Definition & Characteristics of HPV

Human papillomaviruses (HPVs) infect epithelial cells of the skin and mucous membranes, and have been linked etiologically to cervical abnormalities including precancers, cervical cancer, warts, and recurrent respiratory papillomatosis. HPVs are also associated with other malignancies such as squamous cell carcinomas of the anus, vulva, vagina, penis, and head and neck.

Virology

Organization of the Papillomavirus Capsid

The papillomavirus capsid (the protein shell that encloses the viral DNA) is composed of 360 copies of the L1 protein (Late 1: the major capsid protein) and approximately 12 copies of the L2 protein (Late 2: the minor capsid protein) [Bonnez, 2005]. Because viruses only have the capacity to produce a few viral proteins, the capsid consists of many copies of the same viral proteins. One copy of the double-stranded viral DNA is located inside the capsid shell.

Figure 1. Assembly of the HPV Particle

Figure 1 shows that 5 copies of the L1 protein combine to form a capsomer, and then 72 capsomers combine with one copy of viral DNA and 12 copies of the L2 protein at the center of each capsomer to form the infectious virus particle. The virus particle is an icosahedron which means that the capsomers are packed symmetrically in a regular arrangement, such that the virus particle can be rotated around several axes, but the appearance of the virus particle remains the same.

Figure 2. HPV Particle (Electron Micrograph)

Figure 2 shows an electron micrograph of the HPV capsid. Note the regular arrangement of capsomers. The diameter of the virus capsid is approximately 55 nanometers.

Characteristics and Significance of HPV Genome and Proteins

The HPV genome is a double-stranded, circular DNA molecule that contains approximately 7,900 base pairs. Figure 3 shows the genetic map of HPV-16 (a high-risk type).

Figure 3. HPV-16 Genome

Note that HPV 16 produces only eight proteins. These proteins are divided into “early” (E1, E2, E4, E5, E6, and E7) and “late” proteins (L1 and L2). There is also a control region in HPV DNA called the Long Control Region (LCR) or Upper Regulatory Region (URR). The functions of the various proteins and genetic elements are summarized in Table 1 [HPV Handbook, 2004].

Table 1. Functions of HPV Proteins and Genetic Elements

Production of HPV Proteins and Viral DNA in Warts and Low-grade Cervical Intraepithelial Neoplasia (CIN)

Cervical intraepithelial neoplasia (CIN) is a term used by pathologists to classify the severity of dysplasia when examining a cervical tissue biopsy specimen. The term CIN along with a number (1, 2, or 3) describes how much of the thickness of the epithelial lining of the cervix contains abnormal cells. CIN ranges from low-grade (mild dysplasia) or CIN 1 to high-grade (severe dysplasia) or CIN 3. CIN 3 encompasses both precancerous lesions and carcinoma in situ.

Low-grade Lesions: CIN 1 and Warts

• HPV initially binds to and infects epithelial stem cells (basal cells) in the basal layer of the epithelium. The cellular receptors for HPVs may be heparan sulfate proteoglycans or alpha-6 integrins which are present on a wide range of cells. Even though the receptor for HPV is found on many cell types, a productive infection only proceeds in human epithelial cells.
• HPV E6 and E7 proteins (along with E1 and E2) are produced as the basal cells divide, begin moving towards the outside of the mucosa, and become the para-basal and squamous layer of the mucosal epithelium. E6 and E7 inhibit p53 and Rb host tumor-suppressor proteins which normally prevent cell division within these layers of epithelial cells. Thus, these cells become free to divide and produce the thickening of the skin which is characteristic of a genital wart.
• HPV DNA replicates as an episome (circular DNA molecule which is separate from the host cell DNA) in the para-basal and squamous cell layers. In these cell layers, the number of copies of the HPV episomal DNA increases greatly.
• In the squamous and mature squamous cell layers, HPV L1 and L2 proteins are produced. Infectious virus particles are produced only in the most superficial layer of the mature squamous cell layer.

The significance of this life cycle is that HPV delays the production of the immunogenic L1 and L2 capsid proteins until the skin cells have terminally differentiated into squamous epithelium which is sloughed off and not accessible to immune cells. Thus, an immune response against L1 and L2 proteins is slow to appear, and does not occur in all individuals infected with HPV. In addition, the level of production of E6 and E7 in the basal epithelium is low and restricted to the cell nucleus which also limits the immune response against E6 and E7 (see section on Immune Response to HPV).

Figure 4. Production of HPV Proteins and DNA Resulting in CIN1, CIN2, or CIN3

Production of HPV Proteins and Viral DNA in High-grade Lesions

Cervical Precancers (CIN 2/CIN 3) and Cervical Cancer

In contrast to the previous section which described productive infection (i.e., infection with HPV that produces new virus particles), the states of CIN 2/3 and cervical cancer are quite different with respect to synthesis of viral proteins, viral DNA and new virus particles.

Only a subset of viral proteins is produced in CIN 2/3, there is little viral DNA replication, and very few infectious virus particles are produced. However, because E6 and E7 are produced at high levels in CIN 2/3, high-grade lesions (cervical abnormalities including precancers) can progress to cervical cancer [Doorbar, 2005]. The next section shows how E6 and E7 proteins may result in cancer.

The Development of Cancer

There are several mechanisms induced by high-risk HPVs that result in cervical cancer. HPV DNA is generally integrated into the host DNA genome in carcinoma in situ. The integration of the circular HPV DNA genome specifically disrupts or deletes the E2 gene [Münger, 2004] (Figure 5).

Figure 5. Integration of HPV-16 Disrupts the HPV E2 Gene

Because E2 normally inhibits the production of E6 and E7 proteins, the disruption of E2 after integration results in a high level of production of E6 and E7 proteins. These high levels of E6 and E7 proteins result in the high rate of uncontrolled cell division seen in cervical cancer.

Classification

Papillomaviruses are very diverse. Based on isolation and sequencing of complete genomes of papillomaviruses, there are currently 118 different types of human and animal papillomaviruses.

Of the 96 HPV types identified, approximately 30 to 40 HPV types infect the anogenital tract. The anogenital HPV types can be divided into approximately 15-20 high-risk types which are frequently associated with invasive cervical cancer and other genital cancers, and approximately 10 to 15 low-risk types which mainly cause genital warts and low-grade or benign cervical lesions [Schiffman, 2003]. Table 2 shows the phylogenetic and epidemiologic classification of HPV types with respect to high-risk and low-risk types. Note that the agreement between the phylogenetic classification (based on DNA sequence of the L1 protein) and the epidemiologic classification (based on presence in cervical cancer) is very good with discrepancies found only for HPV-70 and HPV-73.

Table 2. Phylogenetic and Epidemiologic Classification of HPV Types

Figure 6 shows the phylogenetic relationship (DNA sequence of the gene for the L1 proteins) between high-risk and low-risk HPVs [de Villiers, 2004]. Note that there are 2 main groups of high-risk HPVs (species 5, 7, 6, and 9) and 2 main groups of low-risk HPVs (species 3, 13, 1, 8, and 10). For example, HPV-6 and HPV-11 (both low-risk types) are closely related to each other and are in the same species (10). However, HPV-16 (high-risk) in species 9 is not closely related to HPV-18 (high-risk), which is in species 7. HPV-6 and HPV-11 are the most common low-risk types of HPV, especially associated with warts.

Figure 6. Phylogenetic Tree of the Sequences of High- and Low-risk Types of HPV

The Immune Response to HPV

Production of Antibodies to HPV

Cervical infection with HPV results in the production of antibodies to HPV in one-half to two-thirds of infected women, but the rate of seroconversion is slow, and the antibody responses are low. For example, in one study of 608 female college students (mean age of 20 years), cervical infection with HPV-16 resulted in IgG seroconversion approximately 8 months after HPV DNA was detected, and IgA seroconversion occurred approximately 14 months after infection. Furthermore, only 56.7% of women became seropositive for IgG and 37% became seropositive for IgA; the median duration of both IgG and IgA as measured by ELISA was approximately 3 years [Ho, 2004]. In contrast, another report showed that the levels of anti-HPV IgG were stable over time even after more than a decade of follow-up.

The largest antibody response measured in people infected with HPV is to the L1 protein in naturally occurring virus particles or recombinant virus-like particles.

The role of antibodies in preventing initial infection with papillomaviruses has been demonstrated in animal models of infection. Uninfected animals that received passive administration of immune sera were protected against infection. This confirmed the ability of neutralizing antibodies to prevent infection [Suzich, 1995].

In humans, there are no definitive studies on the role of antibody. However, antibodies to HPV may prevent the initial infection of epithelial basal cells with HPV (Figure 7).

Figure 7. Role of the Immune to HPV

IgG and IgA in mucous secretions which bind to HPV virus particles may prevent viral attachment to mucosal epithelial cells. The levels of antibody needed for protection against infection have not been determined although this is an active area of investigation. It is not clear whether IgA is important in HPV infection. The cervico-vaginal fluid contains more IgG than IgA, and a significant proportion of the IgG in genital tract secretions is derived from the circulation (plasma).

Cross-reaction of Antibodies Between Different Types of HPV

Antibodies formed after infection with one type of HPV usually do not bind to other types of HPV. Prior infection with one type of HPV does not appear to prevent infection with another type of HPV, suggesting that, in general, different types of HPV correspond to different serotypes, and that the immune response to one type of HPV does not protect against other types of HPV [Thomas, 2000]. One exception may be that because HPV-6 and HPV-11 contain shared epitopes on their intact capsids, antibodies formed after infection with HPV-6 may partially protect against infection with HPV-11 (and vice-versa). Another exception is that antibodies raised in rabbits against HPV-33 also inhibited HPV-16 infection. In contrast, antibodies to HPV-6, 11, 18, 31, 35, 39, and 45 did not inhibit HPV-16 infection.

Cell-mediated Immunity to HPV (Cytotoxic T-lymphocytes, CTL)

In contrast to the role of anti-HPV antibodies in preventing initial infection with HPV, the CTL response may be more important in preventing persistence of infection, clearing HPV DNA, and causing regression of abnormalities. The target cells for CTL-mediated lysis are the keratinocytes in the intermediate layer of the epithelium which produce the HPV early proteins (E1, E2, E4, E5, E6, and E7). Women with previous or ongoing HPV-16 infection can produce CTLs that recognize E6 or E7 proteins. In one study, individuals who cleared HPV were more frequently CTL responders to E6 and E7 (63%; 5/8) than patients with CIN 1, 2, or 3 (14%; 1/7).

In addition, the lack of a CTL response to HPV E6 appears to be associated with the persistence of HPV-16 infection. Studies have shown that coinfection with HPV and HIV-1 results in a higher incidence of HSIL (high-grade squamous intraepithelial lesions) than that in HPV-infected, HIV-uninfected females (21.5% vs. 4.8%, respectively) [Scott, 2001]. These and other results suggest that a CTL response is important for clearance of HPV infection, and also suggest that the lack of a CTL response may be associated with persistent infection, possible progression to CIN 2/3, and the development of cervical cancer.

Evasion of HPV from the Immune Response

How does HPV evade the CTL response and cause persistent infection?
Why does HPV infection result in slow seroconversion of infected individuals?

HPV has evolved several mechanisms to evade the host’s immune response [Tindle, 2002]:

• HPV does not replicate in antigen-presenting cells, lyse keratinocytes, or have a blood-borne phase of replication. This means that the immune system has little opportunity to detect HPV proteins and to mount an immune response.
• The level of production of E6 and E7 proteins in the basal epithelium is low and is restricted to the cell nucleus which also limits the immune response to E6 and E7.
• HPV delays the production of L1 and L2 capsid proteins until the skin cells have terminally differentiated into squamous epithelial cells which are sloughed off and inaccessible to immune cells. In addition, HPV intentionally uses an inefficient process to produce its L1 and L2 proteins. This ensures that the levels of L1 and L2 protein production are low, which lessens the immune response to HPV L1 and L2 proteins.
• Infected keratinocytes may be relatively less susceptible to CTL lysis than other cell types.

Diagnostic Tests for HPV DNA

The Hybrid Capture^® 2 HPV Test

The HC-2 test detects the presence of HPV DNA for 18 HPV types. These 18 HPV types consist of 13 high-risk types (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low-risk types (HPV types 6, 11, 42, 43, and 44) [Clavel, 2002]. The HC-2 test was developed by Digene, Gaithersburg, MD, and was approved by the U.S. FDA in March 2000. There is also a version of the HC-2 test that only detects the 13 high-risk HPV types mentioned above. This test is called the “Hybrid Capture^® 2 High-Risk HPV DNA Test™”.

Performing the HC-2 Test: The sample for the HC-2 test consists of cells taken from the cervix. The cell sample can be taken at the same time as a Pap test if the ThinPrep^® Pap test is performed. Figure 8 shows how the test is performed.

Figure 8. Hybrid Capture-2

The steps in the HC-2 assay are:

1. Release DNA from the cervical cells with an alkaline solution (the DNA consists of cellular DNA which may also contain HPV DNA)
2. The DNA solution may be split in half: one part is hybridized to specific RNA probes for the high-risk types of HPV, and the other part of the sample is hybridized to specific RNA probes for the low-risk types of HPV. If the Hybrid Capture High-risk Test is performed, then only the High-risk probes are added.
3. The RNA probe: HPV DNA hybrids are captured onto the surface of a microtiter well by using an antibody specific for RNA:DNA hybrid molecules.
4. Antibodies are added that are connected to an enzyme (alkaline phosphatase) and that also bind to RNA:DNA hybrids.
5. A chemiluminescent substrate (dioxetane) is added which is split by alkaline phosphatase and produces light.
6. The light in each well is measured with a luminometer.
7. The amount of light is measured in RLUs (relative light units).
8. A positive result in the HC-2 test means that the light reading in RLUs was greater than or equal to the mean of the 3 positive control values supplied with the kit. The minimum amount of HPV DNA that gives a positive result is 1.0 picogram of HPV DNA per mL (1.0 pg is equivalent to 5,000 copies of HPV DNA).

The HC-2 test is a non-radioactive, molecular hybridization assay that uses chemiluminescence to detect HPV DNA in microtiter wells. The results from the HC-2 test can be:

• negative or positive for high-risk HPV DNA
• negative or positive for low-risk HPV DNA.

Note that no specific types of HPV are identified by a positive result in the HC-2 test. If the test results are “positive” for high-risk HPV, then the patient could have any one (or more) of the 13 high-risk HPV types detected by the HC-2 test.

Table 3. Strengths and Weaknesses of the HC-2 Test

Strengths of the Test: The sensitivity of the HC-2 test is very high (Table 3). The sensitivity of the HC-2 test was >90% for detecting high-grade squamous intraepithelial lesions (HSIL) in the ASCUS LSIL Triage Study (ALTS) [Castle, 2004]. The HC-2 test had a negative predictive value of 99% indicating that the vast majority of patients with a negative test result by HC-2 were also negative by the Pap test, and that there were very few false negatives (negative by HC-2 but positive by Pap test) in the HC-2 test.

Weaknesses of the Test: The specificity of the HC-2 test is relatively low. In the same study mentioned above, the specificity was low for the HC-2 test compared to the Pap test. The specificity was 87.3% with respect to the conventional Pap test, and 85.6% for the liquid-based Pap test (see section on Cervical Cancer Screening for more information). This means that 13.7% to 14.6% of women who were negative by Pap test were positive in the HC-2 test; the significance of this is that most young women who are positive for HPV DNA but negative for abnormalities by Pap test may have transient infections that will clear. This is also why the HC-2 test is not used as a true screening test, because there would be many more positives, more anxiety, and no significant disease detected.

Although the HC-2 test can distinguish between high-risk and low-risk genotypes of HPV, it cannot identify specific HPV genotypes. The reason is that the HC-2 test uses two probe cocktails (one cocktail for high-risk genotypes and another cocktail for low-risk genotypes).

Another weakness is that the detection limit for the HC-2 test is approximately 5,000 copies of HPV DNA. The HC-2 test is less sensitive than PCR, but PCR tests are not approved by the FDA. PCR tests are used as investigational tests in clinical trials and can identify HPV genotypes (see section on Investigational Tests ).

DNAwithPap™ = Hybrid Capture^® 2 and Pap Test

The DNAwithPap™ test combines a Pap test with the HC-2 high-risk HPV DNA test and is FDA-approved as a primary screening method in women 30 years of age and older [Obiso, 2004]. There are several reasons why age 30 was chosen. The prevalence of HPV DNA positivity peaks in the late teens or early 20s. However, most newly acquired HPV infections that are positive for HPV DNA in this age group clear spontaneously. The incidence of CIN 2/3 peaks in women in their late 20s and early 30s. Therefore, if the DNAwithPap test was used in women less than 30 years of age, a large number of women would be positive for HPV DNA but would not develop lesions, and most of these infections would spontaneously clear. These women might undergo unnecessary intensive follow-up testing, colposcopy, or treatments. In contrast, HPV DNA testing as part of cervical cancer screening in women aged 30 years or more has been shown to be cost-effective.

Performing the DNAwithPap Test: A sample of cervical cells is collected as for the Pap test, but the cells are dispersed in Cytyc ThinPrep^® (PreservCyt) solution. Separated parts of this solution are used for the Pap test and for the HC-2 test. The healthcare professional receives results for both tests in the same report.

Table 4. Strengths and Weaknesses of the DNA with Pap Test

Strengths of the Test: The DNAwithPap test has a negative predictive value of greater than 99% (Table 4). This result means that a negative result for both the Pap test and the HC-2 test gives one confidence that a woman does not have, and is not likely to develop, high-grade cervical disease or cancer within the next 3 years if there is no change in risk factors.

Weaknesses of the Test: Similar to the weaknesses of the HC-2 test, some women may be positive for HPV DNA but negative by Pap test. These results suggest the woman most likely has a transient infection that is likely to clear.

Investigational Tests

HPV Serological Assays

There are no serologic assays that are sensitive enough for clinical diagnosis of HPV disease. In addition, because of the variability in the time to seroconversion, serologic assays are not useful for diagnosis of HPV disease. Investigational serological assays only detect 50% to 67% of infected individuals [Konya, 2001]. These assays have been used for epidemiological studies to detect past infection with HPV.

One of the investigational serologic assays is an Enzyme-linked Immunosorbent Assay (ELISA) using HPV VLPs (virus-like particles) as the source of antigen. The HPV L1 (or the L1 and L2) proteins spontaneously associate to form VLPs which are the target in the ELISA assay.

Polymerase Chain Reaction (PCR) Tests

PCR is used as an investigational test to identify specific HPV types. PCR tests are not currently approved by the FDA.

There are two ways to perform PCR to detect HPV DNA: type-specific PCR or broad-spectrum PCR. With type-specific PCR, primers are chosen that will only amplify a single genotype of HPV. This method is labor-intensive if the goal is to detect many genotypes of HPV. Alternatively, broad-spectrum PCR uses consensus PCR primers that can detect many HPV genotypes. These primers usually target the L1 gene of HPV which is the most conserved part of the HPV genome. There are several systems of broad-spectrum primers such as GP 5/6, GP5+/GP6+, MY09/11, and PGMY09/11 [Iftner, 2003]. Please see Table 5 for a comparison of how many HPV types are detected by each system of primers.

Table 5. Number of HPV Types Detected by Hybrid Capture-2 Compared to PCR Tests

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