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Papillomaviruses

 

The human papillomaviruses are responsible for producing benign warts. Until recently, little clinical or scientific interest has been generated since lesions were a cosmetic nuisance and the viruses were not thought to be involved in serious disease. Now that papillomaviruses have been linked with genital cancers had stimulated interest. However, research is still hampered by the fact that they will not grow in conventional cell culture systems.

A. Properties

Belong to the genus papovaviridae
naked dsDNA viruses, virions 52-55 nm in diameter
icosahedral symmetry with 72 capsomeres
Genome consists of around 7800 bp, exists in a supercoiled circular form in the virion. All the reading frames are on one strand of the DNA.
Little is known about the proteins produced by the papillomaviruses, in contrast with the polyomaviruses.
7 early proteins are known; E1 to E7, and 2 late structural proteins; L1 and L2.
There is evidence that virus persistence occurs in the squamous epithelium without producing any lesions
HPVs have not been grown in vitro.
At least 57 HPV types are known. The typing is not based on serology but on DNA homology. To be classified as a new HPV type, there must be less than 50% homology with other subtypes

Electron micrograph of papillomavirus particles. (Courtesy of Linda M. Stannard, University of Cape Town)

 

Papillomaviruses have not been grown in vitro, this appears to be due to the fact that the replicative cycle occurs in squamous epithelium with the full cycle occurring only in the most differentiated cells. The episomal form of DNA is generally the usual form of virus persistence for papillomaviruses. Where HPV is integrated, the process appears to be random. As integration occurs, the circular form is disrupted, the breakage occurs invariably in the E1 and E2 region. the E1 and E2 genes encode proteins which have a number of cellular functions. One of the functions of E2 is to repress E6 protein. It is thought that the disruption of E1 and E2 may derepress E6 and E7. E6 and E7 are thought to play a key role in tumourigenesis. E6 binds to p53 and E7 to the Rb protein.
 

B. Epidemiology

HPVs infect and replicate in squamous epithelium on both keratinized and non-keratinized (mucosal) cells. Most people are infected with the common HPVs types 1, 2, 3, and 4. People with the rare autosomal recessive disorder epidermodysplasia verruciformis harbor a number of virus types not found in normal people. Mucosal surfaces can also be infected with HPVs such as the genital tract and the larynx. The oral cavity can also be infected.
   

C. Pathogenesis

1. Clinical Manifestations

  1. Benign warts - HPVs are responsible for the numerous types of warts such as plantar/common hand warts, juvenile/flat warts, genital warts (condylomata accuminata), macular lesions in immunosuppressed patients and patients with EV, and various premalignant lesions e.g. CIN1, CIN2 etc. Uncommon presentations include conjunctival warts that may be occupational hazards to obstetricians and gynaecologists.

  2. Juvenile laryngeal papillomatosis - laryngeal warts are usually caused by types 6 and 11 which have a tropism for mucosal surfaces. (The same types infect genital areas as well). It is considered to be a life-threatening condition in children because of the danger of airways obstruction. Repeated surgery of laser treatment may be required. There have been documented cases where laryngeal carcinomas developed when laryngeal papillomas were treated by X-radiotherapy.

  3. Epidermodysplasia verruciformis - EV is a rare autosomal recessive condition characterized by the appearance of widespread macular lesions and warts on the body. Some of the HPV types are not normally found in normal people. One-third of EV patients develop squamous cell carcinoma, usually in areas exposed to sunlight.

2. Association with Cervical Cancer

The association between certain strains of HPV and cervical cancer is well known. In the West, cervical cancer is the fourth most common cancer in women. In developing countries though, it is the most common cancer, with 1/2 million new cases per year. Overall, it is the most common virus associated cancer worldwide, followed by hepatocellular carcinoma, NPC and Burkitt's Lymphoma. There is now compelling evidence for an aetiological role of HPV in cervical and other cancers.

a. Epidemiological Evidence ;- it has been known for a long time that the incidence of cervical Ca. is related to sexual activity. Promiscuous women are far more likely to develop cervical Ca. Nuns rarely get cervical Ca. Several sexually transmitted agents have been implicated including Herpes Simplex. It is now fair to say that evidence of a role for HSV is very slim and now very much discounted.

b. Virological Evidence ;-

  1. It has now been proven that certain precancerous conditions for cervical and vulval Ca, namely CIN and VIN, are caused by HPV. Normal human epithelium from the cervix and epithelium purposefully exposed to HPV 16 were transplanted to nude mice. (immunologically immature mice which are not able to reject the graft). Weeks later, lesions that resemble CIN histologically were detected in the epithelium which was infected by HPV.

  2. HPV genomes can be detected in 90 - 95% of tumour cell lines. HPV genomes can be found at a lower frequency in pre cancerous state such as CIN1, CIN2 and CIN3. HPV 16 and 18 are the most commonly found. However, other strains may be present, especially in the less dysplastic lesions. Types 6 and 11 are commonly present in the less dysplastic lesions but not in the more dysplastic ones.

  3. HPV DNA persists in an episomal form in normal infected cells but is integrated in the host cell chromosome in malignant cells. The integration of HPV DNA appears to be random. It is thought that as integration occurs, the breakage always occurs in the E1 region. One of the functions of the E1 region is to negatively control the expression of the E6 and E6 genes. Therefore, it is thought that as a result of the integration, the E1 function is lost and the E6 and E7 genes are derepressed. This leads to an overproduction of the E6 and E7 gene products. E6 is shown to inactivate p53 and E7 the Rb proteins.

  4. HPV DNA is biologically active in vitro. It is able to transform human fibroblasts and keratinocytes. It has effects on epithelial cell differentiation and induces DNA replication in the fibroblast. All the above effects can be nailed down to the E7 reading frame. The constant expression of the E7 and perhaps the E6 reading frames products are essential for the maintenance of transformation.

  5. Antibodies to HPV are more prevalent in patients with CIN or invasive Ca. Gissman et al. looked at the prevalence of antibodies to the E7 and E4 HPV proteins in normal people and those with Ca.Cervix. Although the prevalence of Abs to E4 and E7 were increased in patients with Ca.Cervix compared to the controls. The prevalence of Ab to E7 was much higher ; There was only a 2 fold difference in the prevalence of Ab to E4 between the cancer patients and the normal controls. However there was a 7-fold difference in the prevalence of Ab to E7. This suggest that there may be a role for testing for Ab to the E7 protein in future for use as a marker in monitoring the effectiveness of treatment. However, it is very important to have the correct protein antigen of the particular strain of HPV involved.

It must be borne in mind that other factors are involved in the tumourigenesis of cervical Ca ;-

  1. The actual site in the body
  2. The HPV type
  3. The immune status of the patient; immunocompromised patients have a much higher risk of developing Ca.Cervix.
  4. Smoking
  5. Hormones
  6. Vitamin A deficiency

3. Association with tumours in immunocompromised individuals

10% of the general population have warts at any one time but warts and skin cancers are much more common and of a greater problem in immunocompromised patients, in particular renal transplants and people with AIDs. The actual number of people undergoing renal transplant operations have increased gradually in the last 20 years. As survival times increased, it became apparent that many of them are developing serious problems with warts and skin cancers. The problem seems to be related to the maintenance immunosuppressive therapy, which is prescribed to patients as long-term therapy after their operation. Prednisolone and Azothiaprine are the 2 most common immunosuppressants given.

Many renal transplant patients appeared with horrific intractable warts in all parts of the body. The warts could not be controlled by any methods but may respond to the withdrawal of the immunosuppressive therapy. Some of these patients with warts progressed onwards to malignancy in many parts of the body at once. Several factors are known to contribute to malignancy ;-

  1. Diminished immunosurveillance
  2. UV exposure
  3. Oncogenic effects of the drugs given
  4. Chronic antigenic stimulation

The longer the graft survives, the more likely that patient will develop warts and skin cancers.

  Warts Kerratoses Cancers
< 5 years 35% 15% 2%
> 5 years 83% 36% 13%

The widespread appearance of warts in renal transplant patients resembles those found in the condition Epidermaldysplasia Verruciformis (EV). EV is a rare autosomal recessive condition characterized by the appearance of widespread warts on the surface of the body and 30% of the development of squamous cell carcinomas (SCC). Over 20 types of HPV are associated with EV and the HPV DNA exists mainly in the episomal form in the lesions of these patients. In renal graft survivors the ratio of basal cell carcinoma to SCC is 1:15 compared to a ratio of 1:5 for a normal population. The female renal transplant patients are also at a much higher risk of developing cervical Ca., CIN and VIN than the normal population. The precise role of HPV in the causation of skin cancers in the renal-transplant patients is not known. The skin lesions do resemble those of EV on appearance and the DNA of HPV 5 and 8 ( the HPV strains commonly isolated from patients with EV ) are found in 50% of SCC of the renal transplant patients. HPV 5 and 8 may act as inducing agents for the development of skin tumours. In a recent study, it was claimed that the prevalence of anti-HPV 8 Ab is increased in patients with skin tumours ;-

Controls 20%
Basal Cell Carcinoma 40%
Squamous Cell Carcinoma 80%

If the above can be substantiated, then HPV may have a far more sinister role in human cancers than previously thought. It may have a role in inducing the development of skin and other epithelial cancers. It is not clear whether a withdrawal of immunosuppressive therapy will have a beneficial effect on skin cancers. Although some patients with intractable cancer problems have been offered a withdrawal of immunosuppressive therapy. There have been no cases of metastasis from a SCC arising in an ex-renal transplant patient to date.  

4. Persistence of HPV infection

There is circumstantial evidence that HPVs may persist in the squamous epithelium, without producing recognizable lesions, in a fashion similar to the persistence of polyomaviruses in the kidney. Up to 42% of allograft recipients develops cutaneous lesions within a year after transplant. This suggests that transplanted patients experience either new infections or reactivation of latent virus. HPV DNA sequences have been found in biopsies of normal areas of the larynx of individuals who have had recurrent episodes of laryngeal papillomas.
   

D. Laboratory Diagnosis

The appearance of warts vary from the scaly flat lesions on the epithelium of individuals with EV, to the aceto-white flat CIN lesion of the cervix. It may be hard to distinguish wart lesions from other lesions such as molluscum. The methods which are in routine use are:

  1. EM of skin scrapings
  2. Detection of the common HPV antigen in the tissue by IF techniques. This is more sensitive than EM even though the antigen is not detectable in 25% of confirmed warts.

DNA-DNA hybridization and PCR techniques have been developed in research laboratories. The Southern blot is the most sensitive and specific hybridization technique but is labor-intensive and time-consuming. Dot-blot is available in commercial kits and is as sensitive as Southern blot but not as specific, being unable to distinguish between cross-reacting closely related viruses. RNA probes are more sensitive than DNA probes because of the stability of the RNA-RNA or RNA-DNA hybrids compared to DNA-DNA hybrids. Commercial high sensitivity hybridization assays such as the Digene hybrid capture will detect HPV types that are associated with cervical cancers from biopsies of genital lesions. However, the usefulness of these tests in a histopathological setting remains controversial and more research is required.

 

E. Management and Prevention

Although they are a cosmetic nuisance in most cases, warts are notoriously difficult to treat. The treatments which could be used include cryotherapy with liquid nitrogen, podophyllin (an antimitotic agent), lasers (for mucosal lesions such as CIN), electrodiathermy, and surgery. Interferon has been used successfully to treat recurrent laryngeal warts and CIN. However, the cost of the treatment may prove to be prohibitive. Precancerous CIN-2 and CIN-3 lesions should be treated by lasers, electrodiathermy, or surgery. In the case of immunocompromised patients with intractable warts, improvement is often seen. There are mass screening programs for CIN and cervical cancers in a number of countries.

HPV Vaccination

Being one of the most common cancers in women, a vaccine against HPV types associated with cervical cancers would be desirable and a lot of research has been carried out in this area. To date, there are two licensed vaccines available: Gardasil and Cervarix. They are made of VLP (virus-like particles) from the major capsid protein L1 of HPV strains. Cervarix contains L1 proteins of the cancer-associated strains HPV 16 and 18, which are associated with 70% of cervical cancers. Besides HPV 16 and 18, Gardasil contains HPV 6 and 11 which are associated with 90% of genital warts. The vaccines are designed to elicit the production of virus-neutralising antibodies and prevent the initial infection by those strains. The vaccines have been shown to offer 100 percent protection against the development of precancertous cervical cancer lesions and genital warts caused by the HPV types in the vaccine, with little or no side effects. The protective effects of the vaccine are expected to last a minimum of 4.5 years after the initial vaccination.

The target group for vaccination are women between 9 to 25 years of age who have not contracted HPV. Although HPV 6 and 11 do not cause cervical cancers, they cause genital warts that may result in considerable pain and discomfort. It is thought the inclusion of HPV 6 and 11 would make the vaccine more attractive, particularly to men who are not at risk of cervical carcinoma. Because of the high cost-benefit that may result from the prevention of cervical carcinoma, there are ongoing studies and proposals that aim make HPV vaccination available to schoolgirls in a a number of countries including the US, Canada, and Australia.

 

 

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