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

E6

E7

E2

E4

E5

L1

L2

E1

URR transformation

transformation

episomereplication

late cytoplasmicprotein

transcriptionalregulator

capsidproteins

P97

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 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

Duration of HPV Infection

HPV Types Months, median (95% CI)

Franco (1999) “Oncogenic” 8.1 (6.0 -12.6)

Woodman (2001)

HPV 16 10.3 (6.8 - 17.3)

HPV 18 7.8 (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