Human papillomaviruses (HPVs), the most common sexually transmitted infections in the United States, are naked capsid, icosahedral, double-stranded circular DNA viruses that replicate in the nucleus of the infected cell by using host RNA polymerase for transcription and host DNA polymerase for genome replication. More than 100 genotypes of HPVs have been identified in human specimens. The genotypes are antigenically different, and groups of genotypes are associated with specific lesions, and low-risk or high-risk genotypes for cancers. HPVs are transmitted through skin-to-skin contact and through vaginal, anal or oral sex. HPVs have been identified in common hand warts, plantar warts, flat cutaneous warts of other skin areas (HPV 1-4, 7, 10); in juvenile laryngeal papillomas (HPV 6, 11); and in a variety of genital hyperplastic epithelial lesions, including cervical, vulvar, and penile warts and papillomas (HPV 6, 11, 16, 18). In addition, they are associated with premalignant cervical intraepithelial neoplasia (CIN) and malignant disease, cervical cancer (HPV 16, 18). Lesions comparable to those occurring in the cervix are now recognized in the anus, especially among men who have sex with men (MSM) and those who are infected by HIV. HPV 6 and 11 (low risk) are most common genotypes associated with genital infections and cause benign condylomas and condylomata acuminate, HPV 16 and 18 are considered the high-risk genotypes because of their potential to cause malignant cancers such as cervical cancer in women and oropharyngeal cancer mainly in men. While a majority of HPV-associated infections are benign and cleared by the immune system over time, some progress to malignancies. HPV can be detected on regular pap smear test, which is a screening test for cervical cancer recommended in women ages 21 to 65 years. Three recombinant safe, effective, and long-term protection vaccines including Cervarix (HPV 16, 18), Gardasil-4 (HPV 6, 11, 16, 18), and Gardasil-9 (HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58) are licensed and recommended for routine immunization in 11- or 12-year-old girls and boys and until age 27 years in the United States. Gardasil-9 is routinely used in the United States.
Papillomaviruses are small, naked capsid, icosahedral, double-stranded, circular DNA viruses of 55 nm in diameter (Figure 19–1). The icosahedral capsid comprises two capsid (structural) proteins, L1 (major capsid protein) and L2 (minor capsid protein). The 8 kb, circular, double-stranded DNA genome of human papillomavirus (HPV) encodes seven or eight early genes (E1-E8) and two late structural capsid genes (L1 and L2). The early genes are required for regulation of viral replication and transformation. The virus does not encode any polymerases and, therefore, is dependent on host cell transcription and replication machinery. L1, the major capsid protein, is involved in binding to the receptor on host cells. L1 is also a highly immunogenic protein and contains epitopes that induce neutralizing antibodies, and assembles into virus like particles (VLPs) and is therefore, the antigen for HPV vaccine. L2, the minor capsid, may transport viral DNA to nucleus. Among the early E1-E8 proteins, E6 and E7 are involved in transformation and oncogenesis. Based on DNA homology, there are over 100 genotypes of HPVs. Papillomaviruses cause epidermal papillomas and warts in a wide range of higher vertebrates. Different members of the group are generally species specific. For example, bovine papillomaviruses and HPVs infect only the hosts reflected in their names. In some cases, lesions caused by these agents can become malignant, and the role of these agents as causes of certain human cancers is increasingly recognized. While papillomaviruses were difficult to grow in tissue culture and many virologic information were derived from molecular and gene expression studies, several cell lines based culture, including keratinocyte cell line and new differentiating skin system have been developed that are being used to study the biology of HPV.
Electron micrograph of human papillomavirus (HPV) particles isolated from a plantar wart (×300 000). (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford, CT: Appleton & Lange, 1997.)
✺ Naked capsid, icosahedral, double-stranded, circular DNA viruses
✺ L1 major capsid protein interacts with receptor on host cells, neutralizing antibodies are made against L1 and L1 is the antigen for HPV vaccine
While difficult to propagate HPV in tissue culture, new cell cultures have been developed
Devoid of viral RNA or DNA polymerase, uses host RNA and DNA polymerase
The genomes of many of the papillomaviruses have now been cloned and compared by restriction endonuclease and DNA homology procedures. These studies have shown a wide genomic diversity among papillomaviruses that infect different species and also among those that infect humans. This has led to the allocation of numbers for the different genotypes important in human diseases.
Great genomic diversity, important is human diseases
✺ High-risk HPV-associated cancers associated with HPV genotypes
HPV targets stratified squamous epithelium through the damaged area of the epithelium and infects the basal cells. The replication cycle of HPV was reproduced in cultured cells by using a raft culture system made up of stratified squamous epithelial cells. Moreover, in infected human tissue, infectious particles are found. HPV infects the basal layer of squamous epithelium by the initial interaction of L1 major capsid protein probably to heparin sulfate proteoglycan on the basal membrane and then transfer to the receptor expressed on keratinocytes moving on the basal membrane in wound healing process of the damaged epithelium. After the virus is internalized by viropexis and uncoated, the viral DNA is transported to the nucleus probably aided by L2 minor capsid protein. Host RNA polymerase transcribes early (E) genes followed by early protein synthesis. Some of the early genes, E6 and E7, are involved in transformation that causes an increase in cell division. E6 binds to p53 (tumor suppressor) and E7 p105RB (retinoblastoma) proteins and abrogate cell cycle regulation. The dividing cells carry viral genome, as extrachromosomal DNA (epiosmal DNA), allowing HPV genome to persist in these cells. As the infected cells differentiate to early terminal stages, other viral early genes are expressed, namely, E1 and E2, which are involved in regulation of viral transcription and replication. Viral DNA synthesis occurs at two levels directed by host cell DNA polymerase: (1) in the lower portion of the epidermis to maintain a stable multicopy viral DNA for latent infection, and (2) as vegetative DNA replication, which occurs in the more differentiated epithelial cells. In some cases, papillomavirus DNA can integrate into the host chromosomes. The infected cells further differentiate to a terminal stage (keratinocytes) wherein late gene expression synthesis of late (L) capsid structural proteins and vegetative DNA synthesis take place. At this stage, there is a burst of viral DNA synthesis followed by virus assembly in the nucleus and virus release by cell lysis.
HPV infects and initially replicates in the basal layer of the epidermis
✺ E6 and E7 early proteins transform cells to increase cell division and oncogenesis
✺ Host RNA polymerase directs viral mRNA transcription and host DNA polymerase viral DNA replication
Vegetative DNA replication and viral assembly in terminally differentiated epithelial cells (keratinocytes)
Latent viral DNA maintained in the basal layer of epithelium
How does HPV infect basal squamous epithelium when it transmitted through skin-to-skin contact?
HPV is the most common sexually transmitted infection in the United States. More than 79 million Americans are already infected with HPV and more than 14 million individuals are newly infected every year. Before HPV vaccination, about 340 000 to 360 000 genital warts in women and men were reported every year in the United States. In addition, more than 31 000 women and men are diagnosed with cancer caused by HPV, including 11 000 cases and 4000 deaths due to cervical cancer in women and 9 000 cases of oropharyngeal cancer in men every year in the United States. The rates of cervical cancers are higher in black women than white woman as well as in Hispanics than non-Hispanics. On the contrary, the rates of oropharyngeal cancer are higher in whites than blacks and in non-Hispanics than Hispanics. Many types of cancers are caused by HPV including 70% of vulvar and vaginal cancer, 60% of penile cancer, 90% of anal and cervical cancers, and 70% of oropharyngeal cancer. It is believed that tobacco and alcohol also play a role in oropharyngeal cancers. Globally, an estimated 529 000 new cases and 274 000 deaths due to cervical cancer occur every year, and more than 85% of these cases/deaths are in developing countries, which account for 13% of all female cancers.
✺ Most common sexually transmitted infection in the United States
More than 79 million Americans are infected with HPV, 14 million new infections each year
✺ Major cause of cervical cancers in women
✺ In the United States, about 12 000 cases and 4000 deaths due to cervical cancer every year
✺ Globally, about 529 000 cases and 274 000 deaths due to cervical cancer every year, mostly in developing countries
✺ More than annual 9000 cases of HPV-associated oropharyngeal cancer in men in the United States
Think ➮ Apply 19-1. After skin-to-skin contact, HPV may reach the basal squamous epithelium probably through some damage or abrasions.
HPV GENOTYPES, RISK FACTORS, AND DISEASES
HPV genotypes are important in disease spectrum and severity. The genotypes causing genital lesions are different from those causing cutaneous, nongenital warts. Cutaneous, nongenital warts usually occur in children and young adults; presumably immunity to the HPV genotypes causing these lesions develops and provides subsequent protection. Common warts that grow generally on hands are caused by HPV types 1 and 2; plantar warts that grow on soles of feet are caused by types 1, 2, 4; flat cutaneous warts by types 3 and 10; meat and fish handlers are prone to HPV type 7. Over 40 HPV genotypes have been identified in genital lesions of humans, and there are many apparently silent infections with these viruses. Cross-immunity does not occur, and sequential infection with multiple genotypes does take place. A single sexual exposure to an infected person may transmit the infection 60% of the time; usually the infected person is asymptomatic. Having multiple sex partners is the major risk factor for acquiring HPV infection. From 20% to 60% of adult women in the United States are infected with one or another of the genotypes. In addition, more than 50% of sexually active people become infected with HPV at least once in their lifetime. HPV types 6 and 11 are most commonly transmitted HPV genital infection mainly associated (about 90%) with benign genital warts in males and females and with some cellular dysplasias of the cervical epithelium, but these lesions rarely become malignant. These genotypes are considered low risk. HPV types 6 and 11 have been associated with nasal, oral, conjunctival, and laryngeal warts, although high-risk genotypes (types 16 and 18) are associated with oropharyngeal cancer. They can be perinatally transmitted from mother-to-child and cause infantile laryngeal papillomas. HPV types 16, 18, 31, 33, 45, 52, and 58 may cause lesions of the vulva, cervix, and penis. Several others types have also been implicated such as 35, 39, 56, 59, 66, and 68 in dysplasia and carcinoma. Clinically, these HPV genotypes are considered high risk for the development of cervical cancer and its precursor lesion, cervical intraepithelial neoplasia (CIN). Infections with these viral types, especially types 16 and 18, may progress to malignancy. In addition, HPV-16 is probably the most carcinogenic genotype because of its association with 60% of cervical cancers, whereas HPV-18 association is about 10-15%. Furthermore, 80% of the HPV-associated cancers are caused by HPV-16 and 18 and 12% by HPV-31, 33, 45, 52, and 58, and these genotypes are part of Gardasil 9-valent vaccine. Viral genomes of at least one of these genotypes are found in the majority—but not all—of markedly dysplastic uterine cervical cells, in carcinoma in situ, and in cells of frankly malignant lesions.
HPV types 6 and 11 common; mainly benign, rarely lead to malignancy
✺ Types 16, 18, 31, 33, 45, 52, and 58 are associated with dysplasia and malignancy
Type 16 associated with 60% and type 18 with 10% to 15% cervical cancers and CIN
HPV-associated oropharyngeal cancer is on rise, especially in men. While 7% people have oral HPV, only 1% have HPV-16 associated with oropharyngeal cancer in the United States. The high risk (HPV-16) is associated with cancers of head and neck and low risk (HPV 6, 11) with mouth or oral warts.
Type 16 associated with head and neck (oropharyngeal) carcinoma
Why HPV 16 and 18 more oncogenic than HPV 6 and 11?
Human papillomavirus infection is now considered to be a contributory cause of most carcinomas of the cervix. Papillomavirus infection of the anus is a clinical problem in men having sex with men (MSM), especially those with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), and it is related to the subsequent development of anal neoplasia in these individuals.
Think ➮ Apply 19-2. E6 and E7 are the major oncogenic proteins of HPV. The probability could be that E6 and E7 of high risk (HPV 16 and 18) have a higher affinity for pRb and p53 leading to abrogate cell cycle than E6 and E7 of low risk (HPV 6 and 11).
HPV causing common warts are transmitted through skin-to-skin contact and spreads through damaged, broken skin, finger nail biting, etc. People can spread the virus to other parts of their body. The virus can also be transmitted by touching anything that was touched by a person with wart, including public showers, swimming pool, occupational tools, recreational and sports tools. In addition, meat and fish handlers are prone to hand warts. HPV is transmitted sexually during intimate sexual contact through vaginal and/or anal sex. It can also be transmitted through oral sex or other sex play. HPV is the most common sexually transmitted infections in the United States. HPV can be transmitted perinatally from mother-to-child causing recurrent respiratory papillomatosis (RRP) in the baby.
Common hard warts transmitted through skin-to-skin contact, public showers, swimming pools, occupational tools suspected
Sexual transmission through anal and/or vaginal sex; oral sex also transmits HPV
Papillomaviruses have a predilection for infection at the junction of squamous and columnar epithelium (eg, in the cervix and anus). Papillomaviruses were the first DNA viruses linked to malignant changes. In the mid-1930s, Shope demonstrated that benign rabbit papillomas were due to filterable agents (older terminology for viruses) and could advance to become malignant squamous cell carcinomas. External cofactors, such as coal tar, could hasten this process. However, work on the biology and mechanism by which these agents foster malignant transformation has been impeded by the inability to cultivate papillomaviruses in vitro. Molecular probes to detect viral products in vivo indicate that replication and assembly of these viruses take place only in the differentiating layers of squamous epithelia, a situation that has not been reproduced in vitro.
Replication in squamous epithelium
The first evidence that HPVs could be associated with human malignant disease came from observations on epidermodysplasia verruciformis. This disease has a genetic basis that results in unusual susceptibility to HPV types 5 and 8, which produce multiple flat warts. About one-third of affected patients develop squamous cell carcinoma from these lesions.
HPV 16 and 18 are associated with most of the cervical cancers in women. However, the mechanism of oncogenicity of HPV is less clear. Cells infected with genomes of several papillomaviruses can transform cells and produce tumors when injected into nude (T lymphocyte-deficient) mice. The viral genome exists as multiple copies of a circular episome within the nucleus of transformed cells, but is not integrated into the cellular genome. This appears also to be the case with benign human lesions. In malignant tumors, part of the viral genome is found integrated into the cellular genome, but integration is not site specific. Both the integrated viral genome and the extrachromosomal form carry their own transforming genes. Host cells normally produce a protein that inhibits expression of papillomavirus transforming genes, but this can be inactivated by products of the virus and possibly by other infecting viruses, thus allowing malignant transformation to occur. HPV early gene products, E6 and E7, have been implicated in oncogenicity. E6 accelerates the degradation of p53, a tumor suppressor protein, and reduces its stability. E7 interacts with pRB, retinoblastoma protein, to abrogate cell cycle regulation. The inhibition of p53 and pRB functions results in cell transformation by E6 and E7, causing tumors. Another HPV gene product, E5, has been found to function in benign papillomas. HPV DNA is found in more than 95% of cervical carcinoma specimens when tested by polymerase chain reaction (PCR). The discovery that HPV causes most cervical cancers earned the 2008 Nobel Prize in Medicine for the German researcher, Harald zur Hausen.
✺ Viral genomes carry their own transforming genes, E6 and E7
✺ E6 degrades p53 and E7 interacts with pRB to abrogate cell cycle causing transformation
✺ HPV is the major cause of cervical cancer
Immunity is generally limited due to localized infection in basal epithelial cells that are probably shielded by circulating immune cells and the nonlytic nature of virus replication. Innate immunity controls infection to some extent but down regulated by early protein of HPV. In the later stages of infection, immune cells can detect viral proteins when the virus replication moves to suprabasal keratinocytes, leading to a strong localized cell-mediated immunity and in most cases clearance of viral infection. Antibody response against L1 major capsid protein is detected in infection. In regressing warts, infiltrating T lymphocytes and macrophages are seen. However, in some people the virus is not cleared and persists, which increases the risk of cancer.
Effective localized cell-mediated immunity eliminates infection, whereas depressed immunity allows persistence
Antibody response generated during infection
Cutaneous warts develop at the site of inoculation within 1 to 3 months and can vary from flat to deep plantar growths (Figure 19–2). Although they can persist for years, they ultimately spontaneously regress. Respiratory papillomatosis due most often to types 6 and 11 occurs as intraoral or laryngeal lesions. These tend to occur in infants as a result of natal exposure, or in adults. Treatment is varied and complex.
Warts. A. Common warts on fingers. B. Flat warts on the face. C. Plantar warts on the feet. D. Perianal condylomata acuminata. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008.)
Oral or laryngeal papillomatosis in infants infected during delivery
External genital HPV infection occurs as exophytic genital warts (condylomata acuminata) caused most often by types 6 or 11 HPV (Figures 19–2 and 19–3). They are often found on the head or shaft of the penis, at the vaginal opening, or perianal 4 to 6 weeks after exposure. Lesions may increase in size to cauliflower-like appearance during pregnancy or immunosuppression. Genital HPV infection is most often benign, and many lesions reverse spontaneously. However, they may become dysplastic and proceed through a continuum of cervical intraepithelial neoplasm (CIN), CIN 1 (mild dysplasia), CIN 2 (moderate dysplasia) to CIN 3 (severe dysplasia) and/or carcinoma (Figure 19–4). The most common HPV in the malignant lesions is type 16, although this genotype, as well as the others, is most pertinent to cause lesions that regress spontaneously. Higher-grade malignancy is most pertinent to occur in the cervix, but the rate of anal carcinoma related to HPV appears to be increasing, especially in AIDS patients. In oral HPV that is mainly HPV 6 and 11, the oropharyngeal cancer of head and neck (in the back of the throat and base of tongue and tonsils) is caused by HPV 16, which is four times more common in men than women, and the initial symptoms in some people may include persistent sore throat, ear pain, hoarseness, enlarged lymph nodes, pain when swallowing, and unexplained weight loss. In most instances these symptoms may go, whereas in some cases it may lead to malignancy. Several factors such as tobacco chewing, smoking, and alcohol may increase the risk of HPV-associated oropharyngeal cancer.
Extensive condylomata of vulva caused by HPV-6. (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford, CT: Appleton & Lange, 1997.)
Colposcopic photograph of cervical transformation zone with diffusely scattered acetowhite staining, characteristic of HPV infection. (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford, CT: Appleton & Lange, 1997.)
Anal carcinoma due to HPV is on rise
Oropharyngeal cancer four times common in men than women
HPV is not routinely propagated in tissue culture, and antibody tests are rarely used, since results remain positive after the first HPV genotype infection. Papillomavirus infection leads to perinuclear cytoplasmic vacuolization and nuclear enlargement, referred to as “koilocytosis,” in epithelial cells of the cervix or vagina. These changes can be seen in a routine Papanicolaou (Pap) smear (Figure 19–5). The use of immunoassays to detect viral antigen and nucleic acid hybridization or PCR to detect specific viral DNA in cervical swabs or tissue is more sensitive (Figure 19–6) than Pap smear. Four diagnostic tests have been approved by the FDA in the United States, including HC II High-Risk test (Qiagen), HC II Low-Risk HPV test (Qiagen), Cervista HPV 16/18 test, and Cervista HPV High-Risk test (Hologics). Detection of an abnormal cytology due to HPV should prompt colposcopy to assist in following up or treating patients with abnormal lesions.
Abnormal Pap smear. The pink and blue objects are squamous epithelial cells; abnormalities include the doubling of the nuclei and a clear area around them. Most abnormal smears in young women are due to human papillomavirus (HPV) infection; when persistent, it is considered an important factor in the development of cancer of the cervix.
Human papillomavirus (HPV) type 16 DNA demonstrated in a cervical smear by in situ hybridization. The dark dots represent detection of HPV DNA sequences by the DNA probe.
✺ Koilocytosis can be seen in cytologic specimens
Molecular methods to detect specific genotypes in biopsies of cervical swabs are available
Current treatment of HPV is usually either cytotoxic or surgical. Among the topical cytotoxins are podophyllin, podophyllotoxin, 5-fluorouracil, and trichloroacetic acid. Systemic and local interferon-alpha; may provide some benefit. Warts can also be removed by laser or freezing with liquid nitrogen. Loop electrosurgical excision procedure (LEEP) can be used to remove abnormal cells with an electric current. Another procedure called conization, also known as cone biopsy, removes abnormal cells. Recurrences are common after cessation of treatment because of survival of virus or viral DNA in the basal layers of the epithelium. Cervical and anal lesions may be treated with electrocautery, but carcinoma may require chemotherapy, radiation therapy or radical surgery.
Recurrences are common after topical treatment
Removal of warts by cryosurgery or other methods
Condom usage is encouraged to prevent transmission
✺ Cervical Pap smears should be done regularly to detect early lesions due to HPV
Why HPV vaccines have added more HPV genotypes over time?
Three recombinant virus-like particle (VLP) vaccines comprising viral major capsid L1 polypeptide namely bivalent vaccine, Cervarix (HPV 16 and 18), Gardasil quadrivalent vaccine, (HPV types 6, 11, 16, and 18), and Gardasil 9-valent vaccine (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58) are licensed in the United States. Both vaccines are subunit recombinant vaccines that are noninfectious and elicit neutralizing antibodies that provide protection against commonly prevalent HPV that cause cervical cancers and genital warts. HPV vaccine is recommended for routine vaccination at age 11 or 12 for girls and boys, given in 2 shots 6 to 12 months apart. For age above 14 years, 3 shots over a 6-month period should be given. HPV vaccine is also recommended for females aged 13 to 26 and males 13 to 21 not vaccinated previously. Vaccination is also recommended through age 26 years for men who have sex with men and for immunocompromised persons (including those with HIV infection) if not vaccinated previously. Cervarix and Gardasil are given intramuscularly as a 3-dose series over a 6-month period. The vaccine is not recommended during pregnancy. These vaccines are safe, effective and provide long-term protection against cancer causing HPVs. Vaccinated females should continue Pap smear and HPV screening, because other HPV genotypes than those included in the vaccine can cause cervical cancer. Pap smear test for cervical cancer screening is recommended in women aged 21 to 65 years in the United States. The National Health and Nutrition Examination Survey data on the impact of HPV vaccines demonstrate reductions of the prevalence of HPV types 6, 11, 16, and 18 and the prevalence of anogenital warts.
Three recombinant vaccines, Cervarix and Gardasil-4 and Gardasil-9, licensed in the United States
Vaccine is recommended at ages 11 or 12 for girls and boys
Think ➮ Apply 19-3. Different HPV genotypes are associated with different lesions and cancers. The vaccine started with the high-risk genotypes for cancer (types 16 and 18) and then additional genotypes were based on epidemiological evidence of their prevalence with HPV-associated diseases. The current vaccine Gardasil-9 has types 6, 11, 16, 18, 31, 33, 45, 52 and 58.