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

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

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

HPV Genome: Episome

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

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

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

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

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

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

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

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

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

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 Variants best characterized for HPV 16 E6/E7 polymorphisms could modify oncogenicity Cross-reactive in ELISA assays

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

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

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

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

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

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

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

Questions about HPVInfection 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

Duration of HPV Infection HPV TypesMonths, median (95% CI) Franco (1999)“Oncogenic”8.1 ( ) Woodman (2001) HPV ( ) HPV (6.0 – 12.6)

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

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

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

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

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

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

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)

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

Typing Consensus PCR Product: Roche Line Blot Assay

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

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

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

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