Human papillomavirus (hpv) infection is the most common sexually transmitted disease, which causes different symptoms such as genital warts, intraepithelial lesions, and even cervical cancer (99.7%)1,2 depending on type of HPV. Particularly, HPV-16 and HPV-18 are the most prevalent types3 leading to cervical cancer development, which is the leading cause of cancer death among women in developing countries and the second cause of cancer among women around the world.4 But even though HPV infection by an oncogenic viral type is a necessary step for developing cervical neoplasia, other factors such as a persistent infection and high viral loads are strongly associated with an increased risk of progression to cervical lesion or cervical cancer.5–8 Such risk is further increased by behavioral aspects including multiparity, smoking habits, the use of oral contraceptive methods and having had other sexually transmitted diseases (including HIV-infection),9,10 whereas the probability of acquiring a HPV-infection is strongly related to age at sexual debut and number of sexual partners.10,11
It has been reported that the detection of anti-HPV antibodies using HPV-16 virus-like particles (VLPs) as ELISA antigens is associated to high viral loads and persistent infection. Furthermore, a high frequency of these antibodies has been reported in 70% to 90% of patients with bad-prognosis lesions [high-grade squamous intraephitelial lesions (HSIL) or cancer].12–16 In fact, it has been reported that 57% to 60% of HPV-infected women are seroconverted 6 to 18 months following HPV-DNA detection and that this seroconversion rarely occurs in women having transitory HPV infections.17,18
Linear epitopes of the L1 capsid protein have been identified by using synthetic peptides spanning the entire protein. The 473GLAKPKFTLGKKATPTTS491 peptide has reported a 91% detection rate among cervical intraepithelial neoplasia (CIN) patients with HPV-16-DNA positive biopsies compared with a 24% detection in children’s sera and 66% of CIN patients with HPV-16-DNA negative biopsies.19 Furthermore, the nona-peptide (IHSMNSTIL) allows discriminating between HR-HPV and LR-HPV (high-risk and low-risk HPV-types, respectively) among women diagnosed with low-grade squamous intraepithelial lesions (LSIL).20
Previous works done in our Institute have demonstrated that peptides 18283 55PNNNKILVPKVSGLQYRVFR74, 18294 275LYIKGSGSTANLASSNYFPT294, and 18301 414EDTYRFVTSQAIACQKHTPPA434 are specifically recognized by antisera from cervical cancer or CIN patients with a 92% to 97% sensitivity and 89% to 95% specificity.21–23
The current report assessed the humoral immune response against HPV infection in women from Girardot, Colombia by using HPV-16 VLPs and L1-peptides 18283, 18294, and 18301 as antigens; and evaluates its relationship with HPV-DNA detection, socio-demographic background and/or presence of cervical lesions.
MATERIALS AND METHODS
Study Population
Cervical cell and blood samples were collected for HPV-DNA and anti-HPV antibody detection, respectively, from 953 women attending their cervical cancer prevention program between January 2006 and January 2007 at the San Rafael Hospital in Girardot (a small-sized city visited by many tourists). All women who voluntary accepted to participate in this study gave their signed informed consent and answered a questionnaire about their sexual behavior, births, smoking habits, and some socio-demographic characteristics. Each women underwent gynecological examination, which included a Papanicolau smear test (classified according to Bethesda protocol),24 collection of cervical cells taken with a cytobrush and kept in 95% ethanol for DNA analysis by PCR as well as a blood sample taken by venipuncture for ELISA test.25–27
DNA was obtained from 766 women (80.4%) amplifying for the β-globin gene. Mean women age was 41.58 ranging between 14 to 80-years and all had a history of sexual activity. This range was chosen taking into account that most of these women had had their sexual debut at a very young age (22.66% and 78.54% at the age of 15 and 20, respectively) and some women had underwent their first Papanicolau examination after their sixties. Women in this study had a family average income of US $100 to 300 per month and were mainly married housewives (70.90%). 49.5% stated having had only 1 sexual partner during their lifetimes and 78.54% having had their sexual debut before the age of 20. The mean age at sexual debut was 18.44 years (ranging between 7 and 47 years), 82 women said never having given birth and 44% did not use any birth control methods.
Methods
Peptide Synthesis.
20-mer peptides spanning the HPV-16 L1 protein28 were synthesized by the solid-phase multiple peptide system29; using a methylbenzhydrylamine resin (0.7 meq/g), t-Boc amino acids, and low-high cleavage techniques. Peptides were analyzed by MALDI-TOF mass spectrometry and reverse phase-high performance liquid chromatography.
HPV-16 VLP Production.
HPV-16 VLPs were produced in Sf21 (insect cells) following a previously described procedure.30 Recombinant baculoviruses encoding the HPV-16 L1-protein were used for infecting SF21 cells at a 20 multiplicity of infection. Cells were harvested 4 days postinfection; cytoplasmic, and nuclear fractions were separated by 0.5% Nonidet P-40 treatment followed by spinning (10,000 × g, 15 minutes). CsCl gradient fractions were collected and their densities were determined by refractometry. Fractions having a density of around 1272 g/cm3 were pooled in 1× phosphate-buffered saline (PBS) and ultracentrifuged (4°C, 1 hour, 130,400 × g). VLP assembly was verified by electron microscopy.
Enzyme-Linked ImmunoSorbent Assay.
Twelve-well enzyme-linked immunosorbent assay (ELISA)-plates were coated with each of the 4 antigens to be assessed in duplicate using 5 μg/mL for L1-peptide 18301 and VLPs, and 1.25 μg/mL for L1-peptide 18294 and 18283, all previously diluted in PBS. Plates were incubated at 37°C for 1½ h, washed 3 times with PBS containing 0.05% Tween 20 (PBS-T), blocked with 200 μL PBS-T with 0.01% bovine serum albumin for 20 minutes at 37°C and then washed. Wells were loaded with 100 μL of the serum sample being tested diluted 1:200 in blocking buffer and incubated for 1 hour at 37°C, washed and then added 100 μL peroxidase-conjugated rabbit antihuman IgG F(ab′)2 fragment antibodies (VECTOR) at a 1:5000 dilution. The plates were incubated for 1 hour at 37°C before revealing the immune reaction by adding 100 μL peroxidase substrate (TMB KPL) per well. The reaction was stopped by adding 50 μL 1N sulfuric-acid and read at 450 nm. Sera samples were tested in triplicate and were only considered as valid when variation between replicas did not exceed 10%. Seroreactivity was calculated as the (S-N)/(P-N) ratio; where S is the optical density (OD) of the tested serum obtained with each antigen, P is the OD of each serum obtained with antihuman antibody and N is the OD obtained with no antigen. The cut-off point for defining women seropositivity was 0.20 OD for peptides and 0.25 for VLPs, which was determined by analyzing the receiver operating characteristic curves of 200 women (120 having HPV-DNA(−) normal biopsies and 80 diagnosed with HPV-DNA(+) HSIL or cervical cancer). Receiver operating characteristic curves are a graphic representation of the relation existing between the sensitivity and specificity of a test, that takes into account epidemiologic and medical-economical data of the disease.31
PCR.
Each sample was incubated in 100 μL lysis buffer [10 mmol/L Tris–HCL (pH 7.9), 0.45% Nonidet P-40, 0.45% Tween 20 and 60 μg/mL proteinase K] at 60°C for 1 hour and then at 95°C for 10 minutes.32 DNA integrity of all samples was checked by PCR amplification of the human β-globin gene using GH20/PC04 specific primers.33–35 Samples supernatant underwent PCR amplification using a HR-HPV generic GP5+/GP6+ primer set annealing in the L1 gene. PCR amplification was carried out as described elsewhere.35 DNA extracted from HPV-16 L1-transfected Sf21 cells were used as positive control, whereas ultrapure distilled water was used as negative amplification control. All PCR products were visualized on a 2% agarose gel stained with ethidium bromide using a Molecular Imager Fx (BIORAD). Positive samples were further analyzed by PCR amplification using HPV-16 and HPV-18 type-specific primers, which anneal in the E6 and E7 gene regions.36 Samples amplifying with generic primers but not with the HPV-16 or HPV-18 type-specific primers were then tested for presence of other HR viral types such as 31, 33, 45, and 58 using primers annealing in E6, E6, and E7 regions.9,36,37
Statistical Analysis
Group values were presented as percentages, means, ranks, and SD. Differences between the percentage of positive samples found by each antigen (peptides or VLPs) regarding cytology result and HPV-DNA status were assessed using the Fisher exact test. Adjusted odds ratios (ORs) were calculated at a 95% CI using logistic regression models for evaluating association between antigen seropositivity, risk factors, and HPV-DNA status. The statistical significance was set at a 0.05 level (2-tailed) using STATA 9 software for this component of the statistical analysis.
RESULTS
The Pap smear results revealed cervical cell abnormalities in 26 of 953 women (2.73%, CI 95%: 2%–4%), 5 of which were classified as having atypical squamous cells of undetermined significance, 15 as LSIL, and 6 as high-grade squamous intraepithelial lesions (HSIL). All 26 women with cervical cell abnormalities were referred for colposcopy/biopsy, but only 17 attended this examination; 13 of them needed to be taken biopsy samples, of which 6 were negative for cervical lesions, 8 had LSIL, 2 had HSIL, and 1 woman had cervical cancer.
The frequency of anti-HPV serum antibodies was determined by using Type 16 VLPs and 18283, 18294, and 18301 L1-peptides. Seropositivity frequency in this population was 36.62% for peptide 18301 (95% CI: 33.52%–39.7%), 35.36% for 18283 (95% CI: 32.3%–38.4%), and 32.95% for 18294 (95% CI: 29.9%–36%), whereas VLPs reported a 43% seropositivity (95% CI: 39.8%–46.2%). Based on cytology reports, antibody frequency in women with cervical abnormalities was higher than in women having normal cytology with all tested antigens, the highest frequency was obtained with peptide 18301 (80.46% seroprevalence) and the lowest with VLPs (53.8%). Such antibody frequency difference between normal and abnormal cytologies was statistically significant with the 3 peptides for women having atypical squamous cells of undetermined significance, (18301 P = 0.006, 18294 P = 0.003, and 18283 P = 0.005) and HSIL (18301 P = 0.002, 18294 P = 0.001, and 18283 P = 0.002), as shown in Table 1. It is worth mentioning that peptide 18301 was also capable of recognizing LSIL patients (P = 0.026).
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TABLE 1: Anti-HPV Antibody Frequency in Patients Classified According to Cytology Report
The 766 cervical samples (80.38%) whose DNA integrity had been confirmed by amplification of the β-globin housekeeping gene were tested for the presence of HR-HPV-DNA. It is important to note that PCR inhibition on cervical samples is reported to range between 6 to 19% and occur mainly because of low cellular quality and/or the method’s low performance for removing PCR inhibitors.38,39 Therefore, the percentage of unsatisfactory samples found in this study is close to the one reported. A total of 157 women (20.50%) were positive for HR-HPV DNA, of which 14.2% had HPV-16, 5.2% had HPV-18, and 18.4% did not amplified with the HPV-16 or HPV-18 type-specific primers. These later samples were further tested for the presence of other HR viral types; detecting HPV-31 in 13 women, HPV-33 in 6, HPV-45 in 11, and HPV-58 in 18. The seropositivity reported by the three peptides among this positive HR-HPV-DNA group ranged between 32.95% and 36.62%, whereas VLPs reported 43.02% reactivity.
When women were stratified by lesion status and HPV-DNA presence was taken into account, none of the antigens reported seroprevalence differences between women with HPV-DNA(+) and HPV-DNA(−) Pap smears. However, a statically significant higher frequency of antibodies was detected by the 3 peptides in HPV-DNA(+) women having cervical abnormalities compared to HPV-DNA(−) women and/or having normal cytology findings (18301 P = 0.002; 18294 and 18283 P = 0.011), whereas VLPs only detected a 55,56% seropositivity in the same group (P = 0.495), as shown in Table 2.
TABLE 2: Anti-HPV Antibody Frequency in Patients According to Cytology Report and HR-HPV DNA
Regarding socio-demographic characteristics, seroreactivity against peptide 18283 (35.71%) was significantly higher among women >44 years compared with other age groups (OR = 2.309), whereas it was significantly lower (18.84%) among women using an intrauterine dispositive (IUD) (OR = 0.270) compared with those using other birth control methods. Peptide 18301 (OR = 2.197) and peptide 18294 (OR = 2.558) detected a significantly lower frequency of antibodies among women who had never given birth compared with women who had given birth once or more than 3 times.
There was a statistically significant trend toward higher levels of seropositivity for peptide 18283 among separated women (OR = 1.905) and those living together with a partner (OR = 1.614). Statistically, there was no difference between the seropositivity of women according to smoking habits or factors related to sexual behavior such as age at sexual debut and lifetime number of sex partners. Regarding VLPs, there was a significant association between women who used surgery as a birth control method and seropositivity (OR = 1.498), as shown in Table 3.
TABLE 3: Adjusted Association (OR) Between Seropositivity For the 3 Peptides of Interest and VLPs, Sociodemographic Variables, and DNA-HR-HPV Detection
DISCUSSION
Girardot is a small tourist city with a population of around 180,000 inhabitants, visited especially on weekends by people coming from the country’s capital (Bogotá). Although a high prevalence of sexually transmitted diseases has been reported in this city by the health secretariat of Cundinamarca, no studies have been conducted regarding HPV-infection prevalence. Most of the population involved in this study came from nearby rural areas and belonged to low-income families with low levels of education (data not shown), all of which have been identified as important socio-demographic factors related to a high frequency of HPV-associated cervical lesions.
The 20% HPV-DNA frequency found among women participating in this study is similar to the one reported by other studies carried out in Colombia.40–42 Similarly, the frequency of cervical lesions found by cytology examination, even thought being low (2.73%), fall into the range of 2.2% to 7.11% reported in other Colombian populations.42,43 These results suggest that most HPV-infected women’ immune systems raise humoral responses for controlling this infection. However, several recent studies including 1 in an industrialized country involving more than 10,154 women have reported low sensitivity of Pap testing (55%) for detecting cervical lesions,44 which suggests that most women suffering cervical HPV-associated lesions are probably not being detected by cytology screening.
In agreement with other studies, we have found a high frequency of anti-HPV IgG antibodies by using L1 peptides and VLPs in patients with cervical lesions, which could be related to the presence of persistent HPV-infections20; the antibody detection increased with all tested antigens as lesion severity increased. Specifically, women having cervical lesions showed higher frequency of anti-HPV antibodies than women with normal cytology, thus indicating that most women having HPV-associated cervical lesions are being included within the group of seropositive women by screening with these peptides, whereas most women having no cervical lesions are being included within the seronegative group of women. The results also suggest that these peptides contain epitopes capable of inducing antibody production, mainly after developing a cervical lesion which is probably related to a high viral load during a natural HPV infection. Therefore, detecting antibodies against peptide 18301 could be related to progression of HPV infection or cervical lesion development.
Regarding peptide 18283, a higher seropositivity was detected among women whose marital status was separated or living together with a partner as well as among those who had given birth vaginally; whereas it decreased among women having no children and IUD users. This data suggests that changes in these women’ immune responses could have occurred as a result of an immunosuppression effect induced by pregnancy and/or persistent local inflammation because of the use of IUDs, which is evidenced by the low detection of HPV-DNA among women using this contraceptive method. The results also suggest differences regarding peptide 18283 immunogenicity compared to that of peptide 18301, 18294, and VLPs, which was higher among women using other contraceptive methods. Furthermore, no statistically significant anti-HPV antibody frequency was found with the 4 antigens evaluated regarding HR-HPV DNA status adjusted according to the socio-demographic variables contemplated in this study, possibly because of a brief exposure to the virus, low viral load, or nonpersistent infection hampering the development of an IgG-mediated immune response against L1 (this kind of response arises approximately 8–12 months after infection). These results agreed with recent reports by Urquiza et al., attributing such behavior to differences in the antibody-induction kinetics of these B epitopes.23
The results of this study evidence the importance of gynecologically following-up women who have been detected with a HPV-DNA infection and/or who have anti-HPV antibodies because of the low detection rate of cervical lesions by cytology examination. Indeed, a more recent epidemiologic analysis of 2007 evaluating the prevalence of cervical lesions in the city of Girardot reported 66 HSIL patients and 8 cervical cancer cases (data not shown), which are alarming statistics for a small-sized population, thus highlighting the importance of implementing serological screening as a supportive test to cytology examination. In conclusion, 18283, 18294, and 18301 L1-peptides could be useful in the design of a serologic test for detecting women with cervical lesions at risk of progressing to cervical cancer; since they are more specific than VLPs and have a similar sensitivity for detecting HR-HPV DNA positive cervical lesions.
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