Mucosal IgG and IgA Responses to Human Papillomavirus Type 16 ...

VIRAL IMMUNOLOGY Volume 16, Number 2, 2003 © Mary Ann Liebert, Inc. Pp. 159–168

Mucosal IgG and IgA Responses to Human Papillomavirus Type 16 Capsid Proteins in HPV16-Infected Women without Visible Pathology L. ROCHA-ZAVALETA,1 A.L. PEREIRA-SUAREZ,2 G. YESCAS,3 R.M. CRUZ-MIMIAGA,3 A. GARCIA-CARRANCA,1,4 and F. CRUZ-TALONIA5

ABSTRACT Human papillomavirus type 16 (HPV16) may infect the cervical epithelium without producing pathological changes for a long time. To investigate if mucosal antibodies are induced in HPV16-infected women without visible pathology, cervical mucus from HPV16-infected patients with and without evident pathology, along with mucus from uninfected women were analyzed for the presence of mucosal IgG and secretory IgA (sIgA) antibodies to HPV16 capsid proteins by ELISA. sIgA and IgG antibodies were found in a significantly higher proportion of infected patients compared with uninfected women (p , 0.0001). sIgA antibodies were present in 13.1% of infected patients without visible pathology, the proportion of positivity increased to 27.0% in patients with visible pathology (p 5 0.001). Mucosal IgG response was observed in 6.5% of patients without and 27.5% of patients with visible pathology (p 5 0.00005). The antibody mean signal strength was significantly higher in patients with than in patients without pathological evidence (p , 0.005). In conclusion, both sIgA and IgG are found in patients without pathological signs of infection, however, the response increases significantly in patients with pathological evidence, suggesting that the appearance of these changes might be associated with a more vigorous antibody-mediated mucosal reaction. INTRODUCTION

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(HPV) infection of the lower female genital tract is a common sexually transmitted disease. Oncogenic HPV types are strongly associated with the development of cervical cancer (34). HPV type 16 (HPV16) is the most prevalent oncogenic genotype in both, cervical tumors (4) and advanced precursor lesions (19). Furthermore, recent observations suggest that HPV16 is also highly prevalent in early HPV-associated cervical lesions (26). UMAN PAPILLOMA VIRUS

Departments of 1Molecular Biology and Biotechnology, 2Immunology, Institute of Biomedical Research, National University of Mexico (UNAM), 3 Clinic of Dysplasias, Mexican College of Colposcopists, 4Research Division, National Cancerology Institute, and 5National Center for Dysplasia Clinics (CENACLID), General Hospital of Mexico, Mexico City, Mexico.

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ROCHA-ZAVALETA ET AL. Genital HPV infections are predominantly subclinical. These infections are characterized by a lack of macroscopic or histologic abnormalities, that cannot be detected by the naked eye and are asymptomatic. The presence of HPV DNA in subclinically infected cells can be determined with molecular techniques, like amplification techniques (PCR) and liquid hybridization (Digene Hybrid Capture). Early clinical manifestations of HPV infection can be visualized by cytologic and colposcopic examination. The Papanicolaou test is the most frequently used method of evaluating cytological evidence of HPV infection, it is based on the detection of koilocytic cells as a pathognomonic sign of HPV biological activity. On the other hand, the application of a diluted solution of acetic acid causes transient, detectable changes in HPV-infected epithelial surfaces, which turn white. Colposcope-mediated observation of acetic acid-reactive epithelium allows the clinician to detect and to determine the extent of the infection. HPV16 enters the body through the genital mucosa, a barrier whose defense relies on the components of the local immune system. Secretory IgA (sIgA) is believed to be an important component of humoral protection of the genital mucosa. However locally produced IgG along with serum-derived IgG, which leaks through the surface epithelium, also contribute to the neutralization and removal of invader pathogens (23). Systemic (6,9,10,37) and mucosal (3,12,32,35) antibodies against the viral capsid proteins are frequently produced in HPV16-infected women. Moreover, some of these antibodies may be able to bind the virus in vitro (25). Previous data suggest that mucosal IgA but not IgG antibodies to HPV16 capsid proteins are associated with the presence of HPV16 DNA in women with abnormal cytology (3). Additionally, cervical IgG and IgA antibodies have been found to be associated with the prior detection of squamous intraepithelial lesions (12). The present work aimed to investigate whether mucosal antibodies against HPV16 capsid proteins are produced in HPV16-infected women in the absence of pathological signs of infection. sIgA and IgG antibodies to HPV16 capsid proteins were measured by ELISA in cervical mucus obtained from HPV16-infected patients with and without cytological and colposcopical signs of infection, uninfected women were included as controls.

MATERIALS AND METHODS Study population. Study groups were selected from a population of patients (n 5 3,647) who participated in an HPV prevalence study. Samples were collected between 1996 and 2001 at the National Center of Dysplasia Clinics (CENACLID), General Hospital of Mexico, and the Clinic of Dysplasias, Mexican College of Colposcopists, Mexico City. Informed consent was obtained from all women included in this work. Human material was handled according to institutional experimentation and safety guidelines. Three groups of women were studied. The first group was composed of patients who presented abnormal cytological and colposcopical findings, and were positive for the presence of HPV16 DNA; this group is referred to as “infected with visible pathology” (mean age: 30.8 years; range: 19–48 years). The second group included patients with normal cytological and colposcopical findings, who were proved positive for the presence of HPV16 DNA; they are referred to as “infected without visible pathology” (mean age: 31.6 years; range: 20–49 years). Finally, a control group was selected of healthy women with normal cytology and colposcopy who were negative for the presence of HPV16 , or any other HPV type, by molecular means (mean age: 31.0 years; range: 20–46 years). Cytological and colposcopical evaluation. Cervical cells from the uterine cervix were collected by scraping the transformation zone with a cytobrush. Cervical cells were used to prepare Pap smears of all women who participated in a previous HPV prevalence study (n 5 3,647). All Pap smears were examined independently by two experienced cytotechnologists. Samples with inconsistent diagnosis were excluded from the study. Cytology diagnoses were classified according to the Bethesda system into normal, atypical squamous cells with undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), highgrade squamous intraepithelial lesions (HSIL), and cervical carcinoma (31). Patients with cervical carcinoma were excluded. Patients with normal and abnormal cytology underwent colposcopy. Acetowhite changes of the cervix were recorded 30–60 sec after the application of a 15% acetic acid solution. Abnormal colposcopical findings within the transformation zone included acetowhite epithelium, punctuation and mosaicism. 160

MUCOSAL IgG AND IgA RESPONSES TO HPV16 CAPSID PROTEINS Human samples. Colposcopy-directed biopsies were obtained from patients with and without acetowhite changes. In both cases, biopsies were taken from the transformation zone, because this area is the most frequent target for the high-risk HPV infection (11). Tissue samples were always obtained from the acetowhite area of colposcopy abnormal patients. Biospsies from women without an acetowhite reaction were always taken from two opposite quadrants of the transformation zone. Biopsies were transported in tubes containing sterile, contaminant-free phosphate-buffered saline (PBS; Roche Applied Science, Switzerland) and processed the same day. Cervical mucus was collected by washing the uterine cervix with 1 mL of sterile PBS (Roche Applied Science). Cell debris was eliminated by centrifugation at 13,000 rpm for 5 min. Mucus samples were stored at 270°C until tested. HPV DNA amplification by PCR. All reagents used for the isolation and amplification of DNA were purchased from Gibco BRL (Rockville, MD). Tissue samples were treated with Proteinase K as described elsewhere (16). DNA was extracted with phenol-chloroform and precipitated with ethanol. HPV DNA was amplified using the general MY09 (59 CGT CCM ARR GGA WAC TGA TC 39) and MY11 (59 GCM CAG GGW CAT AAY AAT GG 39) primers (20), that amplify a conserved 450-bp fragment from the L1 gene. 100 ng of genomic DNA were denatured by heating the reaction to 95°C for 30 sec. Annealing of primers was performed at 45°C for 30 sec and extension at 72°C for 60 sec. The cycle was repeated 30 times. PCR products were electrophoresed in 2% agarose gels, stained with ethidium bromide and visualized in a UV transilluminator. Internal control to assure DNA integrity was performed by amplifying the b-globin gene using the PC03 and PC04 primers as described previously (27). As positive control, DNA from SiHa, a cervical cancer–derived cell line containing 1–2 HPV16 copies per cell, was run concurrently with each reaction. Additionally, negative controls to assess the presence of contaminants were set using purified water (Gibco BRL) and PBS (Roche Applied Science) instead of template DNA. HPV16 DNA amplification by PCR. Specific amplification of HPV16 was achieved by using the Pr3 (59 GTC AAA AGC CAC TGT GTC CT 39) and Pr4 (59 CCA TCC ATT ACA TCC CGT AC 39) primers (15), that amplify a 499 fragment covering the HPV16-E7 plus fragments of the E6 and E1 genes. 100 ng of template DNA was denatured at 95°C for 30 sec. Primers were annealed at 57°C for 60 sec and then extended at 72°C for 60 sec. The cycle was repeated 30 times, and the PCR products were analyzed as described above. Hybrid Capture II assay. The Digene HPV Test Hybrid Capture® II (Digene Corp., Beltsville, MD) for the detection of high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 was used. 20 mL (250–500 ng) of DNA extracted from cervical tissue were placed in a tube containing 30 mL of specimen transport medium. 25 mL of NaOH-based denaturation reagent was added to each sample, the tubes were vigorously mixed and incubated at 65°C for 45 min. Hybridization and hybrid detection were performed according to the manufacturer’s instructions. Carrier DNA and constructed HPV16 DNA were respectively used as negative and positive calibrators, and were run in triplicate with each test. An assay was considered valid only when the results from the negative and positive calibrators showed a coefficient of variation #15%, and the positive calibrator: negative calibrator mean values ratio was $2.0. Cut-off value for positivity was calculated for each assay and was defined as the mean RLU (relative light units) value of the positive calibrator. Detection of mucosal IgG and sIgA antibodies by ELISA. Antibodies were detected with HPV16 viruslike particles (VLP) as target antigen in an ELISA. Recombinant HPV16 VLP-expressing Baculoviruses were kindly donated by Dr. John Schiller (National Institute of Health, Bethesda, MD). VLP were produced, extracted and purified as described previously (8). ELISA plates (Maxisorp, Nalge Nunc Int. Co., Naperville, IL) were coated with 500 ng/well of purified VLP diluted in PBS at 4°C overnight. A standard ELISA was performed, using 100 mL of undiluted cervical mucus. Rabbit-anti human IgA-secretory component (Dako Co., Carpinteria, CA), which specifically reacts with free human secretory component and secretory component bound to secretory IgA, or rabbit-anti human IgG (Dako) were used as secondary antibodies, followed by an anti-rabbit IgG-alkaline phosphatase conjugated antibody (Sigma Chemical Co., St. Louis, MO). Alkaline phosphatase substrate Sigma 104 was diluted in a 10% diethanolamine (Sigma Chemical Co.) solution and added to the plates. Reaction was read at 405 nm in an ELISA plate reader. All samples were tested in triplicate for each antibody class, the assay was considered valid only when the coefficient of variation of the triplicates was $10%. Additionally all samples were tested on two wells not 161

ROCHA-ZAVALETA ET AL. coated with VLP to define non-specific reactivity. The final ELISA value was calculated by subtracting the non-specific reactivity mean absorbance from the triplicate mean absorbance. Cut-off value for positivity was calculated on the bases of distribution of absorbances of the control group, and was defined as the mean absorbance 1 3 standard deviations after exclusion of outliers. Calculated cut-off values for IgG and IgA were 0.861 and 0.803, respectively. Statistical analysis. To evaluate the differences between the proportions of positive samples in the different groups, data were arranged in the form of 2-by-2 contingency tables and analyzed by Fisher’s exact test to calculate Odds ratios, 95% confidence intervals and p values. The Wilcoxon signed rank test and the t-test were used to compare the mean signal strength (optical density) of the various groups. All tests were two-tailed and considered a basic significance level of p 5 0.05.

RESULTS Presence of HPV16 in human biopsies. The aim of this study was to investigate the mucosal humoral immunity against HPV16 in HPV16-infected women with and without detectable pathology. To identify HPV16-infected women, a set of DNA samples from patients who were positive for both, a universal primer-HPV PCR and a Hybrid Capture Test for high-risk HPV types, as well as women without cytological and molecular evidence of HPV, was analyzed using a HPV16-specific PCR. DNA isolates from 349 patients with visible pathology, 277 patients without visible pathology, and 171 uninfected women were studied. HPV16 was highly prevalent in the high-risk HPV-infected patients. 137 (49.4%; 95% CI: 43.4–55.5) of the patients without visible pathology and 174 (49.8%; 95% CI: 44.4–55.2) of the patients with visible pathology were positive for the presence of HPV16. These results seem to indicate that HPV16 is a highly prevalent genotype among high-risk HPV-infected patients, regardless of the absence of apparent pathology. Detection of mucosal sIgA antibodies. sIgA antibodies are the first line of defense at mucosal surfaces. To investigate if sIgA antibodies are produced as a response to cervical HPV16 infection in the absence of pathological changes, the proportion of sIgA positivity in infected patients without visible pathology was evaluated and compared with the presence of sIgA in patients with pathological changes and uninfected control women (Table 1). sIgA positivity against HPV16 capsids was 2.5–5.1-fold more common in HPV16-infected patients than in uninfected controls. The proportion of infected patients without visible pathology who were positive for the presence of sIgA, was significantly higher than the proportion in the uninfected group (Table 1), suggesting that sIgA antibodies are developed even in the absence of pathological manifestations of infection. The proportion of positive samples increased in the infected group with visible pathology (Table 1). In fact, the probability of presenting sIgA antibodies was significantly higher for the infected women, when compared with the probability for the uninfected women. Interestingly, a significant difference in the mean signal strength among the three groups was found (Fig. 1). The signal intensity was significantly higher in both infected groups as compared with the uninfected control (Wilcoxon signed rank test p , 0.0001; t-test p , 0.05). The difference between the infected groups also proved to be significant, being higher in the patients with visible pathology (Wilcoxon signed rank test p , 0.0001; t-test p , 0.0001). These results seem to indicate that the humoral response is stronger when the pathological changes emerge. Detection of mucosal IgG antibodies. Although IgA antibodies are the dominant immunoglobulin in mucosal secretions, IgG-class antibodies have been found to participate in mucosal immune reactions. Here, the presence of IgG in cervical mucus of HPV16-infected patients and uninfected controls was investigated. Results are reported in Table 1. IgG anti-HPV16 antibodies were not detected in uninfected women, however 9 (6.5%; 95% CI: 3.0–12.1) infected patients without visible pathology were positive for the presence of IgG antibodies. The number of responders increased to 48 (27.58%; 95% CI: 21.0–34.8) among the infected patients with visible pathology (Table 1). The probability of being positive for IgG antibodies was significantly higher for the infected patients with visible pathology than for the infected patients without visible pathology (Table 1). In accordance with the observations for the sIgA antibodies, the mean signal strength of the IgG response was different among the groups (Fig. 2). Again, the lowest signal strength was 162

MUCOSAL IgG AND IgA RESPONSES TO HPV16 CAPSID PROTEINS TABLE 1. PROPORTION OF CERVICAL MUCUS SAMPLES C ONTAINING S IG A AND IG G ANTIBODIES TO HPV16 CAPSID PROTEINS IN RELATION WITH THE PRESENCE OR ABSENCE OF PATHOLOGIC AL SIGNS OF HPV INFECTION Uninfected womena (n 5 171) sIgA positivity (%) Odds ratio 95% CI p value* IgG positivity (%) Odds ratio 95% CI p value*

5.2 Reference

0.0 Reference

Infected without visible pathologyb (n 5 137)

Infected with visible pathologyc (n 5 174)

13.1 1.312 0.828–2.077 0.05 6.5 1.785 1.068–2.982 0.0003

27.0 2.335 1.515–3.599 ,0.0001 48.0 6.716 4.153–10.862 ,0.0001

aWomen with normal cytological and colposcopical findings. HPV DNA could not be detected by PCR or Hybrid Capture II test. b HPV16-infected patients. Presence of HPV16 DNA was detected by PCR using the Pr3-Pr4 primers. cHPV16-infected patients. HPV infection was diagnosed by cytology and colposcopy; HPV16 DNA was detected by PCR using the Pr3-Pr4 primers. CI, confidence interval. *Two-tailed p value calculated by Fisher’s exact test.

observed in the control group, the mean signal strength increased significantly in the infected group without visible pathology (Wilcoxon signed rank test p , 0.0001; t-test p , 0.0001), and it was highest in the group of patients with visible pathology (Wilcoxon signed rank test p , 0.0001; t-test p , 0.0001), suggesting that both, IgG and sIgA responses, become stronger in the presence of conspicuous HPV16-induced lesions.

FIG. 1. sIgA response to HPV16 capsid proteins in cervical mucus from HPV16-infected patients with (n 5 174) and without (w/o) (n 5 137) visible pathology, and uninfected controls (n 5 171). The cut-off value for positivity (0.803) is represented as a dotted line. Solid lines indicate the mean absorbance value of each group: uninfected 5 0.365; infected w/o visible pathology 5 0.430; infected with visible pathology 5 0.573. OD, optical density.

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FIG. 2. IgG response to HPV16 capsid proteins in cervical mucus from HPV16-infected patients with (n 5 174) and without (w/o) (n 5 137) visible pathology, and uninfected controls (n 5 171). The cut-off value for positivity (0.861) is represented as a dotted line. Solid lines indicate the mean absorbance value of each group: uninfected 5 0.210; infected w/o visible pathology 5 0.322; infected with visible pathology 5 0.642. OD, optical density.

DISCUSSION The application of sensitive molecular techniques, such as PCR and the Hybrid Capture test, to the specific detection of HPV genotypes (15,24) has provided an insight into the prevalence of certain oncogenic HPV genotypes. HPV16 has been recognized as the genotype most commonly associated with cervical tumors (4) and precursor lesions (14,19). However, less is known about the prevalence of this HPV type in earlier lesions. Here we found that HPV16 is highly prevalent in patients infected by high-risk HPV types, whose cells do not show visible pathological changes. In fact, it accounted for 50% of the cases analyzed, suggesting that HPV16 is a prevalent high-risk HPV genotype, regardless of the absence of pathological changes. In this work, the presence of mucosal sIgA and IgG antibodies to HPV16 capsids was investigated in uninfected women and HPV16-infected patients with and without visible pathological changes. 5.2% of uninfected women were positive for the presence of sIgA antibodies. It could be postulated that these women were not true-negatives but the result of clinical and/or molecular misclassification. This is unlikely because a number of complementary techniques were used to verify the HPV-negative results. Cytology was performed independently by two cytotechnologists, and the negative cytology results were corroborated by colposcopy. It is also important to mention that the colposcopist was not aware of the cytology results. As to the molecular techniques used, they have been recognized as highly sensitive methods for the detection of HPV infections (7,24), and their individual level of false-negative results is greatly reduced when they are used concurrently to analyze the same samples. In our case, all samples were tested by PCR using two different primer sets (MY09–MY11 and Pr3–Pr4), and they were also analyzed using the Hybrid Capture II test for high-risk HPV genotypes. A more liable explanation might be that these women had suffered from a previous HPV infection, because the production of antibodies against HPV16 capsid proteins only occurs after HPV transmission (17). IgA antibodies to HPV16 capsid antigens have been found in cervical mucus (33), oral fluid (21), and serum samples (28,36,37) from cytologically normal and uninfected controls. On the one hand, it has been suggested that serum IgA might be a marker for recent HPV infections (36); on the other hand, it has been proposed that mucosal IgA might be due to either a fluctuating HPV infection (33) or a previously cleared infection (21). The present study provides cross-sectional data, therefore we cannot conclude about the 164

MUCOSAL IgG AND IgA RESPONSES TO HPV16 CAPSID PROTEINS meaning of the presence of antibodies in uninfected patients. To clarify this point, follow-up studies are warranted. sIgA is the major effector molecule in mucosal immunity. Here we found an sIgA-mediated reaction against HPV16 capsid proteins in 20.9% of HPV16-positive patients. This is in agreement with the report by Wang et al. (35) who found that 25% of cervical mucus samples from HPV16-infected patients contained IgA antibodies against HPV16 capsids. Interestingly, our data show that sIgA antibodies are developed against the viral capsid in HPV16-positive patients who do not present pathological evidence of infection. In a previous work, a proportion of women with benign cytological diagnosis was shown to be positive for the presence of IgA to HPV16 capsids (35); however, the population studied included women infected by a number of different HPV genotypes. Therefore, the information reported in our work is more specific, since only HPV16-infected women were studied. The proportion of sIgA-positive patients was significantly higher in the group with (27%) compared with the group without visible pathology (13.1%). The proportion of positivity in the group with pathological changes is consistent with the proportion found by Bontkes et al. (3). A more recent report shows that the presence of sIgA against HPV16 capsid antigens is associated with detection of squamous intraepithelial lesions (12), which is in agreement with our results. IgG antibodies are also involved in the female genital tract immune reactions. In the present report it was observed that 18.3% of HPV16-infected patients were positive for IgG antibodies. Similar proportions of cervical IgG positivity have been reported for HPV16-infected women (3,12). In accordance with the sIgA data, the proportion of IgG positive patients was higher in the infected group with visible pathology compared with the group without visible pathology. Contrary to our results, Bontkes et al. (3) found that there was no significant difference in the proportion of cervical IgG positivity between HPV16-infected women with normal and abnormal cytology. We consider that the two studies cannot be adequately compared, because ours is a cross-sectional study, whereas Bontkes et al. performed a follow-up study. Besides, they organized their study groups on the bases of a modified Papanicolaou system, and considered Pap 1 (no cytomorphological abnormalities) and Pap 2 (inflammation) together as “normal cytology,” whereas we used the Bethesda system for cytological classification, therefore, our infected group without visible pathology included exclusively patients with normal cytology. As expected, the mean signal strength produced by both sIgA and IgG antibodies, in the HPV16-infected groups was significantly higher than that observed in the uninfected control group. Interestingly, patients with pathological signs of infection exhibited a significantly higher mean signal intensity than those without visible pathology. This observation might indicate that a more vigorous antibody-mediated reaction is developed in patients suffering from clinically identified HPV16 infections as compared with those infected but lacking pathological changes. The major capsid protein (L1) can be found in all degrees of koilocytosis (18), condylomata acuminata (38), and preneoplastic lesions (2) of the lower female genital tract, which may well explain the presence of anti-HPV capsid antibodies in patients with this type of lesions. Nevertheless, as far as we are aware, there is no information about the expression of capsid proteins in infected tissue whose cells do not present pathological signs of infection. A tentative explanation might be that a low level of capsid protein expression takes place in some cells at this stage of infection, inducing the concomitant production of modest antibody levels. To corroborate this hypothesis, studies of the transcription of the viral capsid genes, as well as immunohistochemical detection of the capsid proteins in human biopsies are necessary. Taken together, these data indicate that mucosal sIgA and IgG antibodies to HPV16 capsids are produced as a response to infection, regardless of the absence of detectable pathology. Besides, the level of response increases with the presence of pathological signs of infection. There is strong evidence suggesting that cellmediated immune responses are important modulators of the natural course of HPV infections (1,13), but the clinical meaning of the antibody response to HPV infections remains unclear. The protective capacity of mucosal antibodies against some genital viruses has been demonstrated (23,30); locally produced IgA antibodies have been reported to neutralize genital viruses (5), and to inhibit intracellular viral replication (22). Interestingly, some evidence suggests that antibodies generated against HPV type 6 may interfere with the carcinogenetic process associated with HPV16 (29); therefore, a potential implication of mucosal antibodies in the defense against HPV cannot be ruled out, instead we consider that more studies are needed 165

ROCHA-ZAVALETA ET AL. to evaluate if naturally occurring antibodies, detected during early HPV infection, contribute to control the infection of new cervical cells.

ACKNOWLEDGMENTS We thank Dr. John Schiller (National Institutes of Health) for the donation of the HPV16 VLP-expressing baculovirus; Miriam C. Guido for technical assistance; and Jorge Ortiz, M.D., Guillermo Gomez, M.D., and Miguel A. Castillo, M.D., for assistance in obtaining the human material. The English version of the manuscript was corrected by Isabel Perez Monfort.

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Address reprint requests to: L. Rocha-Zavaleta, Ph.D. Instituto de Investigaciones Biomedicas Departamento de Biologia Molecular y Biotecnologia UNAM Circuito Escolar s/n. Ciudad Universitaria Apartado Postal 70228 Mexico D.F., C.P 04500, Mexico E-mail: [email protected] Received July 22, 2002; accepted January 14, 2003.

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