Review Article
Clinical Medicine
Infectious Diseases
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This article discusses HPVs, and is hopefully targeted at clinicians wishing to gain insights into HPV infection. It covers briefly salient points about HPV, relating to:-
Background and Provenance;
Vulnerable sites; phenotypes; configuration and genetic structure;
Ingress, growth and infection;
Maturation and reproduction;
Cellular release and discharge;
Neoplastic change and oncogenic progression;
Oral, pharyngeal, head and neck cancer: “Clinical Smarts”;
Prophylaxis and therapy;
Concluding remarks
References.
KeywordsCancer, Condyloma, DNA, HPV, Infection, Papilloma, RNA, Viral-therapies, Vaccine, Virus, Warts
Author info
McGill University, Faculty of Dentistry.
RecievedSep 05 2013 AcceptedOct 02 2013 PublishedOct 23 2013
CitationLouis Z.G.Touyz (2013) Human Papilloma Virus (HPV)-A Biological and Clinical Appraisal: 2013. Science Postprint 1(1): e00001. doi:10.14340/spp.2013.10R0002
Copyright©2013 The Authors. Science Postprint published by General Healthcare Inc.. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 2.1 Japan (CC BY-NC-ND 2.1 JP) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
FundingThis article is a spontaneous contribution from a full-time tenured academic professor; no conflict of commitment nor financial interests exist.
Competing interestsThe article is an objective, neutral balanced appraisal of existing knowledge, to the best of the authors perception, understanding and interpretation. Consequently there are no competing interests.
Corresponding authorLouis Z.G.Touyz
AddressMcGill University, Faculty of Dentistry, Strathcona Anatomy & Dentistry Building 3640 University Street Montreal, Quebec, Canada.
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Viruses are obligatory intracellular parasites, and demand a living cell to reproduce. Papilloma viruses evolved millennia ago and are known to infect lower orders of organisms like amphibians, reptiles, birds and mammals. Most are host specific and rarely cross the species barrier. About a short 150 years ago, scientists and biologists disproved the theory of spontaneous generation. Louis Pasteur explained anaerobic fermentation of sugars by yeasts to produce alcohol, and Koch postulated the necessity of microbes to produce disease. The revelatory microbial organisms were cellular, demonstrable, visible and resultant reactions were manifest. At that time sub-cellular biological entities were scoffed at, although viruses were long suspected to exist as were diseases deriving from them. In 1907 Guiseppe Ciuffo showed human warts could be induced by viruses. As early as 1911, the concept of a biologically viable, macroscopically invisible virus was demonstrated by Peyton Rous, who showed a cell-free filtrate could induce sarcoma tumours in chickens. In 1930 Richard E. Shope initially isolated the influenza virus, then in 1933 demonstrated the cotton-tail rabbit-virus from infected leporidal skin. Subsequently Shope demonstrated this virus could induce skin growths (papillomata) in rabbits. From 1974 to 1977 suspicions that many human neoplastic growths derive from human papilloma-viruses (HPV) was suggested by K. Syrjanen. In 1983/4 Harald zur Hausen described high-risk HPV-16 and HPV-18 from cervical cancers.1,11 Over 95% of cervical cancers correlated with the presence of these HPV genotypes, but excluded a small, but important, subset of other HPV free neoplasms. These observations provided persuasive evidence that HPV was causally related to the aetiology of cervical cancer.
Well over 150 different HPV genotypes have been described, with at least 10% difference in DNA sequencing determining specific characterization. Closely related types tend to have similar biological characteristics, like strong tropism for specific tissues, manifestation of disease, and pathogenicity.
Growing microbes present unique problems in research and demonstrations for evidence are always challenging. Key information was procured by growing viruses in fertilised eggs, but some viruses are inordinately difficult to culture. This precluded gaining useful research information from differentiation processes, such as in maturation of keratinocytes which occurs in the skin. Since epithelial maturation processes are essential for HPV growth, multiplication and re-infection, research progress was stunted till cell-raft culture was developed.
The α-papillomavirus group of HPV (some 15) infects mucosal sites such as the upper aerodigestive tract, the head and neck organs, and ano-genital tract. Each of these sites has abrupt transitional zones from one type of mucosa to another. Proximal linings of body cavities are stratified squamous epithelia, while more internal epithelial linings are typically columnar and some ciliated (the respiratory tract). A circular field of rapidly dividing and differentiating keratinocytes, called the metaplastic zone establishes a squamo-columnar transformational area at each end of the body opening. These anatomic areas are found in close proximity to the anal/rectal junction, the uterine cervix, the urethra, the nasal sinuses, the larynx and the oral cavity. Each metaplastic zone is susceptible to HPV ingress, infection and may support various degrees of viral reproduction. Many HPVs are mucotropic, may be transmitted through sexual activity, and can cause local and or systemic diseases. Accordingly these HPVs are subdivided into low-risk (LR, such as HPV-6 and HPV-11[species 10]) and high-risk (HR, like HPV-16[species 9] , HPV-18 [species 7], HPV-51[species5], and HPV-53 [species6]. HPVs are small DNA viruses and may cause benign or malignant tumours.5 They are responsible for infections of the upper aero-digestive tract as well as causing genital lesions. They are non-enveloped icosahedral structures with L1 and L2 proteins organised as surface pentamers.
The genome is visualised as either a (conventional) linear or circular map with early (E) and late (L) proteins being encoded as promoters, moderators or inhibitors of genetic changes. 3-7
Most HPV infections are acquired from close physical contact, exchange of bodily fluids, or from viable virion exposure to receptive cells. Repeated intimate physical contact, kissing and sexual activity are among the major sources of transmission, though vertical transmission from mother to child is also feasible, particularly from infected mothers to babies born after prolonged labour with difficult natural births. Most skin infections are acquired early in infancy from infected persons. 15 HPV infection is dependant on inflicted breaches in the epithelium (like cuts , tears or abrasions) through the top layers to access and infect dividing basal layers of epithelium. The progressive stages toward replication are referred to as the establishment phase, followed by the maintenance phase and finally the productive phase, with the HPV reproductive cycle being totally dependant on the complete squamous cell differentiation of the host epithelium.
Enormous research progress was achieved when organo-typic raft cell-cultures were perfected. What are these raft cell cultures? Essentially they are fibroblasts embedded on a collagen matrix, and seeded with keratinocytes from human neonates’ foreskins. When this experimental scaffold is cultured in air, the keratinocytes proliferate and differentiate and layer themselves into similar strata of the epidermis. The final resolved experimental model is similar to stratified squamous epithelium, is vital and allows for maturation not only of keratinocytes, but also for genetic development, maturation and expression of HPVs. The HPV protein formations are complex and mRNA may translate into different proteins. These are referred to as polycistronic mRNA. Some of their functions are known, but the totality of their exact functions remains obscure.
The basal cells divide asymmetrically leaving one cell in situ as a source (basal cells), while the other cell migrates toward the epithelial surface. The latter are transit amplifying cells, and undergo different cellular biochemical progressions.
There is HPV-DNA replication with low replicated numbers, in the dividing basal- and para-basal keratinocytes; but increased RNA transcribing and DNA augmentation to high replicated numbers happens mainly in the differentiating middle and spinous layers of stratified squamous epithelia. The capsid protein is scant in superficial cells, because… as these cells mature and differentiate to keratin… they loose those enzymes and substrates needed for DNA replication. Similarly squamous and/or glandular carcinomas do not promote viral reproduction. Intact immunity modulates viral replication, as immuno-suppressed patients show increased viral activity and resultant lesions.4
In HPV infected keratinocytes the double stranded DNA replicates in the nucleus and produces copies, but is located in extra-chromosomal plasmids. RNA polymerase II promotors are designated P1, P2 and P3 as the main promotors of low-risk HPVs, and these P1, P2, and P3 promotors are located in close dependant proximity of expression of oncoproteins E6 and E7.2
Mainly early (and some late) primary transcripts are spliced in genomes to pattern protein transcription for the high-risk HPVs, as in HPV-31.3 P97 splices with an early polyadenylation signal (pAE) to form combinations of E6, E7, E1, E4 and E5, and also E6,E7,E1,E4 and E5.
Late viral gene expression depends on a late-stage viral promoter, a specific late-stage polyadenylation site and a late stage specific splicing site. P99 and P742, late transcripts, splices with AE 4140 and AL4140 and form combinations of E1,E4, E5; and E1,E2, E5; and E1,E4,E5,L2,L1; and E1,E4, L1.3
HPVs are closely associated with cell differentiation in infected epithelium. Protein E1 and E2 (in low titre; at high titres E2 represses E6 and E7 activity) serve as accessory replication complexes, as E1 expresses DNA helicase activity that unravels DNA and interacts with other intra-cellular host proteins to facilitate DNA replication. Not only does HPV proteins E5, E6 and E7 all facilitate viral replication by establishing a conducive intracellular milieu to allow DNA assemblage, but also these proteins induce proliferation of epithelial cells, and simultaneously the production of viral DNA.10
L1 and L2 from late capsid genes are involved in forming pentameric- shaped capsomeres of stable HPVs. Papilloma virions do not have a membrane envelope, but L1 capsid proteins have cross-linked disulfide bonds which give stability to the virus-like particles when released from the host cell. The L1 protein may be used to produce anti-HPV vaccines. HPV binding to extracellular matrices derives from a conformational change on the receptor site, possibly from proteolysis. The reducing environment in the cell fractures the disulphide cross-links and the acidic environment of the endosome, dissociates the capsid to release the viral genome into the cytoplasm, from whence it is transported into the host cell nucleus.
Late HPV molecules E1ˆE4 bring changes in the morphology, width, composition and toughness of the maturing infected keratinocytes which facilitate cell desquamation and virus release. Microtrauma, producing mechanical wounds, tears or abrasions, exposes underlying basal and parabasal cells to concentrated packages of virus, and induce epithelial proliferation. Normal human epithelium is about 8 to 10 cells thick, but condylomas, resulting from HPV infections easily reach 100 cell layers.
Precise reasons for activation of dormant viruses remain obscure, but trauma, reinfection with HR strains of HPV and some carcinogenic chemicals may accelerate oncogenesis with increases in oncogenic molecule production (E6,E7) and reduction of p53.8-11
HPV infected epithelia undergo cellular changes which are reflected as histological changes; changes in nuclear material, disrupted maturation and unco-ordinated mitoses are all part of what is termed dysplasia. Dysplasia is graded from as mild, moderate or severe, or as grade 1, 2 or 3 respectively. Dysplasia grades 1 to 3 display levels of E6 and E7, but expression of these proteins is upgraded most in grades 2&3. 10
Should HPV DNA integrate into the host chromosomes, rather than localizing in the extra-chromosomal plasmid, the host chromosome may progress faster to neoplastic change. 18 The development of warts and with high risk strains of HPV, E6 and E7 protein expression is prime. HR HPV E6 is best known for its ability to degrade the tumour suppressor protein p53. p53 modulates the genome by inducing genes to influence cell-cycle arrest, DNA assemblage, cell senescence and programmed cell death (apoptosis), by affecting host proteins that regulate cell polarity and signal transduction including hDLG, HScribble, MAGI, GAP and telomerase (hTert). 5. Together with HR HPV E7, these molecules destabilize pRB proteins and immortalize growth of cells from mucotropic sites (like foreskin and cervical keratinocytes). 8, 9 HPV E7 protein forms a complex with pRb, inactivates the latter, and encourages cell mitoses by expressing transcription factor E2F, which in turn initiates hyperplasia. Raised titres of p16 are allied to HPV E7 action, and both are increased in cervical neoplasia and high-grade CIN. 12
Chronic infection with high-risk HPV seems to be a necessary pre-requisite for many, but not all, cancers of the cervix. Yet invasive neoplasia do not manifest in all subjects infected with HR HPV. Cancer is uncommon among women (and men) infected with high-risk HPV, because infection with high-risk HPV is frequently self limiting, and High-Risk HPV’s remain dormant in affected cells for decades. Generally at least a decade or more passes prior to developing histologically identifiable neoplastic changes from stage 1 dysplastic lesions (CIN lesions). An HPV-infection will develop in progressive inexorable stages to frank cervical carcinogenesis, as different biological moderators are changed. Data from high-risk HPV in vitro transformation studies and data from clinical specimens reveal a progressive metamorphosing model from dysplasia to neoplasia in cervical cancer. Consequently this model reflects important fundamental observations in deciding at the early stages, after initial HPV-infection, which individuals are susceptible to developing cancer from specific HPV types. This susceptibility is determined by the genetic composition of immune moderating proteins. When an infected HPV infected dysplastic lesion results in changes in DNA, transcription control of the high-risk HPV
factors produce moderation in the E6/E7 oncogenes. This disrupts genomic stability and allows the critical genetic processes for cell changes, from a virus-replicating cell infection, through a stepwise progression toward neoplastic change. Subsequent extra epi-genetic modifications assemble in Grade 3 CIN basal and para-basal cells and produce evident cancers by altering moderators (like E2) and growth factors (like p53) controlling E6 and E7oncogenes. The cellular scaffolds and environments slowly transform to being less reactive to these growth-modulating influences, which are mediated by cell-to-cell cytokines and cell-to-cell membrane receptors, intra-cellular, and extra-cellular chemical cascades.
Also other possible factors affecting cervical carcinogenesis is the effect of female hormones like estrogen and progesterone. Other modifications may derive from inflammation (common in gynecological pelvic inflammatory disease) and associated inflammatory cytokines like tumor necrosis factor (TNF-ά) and metallo-proteases, which enhance invasiveness of malignant cells. Identifying high-risk HPV infections early and implementing aggressive vaccinations, and chemotherapy may minimize malignant transformation of high-risk HPV infections, and also maximize effects of other selected therapies. Almost all laryngeal papilloma are caused by HPV-6 and HPV-11; early detection and treatment could optimize prevention against developing recurrent, respiratory papillomatosis (RRP). Because there are as many as 150 different HPV’s, and not all their patho-biological properties have been elucidated, it is salutary to recall that currently available HPV vaccines (from HPV-16 and HPV-18 for cervical cancers; and HPV-6 and HPV-11 for condylomata) are not absolute in preventing, or curing existing HPV infections.
Head and neck cancer is prevalent worldwide, but those caused from tobacco-and alcohol-abuse are declining, while HPV-induced head and neck cancers are increasing. With the advent of sensitive laboratory techniques to grow and demonstrate HPVs, detection of HPV has highlighted how prevalent the virus is in head and neck cancers. Recent studies concur that HPV is prevalent in the vast majority (around 90percent) of advanced grade 3 CIN and invasive cancer of the cervix, and in most oro-pharyngeal squamous cell carcinomas (70percent OSCC). Finding HPV in OSCCs seems to be increasing in prevalence as in 1980’s it was only 10 percent in the USA, compared to 70 percent reported in decades following till 2010. Certainly six types of cancer namely the oropharynx (including tongue-base and tonsils), vagina, vulva, cervix, penis and anus have definitively been causally associated with HPV.14, 15 Cervical neoplasia biopsies repeatedly show high risk HPV genomes. 11, 13, and high risk HPVs are also associated with anogenital, vulval, penile and even breast neoplasias. HPV 16 and occasionally HPV 18, have been identified. 11, 14, 15, 16,17
Macroscopic changes in the appearance of epithelium, mainly as color, form and texture modifications may be seen clinically as atrophy, hyperplasia, leukoplakia, erythro-leukoplakia, speckled leukoplakia, or erythroplakia.
Sessile or pedunculated growths are recognized as condylomata, papillomata or warts. Any changes in appearance should carry a high level of suspicion, and all mucosal changes showing other demonstrable differences (the ODD principle for diagnosis) from health, should be excised, biopsied and further investigated.18 Viral culturing, typing, cell biopsies, histopathological examination (to detect abnormal cytology, dysplasia and malignant change), genetic, biochemical (PCR) and serological tests (immunofluorescence with antibodies) and investigations could all contribute to refining and defining the infection.24, 25
Mechanisms to block pathways or inhibit growth progressions are ongoing. There is only limited success from therapies discussed below, as no single therapy has proved to be consistently reliable. Concomitant unavoidable damage to adjacent structures occurs, ultimately often producing unsightly scars and recurrence. Type-specific inhibitors would be desirable as for example HPV-11 and HPV-6… the main aetiology in RPP… but type-specific inhibitors for other infected sites (oro-pharynx, vagina, anus, skin etc) are not effort-effective, or reliably successful as a wide number of variable genotypes cause HPV lesions.
Skin warts are most frequently caused by cutaneous HPV 1,2,3,4,27 and 57. Verruca vulgaris are most prevalent (70%) in children, and plantar warts (myrmecias) and flat warts (verruca plana) which are prevalent in older people. Most of these infections are acquired in early infancy through close skin contact. Genital and concomitant oral lesions are common, and given that malignant transformation is likely in both, early prophylactic removal after detection is advisable.
Di-indoylyl-methane (DIM) and Indole-3-carbinol (IC3), are derivative products of cruciferous (green quadripartite leaves) vegetables; IC3 is modified in the stomach to DIM, and renders this molecule similar to β-estrodiol. This similarity to estrogen allows the body to convert DIM to 2-hydroxyestrone which is an anti-estrogenic metabolite and suppresses papilloma mitogenesis and consequently growth. It is useful in RRP but rarely totally cures the condition, only prolonging times needed between surgical removal.
Interferon-α. (IFN) This is an intrinsic molecule produced from innate humoral mechanisms of mammalian anti-viral immune reactions. Interferons have been used successfully to shrink other virally induced lesions, like Kaposi’s sarcoma from Human Herpes Virus 8. There are important complications which arise from prolonged use of interferons; pyrexias, headaches and skin rashes may occur. While a positive response from RRP to interferon with reduction of papillomata, is expected, a florid recurrence may occur after IFN therapy is halted.
Vitamin A administered markedly slows down maturation of keratinocytes, and consequently will also retard most of HPV replication. Excess Vit A induces cutaneous atrophy, and the thin epithelial covering is prone to micro-trauma, secondary infections and persistent open painful lesions.
Acyclic nucleoside analogs: HPMPC (Cidofovir/Vistide).
These acyclic nucleoside analogs are injected as drugs direct into the lesions, and also used for Recurrent Respiratory Papillomatosis (RRP). The drugs are phosphorylated and used by DNA and RNA polymerase. Incorporating HPMPC inhibits the action of DNA and RNA polymerases. As healing is slowed, voice changes from vocal-cord rigidity may be experienced.
Cox II inhibitors: Cyclo-oxygenases (Cox) are essential for prostaglandin production (PG). Besides Cox 2 and PG2 production sustaining and supporting growth of papillomata, both are produced by HPV RRP. Cox2 inhibitor drugs (celecoxib or Clebrex) were originally introduced to reduce inflammatory induced pain, and because they are effective at blocking development of inflammation by inhibiting Cox, they may be effective too at slowing the growth of HPV related papillomata.
Anti-malarial drugs: Artemisinin, artesunate and similar compounds are wormwood derived anti-malarial medications. The active ingredient absinthin, a liquorice flavoured substance from Artemesia absinthium is claimed to have antiviral properties, be toxic to infected HPV cells but not healthy cervical ones. Anecdotal suggestions about production of free radicals precipitating oxidative stress, may hasten involution of papillomata.
Mumps Vaccine: The initial sensitizer is a live attenuated mumps virus which successfully produces cellular and humeral immunity, when reinfected, the anamnestic rebound of immunity boosts both reactions, and anti-viral molecules (like Interferon-α) are increased. Through this mechanism it is theorized much benefit is derived in suppressing HPV activity, and consequently reduction of HPV papillomata. 5, 20
Larger viruses like Human Herpes virus, have enzymes which convert anti-viral molecules into analogues which become substrates to be integrated into the Herpes viral DNA. These analogues then block Herpes viral DNA replication. However, HPV uses cell enzymes and use of anti-Herpes drugs (acyclovir or gancyclovir) would not specifically impact HPV lesions. 5-fluorouracil, another powerful anti-viral drug, produces undesirable genetic mutations in uninfected cells, could successfully provoke permanent DNA damage, and consequently hasten neoplastic change.
Ionizing radiation is totally contra-indicated for all papillomatous lesions; radiation promotes chromosomal DNA disruption, and when the mutated DNA products are included in DNA synthesis of ordinary non-infected cells, speedy growth to neoplastic conversion cannot be stopped.
Oral papillomata derive mainly from HPV 6 or HPV 11. After 1960 when there were major changes worldwide Oral papillomata derive mainly from HPV 6 or HPV 11. After 1960 when there were major changes worldwide with regard to sexual expression and activity, the prevalence of many other HPVs became prevalent in the oro-pharynx. For example also HPV-16 has been isolated from oral lesions, but oral-genital sex was not the only source of infection. Children born after difficult labour are more prone to acquire HPV infection from the mother than children born by caesarean. The mother is usually the main source of infection and horizontal infection of other family members are acquired via saliva or other contacts. Oral and/or genital condylomata may indicate child sexual abuse. Health care workers should be on the alert, follow-up and enquire further regarding the cause for the child’s future well being. 19
Two vaccines are available targeting mainly against squamous cell carcinomas of the head and neck, and uterine cervical regions, by inducing immunity against 4 types of HPV. They both induce highly effective, but not total, partial immunity to protect against most other HR HPVs. The vaccines are not curative against existing HPV lesions, but act as a prophylaxis against most HPV mediated cancer development.
Cervarix® (GlaxoSmith Kline, Brentford UK) is a vaccine against HPV-16 and HPV-18. Gardasil® (Merck Sharp and Dohme; MSD, USA) is a quadrivalent vaccine against HPV-16, HPV-18, and also against HPV-6 and HPV-11, the latter two HPVs (6 & 11) being causally related to genital condolomata and recurrent respiratory papillomatosis.
The recommendation is to administer the vaccine before the start of sexual activity, especially for girls. Because young males are deemed to be reservoirs for sexually transmitted HPVs, young boys should also be targeted for vaccination. The vaccinations through acquired immunity, limit development with consequent reduced prevalence of cervical intra-epithelial neoplasia and infiltrative cervical carcinomas. High-levels of antibodies against HPV 16 and HPV-18 may persist for up to 7 years, and booster vaccines are recommended before that. These vaccinations stimulate immuno-modulating mechanisms, and positively impact on prevention and prognosis for HPV positive head and neck squamous cell carcinomas.
Worldwide, women suffer from cancers; third among the most prevalent female cancers is cervical cancer, with over 85% of cases occuring in developing countries, among which China is the most populace. Spread of HR-HPV is blamed for this high prevalence.21, 22
HPV related cancers are being defined and their management addressed on national levels. For example in China pooled studies have defined the extent of the problem and an active immunization policy is being introduced.21, 22 Consequently vaccines are actively being introduced into China, arguably the most populated nation on the planet. 23
Other screening tests, like cytology smears, biopsies and possible HPV tests are being developed.24-27 These tests may need to be modified and targeted to specific HPV’s, as different world locations have different HPV prevalences and subtypes.
Variable prevalences of HPV have been found in colorectal cancers in Asia, Europe, and South America. An estimate for the overall prevalence is 32% (95%CI: range 19-47.9) ; it was lowest in Europe (14% CI 95% Range 4.9-34.1), and highest in South America (61% CI 95%:range 42.7-76.4). Analysis of 5 case-control human researches revealed an increased risk for colorectal carcinoma HPV (OR = 10.04; 95% CI: 3.7-27.5) . HPV 18 was encountered more in colorectal cancer cases derived from Asia (73.34%, 95% CI: 44.9-90.7) and Europe (47.3%, 95% CI: 34.5-60.4). HPV 16 was more frequent in colorectal cancers from South America (58.3%, 95% CI: 45.5-69.9).30
Many cancers have no established aetiology. Genetic susceptibility, pre-neoplastic conditioning by chemicals, infections or chromosomal damage, may pre-dispose some individuals to certain types of cancer development. Viruses are but one aetiological factor in neoplasia. HPV have firmly been implicated in this process for specific kinds of cancer. HPV related cancers are being progressively defined, and putative HPV genomes are successfully being deconstructed, identified and understood. As different HPV types and their constituents are being elucidated, novel more effective vaccines will appear. Identifying any biological weak point in the genome and molecular physiology of the HPV group, will hopefully provide a safe, reliable and totally effective cure against HPV neoplastic conversion. Much is known about HPV; but precise biomolecular mechanisms relating to mutation, resistance and carcinogenesis still remains obscure. More research into the many aspects impacting HPV biology and biomedical knowledge is needed.
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