# Presentation: 465239

## 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 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