • Also called long control region (LCR) or non-coding region (NCR) • Contains transcriptional and replication regulatory elements

7. Late genes • Region of greatest genetic conservation • L1 is major capsid protein • Capsid is 72 pentamers of L1 • Expressed L1 assembles into viral conformation, viral-like particles (VLPs) • L2 is minor capsid protein • Required for encapsidation of viral genome

8. Early genes • E1: Viral replication; maintains episome • E2: Transcriptional regulation, co-factor for viral replication • E4: Disrupts cytokeratins • E5: Interacts with growth factor receptors • E6: Transforming protein; p53 degradation • E7: Transforming protein; Rb binding

9. Viral Replication • Replication and assembly in nucleus • Infection initiated in basal epithelial cells • Steady-state viral replication, some early-region transcription • Presumed site of latent infection • High-copy viral replication, late gene transcription and virion production limited to differentiating cells

10. Viral Integration • Not part of normal viral life-cycle • Occurs randomly in host chromosomes • Characteristic breakpoint in viral genome • E1-E2 disruption • Abnormally regulated E6/E7 expression • Associated with oncogenesis but not required

11. Immune response • Non-lytic infection minimizes exposure to immune system • Virus released with desquamating cells • Immune system influences outcome of HPV infection • Humoral and cellular responses identified • Not all infected hosts have detectable immune response

12. Human Papillomaviruses • More than 100 types, >80 fully sequenced • Typing based on nucleic acid sequence • >10% sequence variation = new type; 2-10% = subtype, <2% = variant • Types assigned sequential number based on order of discovery • No relation to phylogeny

13. HPV Types • Two major phylogenetic branches, differing affinities for site of infection • Cutaneous: Keratinized squamous epithelium • Mucosal: Non-keratinized squamous epithelium

14. HPV Mucosal Types and Variants • More than 30 types found in anogenital tract • “Low risk” types: rarely found in cancers • “High risk” types: frequently found in cancers or similar to types found in cancer • High risk types most prevalent in population, regardless of disease status • Variantsbest characterized for HPV 16 • E6/E7 polymorphisms could modify oncogenicity • Cross-reactive in ELISA assays

15. Unique features of HPV • No simple in vitro culture method • Antibody methods lack sensitivity • Diagnosing infection requires detection of HPV genetic information • Corollary: requires cellular sample from the site of infection • Only current infections identified

16. HPV Detection • “Infection” monitored by DNA detection • Sample and assay frame view of disease • Complicates definition of latent, occult, persistent or recurrent infection

17. Tissue Samples • Biopsies provide direct correlation between pathology and virus • Includes basal layer of epithelium • Limited area sampled • Not suitable for screening

18. Exfoliated Cytology Samples • Noninvasive approach for population screen • Sampling not directed at “lesion” • Quality dependent on collection device and anatomic site sampled • Swabs, brushes, scrapes, washings • Basal epithelium not commonly included • Cervical sample most commonly used in women • Appropriate sample in males is not clear

19. Estimates of HPV Associated Disease in the US • Genital warts: 1%, 1.4 million • Colposcopic (sub-clinical) changes: 4%, 5 million • DNA positive, no lesions: 10%, 14 million • HPV antibody positive but no DNA or lesions: 60%, 81 million • OVERALL 75% of population exposed

20. Natural History Overview • HPV infection is very prevalent in the population • Genital HPV is acquired around the time of sexual debut • Infection is usually transient and not associated with symptoms • Persistent infection is more likely to be associated with potential for neoplasia

21. HPV and Cervical Cancer • Consistent epidemiologic association of HPV with cervical cancer precursor lesions • Plausible biologic mechanisms for HPV oncogenesis • HPV oncogenesis is a rare event with long interval between infection and cancer • Infection alone is insufficient to cause cancer • Additional factors required for neoplasia

22. Questions about HPV Infection • Is HPV eliminated from the host? • HPV “clearing” is monitored by DNA detection in cytology samples • Negative results indicate shedding below limit of detection but basal compartment of epithelium not sampled • HPV can be detected in histologically normal margins surrounding gross lesion

23. Duration of HPV Infection

24. Persistent Infection? • No consensus on definition • Requires detection of same HPV type on more than one occasion • Time interval varies: 3-6 months • Long intervals: re-infection not excluded • Consistent detection on each occasion versus intermittent detection

25. Latent Infection? • Formal definition: Presence of HPV DNA in the absence of virion production • Practical definition: Detectable HPV DNA in the absence of identifiable lesion • HPV DNA positive, normal cytology • Equated with occult infection

26. HPV DNA Assays • Multiple HPV types complicate assays • Sensitivity and type-specificity vary • Inter-assay comparisons difficult • Direct hybridization • Southern blot, dot blot, in situ, HybridCapture • Amplification (PCR) • Type specific, versus consensus

27. HPV Hybrid Capture Assay • Current FDA approved test • 1995 tube format; 1999 micro-titer format • Liquid hybridization technique • Chemiluminescent detection • Semi-quantitative signal, but no control for amount of input DNA • RNA probes react with DNA targets • RNA-DNA hybrids captured and detected with monoclonal antibody to hybrids

28. Hybrid Capture II Assay • Low risk probe mix • HPV types 6, 11, 42, 43, 44 • High risk probe mix • HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 • Good inter-laboratory comparison • Results not type-specific

29. Hybrid Capture II Assay • Designed to work with exfoliated cervical sample • Recommended collection kit includes brush and sample transport media • Collects endo- and ectocervical cells • 5% of total specimen assayed for each probe group

30. HPV PCR Assays • Small portion of genome targeted • Allows testing of samples with poor quality DNA • Small changes in virus (variants or integration) may give false negative results • Amount of DNA assayed varies (limits number of cells sampled)

31. HPV PCR Assays • Type specific assays • Generally target E6/E7 region • Consensus assays • Generally target L1 region • Type(s) determined by type specific hybridization, restriction digestion or sequencing

32. Typing Consensus PCR Product: Roche Line Blot Assay

33. HPV Quantitation • “ Viral load” difficult to estimate because of uneven tissue distribution and variation in sampling • Requires some measure of number of cells in assay (denominator) • Quantitative PCR assays, usually type-specific

34. HPV In Situ Hybridization • Only method permitting direct visualization of virus in morphologic context • Applicable to formalin-fixed paraffin-embedded tissues • Type specificity is good, but cross-hybridization cannot be totally avoided • Results are very technique dependent • Integration status can be determined

35. HPV Serology • ELISA-based detection of antibodies against L1-VLPs • Serum or mucosal; IgG or IgA • Type-specific, at least at low titers • Reaction indicates past or current infection • Less than 70% of HPV positive subjects develop detectable antibodies; lag-time of several months

36. L1-VLP Assays • Formats vary (direct vs. indirect) • VLP production not standardized • Different expression systems, preparative methods, QC approach • No gold-standard for setting threshold for positive result • Few inter-laboratory comparisons