Human Papillomavirus

General Information:

Human Papillomavirus is a virus from the Papillomaviridae family of non-enveloped circular double stranded DNA viruses with icosahedral capsids, that commonly infects both cutaneous and mucosal cells in humans.
Genome comprises ~8kbp and codes for a total of 8 genes classified by their early (E) or late (L) expression.
HPV is the most common sexually transmitted infection worldwide
They are most known for their roles in both internal and external genital cancer as well as oropharyngeal cancer.
Around 90% of infections are asymptomatic and it is thought that approximately 80% of woman are affected by the virus at some point in their life.
In Scotland, the incidence of HPV is high, with approximately 1 in 5 women attending cervical smears testing positive for HPV.
Herpes

Types of HCV:

HPV 16 and 18 account for ~70% of cervical cancers
- Type 16 is considered the most potent strain of the virus
- Other high risk strains for cervical cancer are 31, 33, 45, 52 and 58 (among a number of others)
HPV 6 and 11 are also a problem, most commonly causing genital warts (90% of cases) and laryngeal papillomatosis

Transmission:

Transmission most commonly occurs via skin-skin transmission, by means of multiple sexual partners, smoking and poor immune function. One of the most significant risks, however, presents in those who begin having sex at an early age, before they have had their HPV vaccination.
HPV can survive for months without a host as long it remains at a relatively low temperature, therefore those with warts on the underside of their feet (e.g. verrucas) can spread the virus by walking barefoot.

Virology Behind Infection:

HPV exclusively replicates in the basal cells of stratified epithelium, mainly affecting the transformation zone.
It cannot infect live tissue and thus can only infect an individual via epithelial abrasions which reveal the basement membrane. Such abrasions occur during sexual intercourse, thus explaining why HPV is typically transmitted in this way
HPV connects with alpha integrin receptors among a number of others, therefore entering the cell via endocytosis.
As the cells mature, the L1 and L2 proteins are expressed by the virus, allowing replication to occur.
Interestingly, HPV infection does not bring about the death of a cell, rather spreading via the natural desquamation process of epithelial cells.

Oncoproteins:

The main oncoproteins expressed by the high risk strains of HIV are E6 and E7, whereby 'E' describes a protein that has been expressed early (L = late expression). These proteins selectively disrupt two critical tumour suppressor proteins. The E6 oncoprotein inhibits p53 by ubiquitinylation, whereas E7 inhibits the active form of the retinoblastoma protein by directly binding to it, so its main substrate (E2F) cannot, leading to disregulated cell replication (thus leading to cancer cells not being apoptosed).

Direct HPV Testing:

In Situ Hybridisation
- Identifies a certain genetic chain in cells by use of a probe containing a complementary nucleotide sequence. The probe is sometimes fluorescently tagged, thus making it FISH, which then visualised used UV light.
- Less sensitive than PCR
Polymerase Chain Reaction
- PCR can be utilised to identify HPV in samples by first amplifying with specific primers before analysis by application of restriction enzymes, amplicon sequencing, or a number of other methods.
- This is more sensitive than ISH and FISH

Indirect HPV Testing:

Detection of Cellular Abnormalities in Histological Samples (CIN Grading)
- Smear testing programme in the UK:
  - under 25 - up to 6 months before you turn 25
  - 25 to 49 - every 3 years
  - 50 to 64 - every 5 years
  - 65 or older - only if 1 of your last 3 tests was abnormal
- Based on identification of nuclear abnormalities in uamous cells. The nucleus becomes a 'raisinoid' shape, hyperchromasia occurs (darker staining of nuclei due to increased DNA production) and chromatin granules are dispersed within the cell. Cells exhibiting these features, that have been infected by HPV, are known as koilocytes.
- CIN 1:
  - Classed as koilocytosis. The abnormal koilocytes are only found on the lower third of the epithelium. Either this, or it is that there are abnormalities in the upper segment of the epithelium implying that HPV maturation has occurred.
  - Koilocytes are larger than usual and an abnormal shape with well defined perinuclear halos and increased thickness of the cytoplasm. Nuclei of these cells are significantly enlarged. Such changes to the nucleus are required for a diagnosis of koilocytosis.
- CIN 2:
  - Dysplasia affecting the lower two thirds of the cervical epithelium.
  - Mixture of low and high grade lesions
- CIN 3:
  - Extreme dysplasia of the entire thickness of the epithelium
  - This is also referred to cervical carcinoma in situ
  - P16 (cyclin-dependent kinase inhibitor) immunostaining for overexpression is used to differentiate a Low Grade Squamous Intraepithelial Lesion (LSIL) from a High Grade Squamous Intraepithelial Lesion (HSIL) - positive staining with P16 = HSIL. This compound is widely overexpressed in cancerous cervical tissue. Staining of Ki67 is also occasionally used to assess the severity of the lesion.
Serological testing
- Antibody testing for antibodies associated with the relevant strains of HPV. Most utilised is a monoclonal antibody against HPV L1, which is the major capsid protein of HPV.
- The best serological test currently in use for HPV comprises ELISA testing using HPV capsids
- Anti-capsid antibodies are however not a perfect means of testing for HCV, as such antibodies are undetectable in approximately 20-40% of woman that test positive for cervical HPV.
Over-expression of certain cellular proteins

The Ideal Test For HCV Must...

Have its efficacy proven by peer-reviewed studies.
Have both high sensitivity and selectivity
- HPV commonly clears, therefore the sensitivity of the test should naturally surpass its specificity
- The negative predictive value is generally higher than the positive predictive value, meaning that individuals who test positive may not actually have any clinical disease
Be available at a reasonable price
Be easily used and interpreted
Detect strains of HPV which leave a patient at high risk of disease
Not give positives for strains of HPV that are not clinically relevant, so as not to cause over-treatment of the population

Cervical Screening in the UK (THEN)

Used to start with Cytology in the form of a cervical smear test. This is the more specific test.
Women with low grade dyskaryosis then had their cells sent for a HPV test which is the more sensitive test. They were also referred for colposcopy. Their appointment would be within 6 weeks.
- The dogma of screening is to do the more sensitive test followed by the specific test, to first identify all those who may have the condition before separating out those who do from those who don't. Therefore, this way of conducting screening didn't make sense.
Women with high grade dyskaryosis a referred for colposcopy to be seen within 2 weeks.
At colposcopy appointments, speculoscopy using stains are sometimes used to help the examiner visualise the cervix.
- If acetic acid is used, areas of cervical intra-epithelial neoplasia (CIN) will stain white
- If iodine is used, areas of CIN appear dark brown
- The individual doing the colposcopy may take a biopsy to confirm any suspected diagnosis

Cervical Screening in the UK (NOW):

Colposcopy system of referral is unchanged.
The Scottish Government changed the order of screening so that it applies to the dogma of screening (Sensitive test followed by specific test). This was fully implemented in March 2020 in Scotland, following NHS England's implementation of this scheme in 2019.
Many countries (Italy, Norway, UK, etc) have started using HPV testing as a method of primary screening instead of cytology which has become secondary screening; basically flipping the order in which things are done. A swab is taken at the time of the cervical smear and tested for HPV instead of the cells being sent for cytology.
- Starts with HPV test by taking swab during a cervical smear test. This is the more sensitive test, so is able to effectively identify those most likely to have HPV-associated epithelial changes
- Women with low grade dyskaryosis have their cells sent for a Cytology which is the more specific test (able to identify those who do not have the epithelial changes associated with HPV e.g. koilocytes).
Main advantages of this change:
- Higher sensitivity means that an increased number of woman that are at risk of cervical cancer will be identified
- Lower false negative rate means that less women will have to attend follow up appointments, which saves the country money.
- This will also lead to a massive reduction in cytology workload, which will on one hand save the country money, but a disadvantage is that it will likely lead to many people losing their jobs

Biological Markers:

The Smear test's main disadvantage is that it has a high false negative rate. Studies have shown some of these false negative samples to contain no trace of abnormal cells. Why? Because it relies on the shedding of epithelial cells which have HPV within them. Therefore, if the virus has not yet expanded to reach outwith the basal epithelium, the result for the smear will be negative.
The identification of certain biomarkers, such as HPV E6, E7 and P16 (cyclin-dependent kinase inhibitor), via specific assays are occasionally used as an adjunct to HCV screening to get around this problem.
Assays which detects hyper-methylated HPV L1 have also been developed and have been shown to indicate viral persistance and an increased susceptibility to oncogenic development

HPV Vaccination In The UK...

Costs around £110 million annually.
Given to girls (and now boys) aged between 12-13. Minimum 6 months between each dose.
Used to be bivalent vaccine (Cervarix, which immunised against HPV 16 and 1. This was changed in September 2012, where Cervarix was replaced by quadrivalent vaccine (Gardacil), which protects against HPV 6, 11, 16 and 18 to encapsulate protection against genital warts also.
In 2017, a bill was passed meaning that men who have sex with men who attended the GUM clinic were now offered the HPV vaccine
In the past year, a decision has been made by the UK government to start immunising boys with the HPV vaccine as well as girls, however in December the WHO called for the suspension of male vaccination, as they feared a shortage would result in a reduced coverage within females.
- Interestingly, Scotland recently changed from a 3 dose system for HPV vaccination to a 2 dose system, following scientific data showing the latter was more effective.
- The WHO recommends 2 dose for girls under 15, 3 doses for girls over 15/those at a higher risk of HIV.
Question: Can both a vaccination and screening programme be justified? This could be an exam question.
- Between 1988 and 1995, the implementation of HPV vaccination was shown to reduce prevalence of HPV 16 and 18 in women by over 25%.
- It has also brought about significant reductions in the rates of CIN in women significantly (>50% reduction in CIN3 cases)
- The same study also showed a significantly increased herd immunity of women who were not vaccinated in 1995 compared to those in 1988.
- It has been shown that only having 1 dose of the vaccine results in levels of antibody that are far higher than those achieved by natural infection, thus possibly suggesting that a future vaccine may only require one dose, thus potentially saving the country money.

Challenges Associated with HPV Vaccination

Claims by anti-vaccine groups regarding Postural Orthostatic Tachycardia Syndrome, as well as chronic fatigue syndrome and complex regional pain syndrome, are associated with the condition have brought about significant challenge in getting some women vaccinated. The EMA have released a paper disproving these theories.
- Despite this worry, it is estimated that since its implementation the vaccine program has had over 80% uptake among women.
An example is Japan, in which the government stopped proactively recommending that women should be vaccinated, resulting in a drop from 70% uptake of the vaccine to 1%.
Another example is the R.E.G.R.E.T. movement in ireland oweing teenage girls 'otherwise unexplainable' health conditions to the Gardasil vaccine which led to a drastic reduction in girls getting their HPV jabs.

New 9v HPV Vaccine

A 2015 study into a new form of vaccine, the 9vHPV Vaccine, was a double-blind randomised controlled trial which displayed a new type of vaccine which took into account strains 16, 18, 6 and 11 as well as 5 new strains (31, 33, 45, 52 and 58).
This study was reliable as it blinded the pathologists and participants involved in the study as to the vaccine type (thus reducing bias). Also, a large number of women of a variety of ethnic background were included in the study, increasing the reliability.
The limitations of the study were that the study group were a lot older than the girls currently vaccinated, so it is not known how the vaccine will affect girls of 12-13. Also, there was no direct comparison made between the abilities of this vaccine and the gardasil vaccine to protect against strains 6, 11, 16 and 18, so in this respect it is not known which is more effective.

	Created by Matthew Coulson over 4 years ago

Honours Degree Microbiology (Medical Virology) Note on Human Papillomavirus (HPV), created by Matthew Coulson on 24/03/2020.

Human Papillomavirus (HPV)