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Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as measured by a novel ELISA based on denatured recombinant HPV16 late (L) and early (E) antigens

Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as... Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV. Objectives: The objective was to assess the clinical and epidemiological correlates of HPV- seroreactivity in a cohort of HIV-negative and HIV-positive women. Methods: Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study. Results: No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women (OR = 0.158; 95%CI 0.036–0.695, p = 0.014; Fisher's exact test). Importantly, this association did not loose its significance when controlled for Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 confounding from age (continuous variable) in multivariate analysis or using Mantel-Haenszel test for age-groups. Conclusion: It is tempting to speculate that HIV-positive current smokers comprise a special high- risk group, with highly impaired immunological response that could prevent eradication of persistent HPV infections and thus contribute to development of CIN3/CC. evaluation of the new HPV vaccines the WHO recom- Background Human Papillomavirus (HPV) infection, known to be mends the standardisation of the different in-house L1- associated with both benign and malignant tumours at based serological methods by the use of reference sera and different mucosal sites, is widespread among female and promoted an international collaborative study with this male populations [1,2]. The oncogenic high-risk HPV proposal [21]. types (HR-HPVs) are the single most important etiological agents of cervical cancer (CC) and its precursor (CIN) To improve the understanding of correlation between lesions [3,4]. Despite global differences in the prevalence infection and antibody response in patients with and of individual HR-HPV types, HPV16 remains the most fre- without cervical lesions, prospective cohort studies using quent HR-HPV type in all geographic regions and the cur- a widespread panel of viral antigens would be needed rent HPV research is mostly focused on it [3,4]. Diagnosis [14,15,26]. Similarly, it would be necessary to study the of HPV infection is based on detection of viral DNA using dynamics of HPV-E and -L antigen-specific antibodies in different PCR-based methods or commercially available relation to their clinical and epidemiological correlates hybrid capture (HC2) test [5,6]. Until now, serological [27,28], including immune-suppression due to HIV infec- studies have had little to offer in diagnosis of HPV infec- tion [25,29]. There are recent growing evidences regarding tions [7]. The first serological assays for HPV antibody the induction of antibodies neutralising across papilloma- detection were introduced in the early 1970's [8], but the virus types after vaccination with VLP displaying L2 more widespread use of HPV serology had to await the epitopes or with fusion protein containing L2E7E6 viral development of more refined assays based on virus-like proteins or synthetic peptides of the HPV16 L2 protein particles (VLP) in the early 1990's [9,10]. A growing body [30-32]. Very recently Rizk and collaborators [33] have of serology data has being obtained using different viral shown that L1 fused to GST protein displays a broad vari- protein-based ELISA to analyse HPV antibodies in differ- ety of epitopes, both conformational and linear; the neu- ent settings, most notably in large-scale seroepidemilogi- tralising ones are conformational and, mostly, type cal- and case-control studies to assess the risk factors of specific while the cross-reacting ones are associated with cervical carcinoma (CC) in HPV exposed subjects [11-17]. linear epitopes. The main conclusion from the plethora of sero-epidemio- logical studies is that antibody response to viral proteins We have recently reported the development of an in- does not invariably occur during a natural HPV infection house ELISA test based on five recombinant HPV16 pro- [14,15]; only half of HPV DNA-positive women with nor- teins expressed in E. coli: L1 and L2 capsid proteins, E6 mal cytology shows antibody response to VLPs [18,19]. and E7 oncoproteins and the non-structural E4 protein, HPV E6/E7 antibodies are predominantly found in all used in unfolded form [34]. The idea of using dena- patients with CC, but they are also detected in healthy tured antigens comes from the observation of a high controls, precluding the use of their detection as predictor amino acid identity, ranging from 36% for E6 to 74% for of CC [14,15,20]. L1, among the same proteins belonging to different HPV genotypes and then the possibility to detect cross-reacting VLP-based assays have been recognized as reliable geno- antibodies directed against linear epitopes. By this assay type-specific assays [14,15,21] and potentially will con- we were able to detect an antibody response in women tribute to the understanding of the natural history of HPV infected with HPV genotypes different from that of the ref- infections [18,19,22]. New L1VLP-based vaccines have erence antigen. In this study we also determined the Avid- been shown to induce levels of HPV type specific antibod- ity Index (AI) [35] of the positive samples. Antibody ies higher than that found in natural infected women response to linear epitopes has been reported in the past [23]. Correlation between anti-L1VLPs antibodies and [36,37] and was correlated to the specific stage of HPV- protection from reinfection in natural infection is still a associated cervical disease [38]; nevertheless this antibody controversial issue [24,25]. An important problem with response can not be related to protection level against serological studies resides in the fact that the results from HPV infection, that is associated to the neutralising com- different laboratories are not comparable for the lack of ponent of humoral response. Our system, revealing anti- reference specific sera [26]. For the serological response bodies only against linear epitopes, could be utilized to Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 evaluate the immunological responsiveness of subjects to Results HPV infection and contribute to estimate the risk of infec- Correlation of epidemiological characteristics to serological response tion progression but it could not provide information about protection level from reinfection in both natural The recorded epidemiological characteristics of the infection and after vaccination. enrolled women have been correlated to their serological response to either all HPV16 antigens, or separately E and In the present study we have correlated the antibody L proteins (Table 1). The statistical analysis shows that response, evaluated with our system, to the baseline epi- antibody response is not related to patient's age or their demiological, clinical and HPV DNA data of women ethnic origin (data not shown); however, the power of the enrolled in a multi-centre cohort study designed to study to detect true differences in the age between the two explore pathogenetic mechanisms and prognostic factors groups is limited, due to the small sample size. Interest- of CIN lesions in HIV-positive and HIV-negative women, ingly, current smokers have significantly (p = 0.009) less known as HPV-PathogenISS study [39]. seroreactivity to L antigens as compared with the non- Table 1: Key epidemiological data of patients related to their serological response to HPV16 antigens Variables Anti-E and -L Anti-E -E and E Anti L and -L 4 6 7 1 2 + - Sign. + - Sign. + - Sign. Mean Age (yrs) 34.9 36.9 p = 0.460 34.8 36.0 p = 0.533 36.1 35.6 p = 0.805 Smoking status % Non-smoker (40) 92.5 7.5 OR = 2.8 (0.7–11.3); 72.5 17.5 OR = 1.4 (0.6–3.6); 85.0 15.0 OR = 3.7 (1.3–10.5); p = 0.211 p = 0.495 p = 0.009 Current smoker (48) 81.3 18.7 64.6 35.4 60.4 39.6 N. cigarette/d 13.4 10.1 p = 0.285 13.8 10.9 p = 0.225 13.3 12.0 p = 0.603 Age start smoking (yrs) 18.9 19.6 p = 0.776 18.2 20.6 p = 0.122 19.5 18.1 p = 0.379 Years smoked 15.2 13.3 p = 0.592 16.5 12.0 p = 0.089 14.0 16.8 p = 0.296 Contraception (%): Yes (53) 92.5 7.5 OR = 1.4 (0.3–6.8); 73.6 26.4 OR = 1.3 (0.5–3.4): 73.6 26.4 OR = 0.7 (0.2–2.1): p = 0.694 p = 0.658 p = 0.560 No (29) 89.7 10.3 69.0 31.0 79.3 20.7 ^New partner (%): Yes (5) 100 - OR = NC; p = 0.508 60.0 40.0 OR = 0.7 (0.2–2.2); 60.0 40.0 OR = 0.7 (0.2–2.1); p = 0.630 p = 0.619 No (76) 86.8 13.2 71.1 28.9 72.4 27.6 Sexually active (%): Yes (79) 89.9 10.1 OR = 5.9 (0.9–40.9); 70.9 29.1 OR = 1.6 (0.3–10.4); 74.7 25.3 OR = 4.4 (0.7–28.4); p = 0.105 p = 0.631 p = 0.124 No (5) 60.0 40.0 60.0 40.0 40.0 60.0 Partner HIV+ (%): Yes (8) 87.5 12.5 OR = 0.9 (0.1–8.7); 87.5 12.5 OR = 3.3 (0.4–28.3); 62.5 37.5 OR = 0.6 (0.1–2.8); p = 1.000 p = 0.679 No (75) 88.0 12.0 68.0 32.0 73.3 26.7 Actual contraception % Yes (37) 86.5 13.5 OR = 0.7 (0.2–2.7); 67.6 32.4 OR = 0.8 (0.3–2.1); 64.9 35.1 OR = 0.4 (0.1–1.1); p = 0.726 p = 0.660 p = 0.093 No (43) 90.7 9.3 72.1 27.9 81.4 18.6 Condom Use % Never (43) 90.7 9.3 72.1 27.9 72.1 27.9 Often (14) 100 - p = 0.138 78.6 21.4 p = 0.334 100 - p = 0.022 Always (18) 77.8 22.2 55.6 44.4 61.1 38.9 STD since last visit % Yes (6) 100 - OR = NC; 100 - OR = NC; 83.3 16.7 OR = 1.7 (0.2–15.7) p = 1.000 p = 0.175 No (74) 89.2 10.8 68.9 31.1 74.3 25.7 1 2 NC, non-computable; Contraception used until past; Contraception in current use; *For linear trend; HPV-NCIN, HPV infection without CIN.... ^New partner since last visit Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 smokers (OR = 0.26; 95%CI 0.095–0.764), whereas the HPV negative women had high-avidity antibodies to E6, difference in reactivity to E and E+L antigens is not signif- as compared with 29.4% of HR-HPV positive women (p = icant. No differences in seroreactivity could be attributed 0.059). Interestingly, women with normal colposcopy to the other smoking variables (age at onset, number of had the highest AI for E7, followed by those with high- cigarettes/day, smoking years) (data not shown), or to any grade colposcopy (77.1%) and low-grade abnormality of the indicators of obstetric history or sexual behaviour, (54.1%) (p = 0.011). AI was not related to HIV/smoking including contraception used in the past. Women's sero- status, being practically identical between the two groups reactivity was not influenced by their being sexually for both L and E antigens. active, their having a new partner since the last visit in the clinic, or their having a HIV-positive sex partner. Interest- Correlation of HIV status to serological response ingly, the actual mode of contraception itself was not sig- When the HIV status was taken into account (Table 2), nificant, but the different practice of condom use was HIV-positive women showed less seroreactivity to E+L or significantly (p = 0.022) related to seroreactivity to L anti- E and L separately than the HIV-negative ones. Interest- gens, being the least frequent among women using always ingly only the difference of seroreactivity to L antigens condom in sexual intercourse. reached statistical significance (Fisher's exact test; p = 0.030). Correlation of clinical data to serological response The key clinical data were then related to HPV seroreactiv- HIV-positive and current smoker women, showed the ity; the results are shown in Table 2. Seroreactivity to E, L lowest L seroprevalence (33.3%), as compared with or E & L antigens seems to be not associated with HPV 75.9% among all other women (p = 0.014; Fisher's exact DNA positivity, either for all HPVs or for LR-HPV and HR- test), with OR = 0.158 (95%CI 0.036–0.695). This associ- HPV genotypes separately. Other baseline clinical data, as ation was not confounded by age, when tested either the reported past HPV infection, abnormality in the refer- using Mantel-Haenszel test for age groups (common OR = ral Pap smear and grade of CIN, were analysed regarding 0.178; 95%CI 0.038–0.832) (p = 0.028) or logistic regres- the sero-status. Only a reported past HPV infection was sion analysis for age as continuous variable (adjusted OR associated with significantly (p = 0.046) higher probabil- = 0.167; 95%CI 0.037–0.736) (p = 0.020). On the other ity of having antibodies to E antigens (OR = 4.8; 95%CI way round, women who are HIV-negative and not smok- 1.1–22.8). No statistical correlation was found between ers are significantly more likely to mount antibody the sero-status and the other considered clinical data, as response (OR = 6.31; 95%CI 1.43–27.71). the presence of external genital warts (Condylomata acu- minata) or their number (data not shown). Mild abnor- Discussion mality on colposcopy was found associated with In a previous study we have reported the baseline data of significantly increased seropositivity to L antigens, as a cohort comprising HIV-positive and HIV-negative compared with normal or high-grade colposcopy (p = women [39]. The present report provides data on HPV 0.015), no such difference was found in reactivity to E or exposure status of a group of these women that gave their to E+ L antigens. blood. The analysis regards the serological response to recombinant HPV16 L1, L2, E4, E6 and E7 proteins eval- The key epidemiological and clinical data were also uated by a new ELISA technique [34]. related to the AI values of L and E antigens determined in a previous study [34]. This is a useful parameter used in a We have correlated the women's epidemiological charac- large number of viral diseases to discriminate between teristics to their serological response to HPV16 antigens, low and high antibody avidity; 40% is considered the dis- using three possible outcomes; i) seroreactivity to either E crimination AI value [40-42]. In our system AI has been or L (= HPV seropositive or not), ii) reactivity to E, and iii) considered as an indirect measure of the specific interac- reactivity to L proteins. Only a few of the recorded epide- tion between antigen and cross-reacting antibodies. Most miological variables proved to be significantly related to reactions with L1, L2, E4 and E7 antigens showed an AI serostatus of the women in this cohort. Interestingly cur- higher than 40%, but not those reacting with E6 antigen. rent smokers showed significantly less seroreactivity to L Few significant associations were observed between high antigens as compared with the non-smokers (OR = 3.7; AI and the different epidemiological and clinical varia- 95%CI 1.3–10.5) (p = 0.009), while the difference in reac- bles. High AI of E7 antibodies was associated with 100% tivity to E and E+L antigens was not significant. This is in of women who did not use contraception in the past, as full agreement with a recent report, where almost identi- compared with 80% among women who used regular cal observations were reported [28]. In this study, smokers contraception (p = 0.044). Similarly, regular use of con- were twice as likely as non-smokers to test negative for dom was marginally (p = 0.076) associated with lower anti-HPV L1 antibodies, even when the effects of other probability of high-avidity antibodies to L1. None of HR- covariates were considered in the analysis (adjusted OR = Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 Table 2: Key clinical characteristics of the patients related to their serological response to HPV16 antigens Variables Anti-E and -L Anti-E -E and E Anti L and -L 4 6 7 1 2 + - Sign. + - Sign. + - Sign. HPV DNA (%): Positive 76 89.5 10.5 OR = 2.8 (0.8–9.9) 71.1 28.9 OR = 1.3 (0.4–3.7) 71.1 28.9 OR = 1.6 (0.6–4.6) p = 0.136 p = 0.604 p = 0.350 Negative 20 75.0 25.0 65.0 35.0 60.0 40.0 HR-HPV (%) Positive 66 87.9 12.1 OR = 1.4 (0.4–4.8) 68.2 31.8 OR = 0.8 (0.3–2.0) 69.7 30.3 OR = 1.1 (0.5–2.9) p = 0.536 p = 0.608 p = 0.767 Negative 30 83.3 16.7 73.3 26.7 66.7 33.3 LR-HPV (%): Positive 10 100 0.0 OR = NC 90.0 10.0 OR = 4.3 (0.5–30.0) 80.0 20.0 OR = 1.9 (0.4–9.7): p = 0.348 p = 0.273 p = 0.720 Negative 86 84.9 15.1 67.4 32.6 67.4 32.6 Previous: HPV (%): Yes 19 94.7 5.3 OR = 2.8 (0.3–23.9); 89.5 10.5 OR = 4.8 (1.1–22.8) 73.7 26.3 OR = 0.9 (0.3–3.1); p = 0.445 p = 0.046 p = 0.961 No 66 86.4 13.6 63.6 36.4 74.2 25.8 HIV-Status (%): Negative 84 89.3 10.7 OR = 4.2(1.1–16.5) 71.4 28.6 OR = 1.8 (0.5–6.2) 72.6 27.4 OR = 3.7 (1.1–12.9) p = 0.032 p = 0.355 p = 0.030 Positive 12 66.7 33.3 58.3 41.7 41.7 58.3 Smokers 9 66.6 33.3 55.6 44.4 33.3 66.7 Previous: PAP (%): Never 10 90.0 10.0 p = 0.793 70.0 30.0 p = 0.111 80.0 20.0 p = 0.445 Irregular 26 92.3 7.7 84.6 15.4 61.5 38.5 Regular (1–3 yrs) 49 85.7 14.3 61.2 38.8 75.5 24.5 Referral PAP (%): ASC-US 7 100 0.0 p = 0.431 85.7 14.3 p = 0.623 100 0.0 p = 0.094 LSIL 48 91.7 8.3 64.6 35.4 75.0 25.0 p = 0.031* HSIL 16 81.3 18.7 68.8 31.2 56.3 43.7 Biopsy Result (%): Normal 1 100 0.0 0.0 100 100 0.0 HPV-NCIN 9 100 0.0 p = 0.262 66.7 33.3 p = 0.526 77.8 22.2 p = 0.564 CIN1 11 90.9 9.1 54.5 45.5 63.6 36.4 CIN2 14 92.9 7.1 71.4 28.6 85.7 14.3 CIN3 15 93.3 6.7 73.3 16.7 73.3 26.7 SCC 2 50.0 50.0 50.0 50.0 50.0 50.0 Genital Warts (%): Yes 20 95.0 5.0 OR = 3.3(0.4–27.7) 80.0 20.0 OR = 2.1 (0.6–7.0) 75.0 25.0 OR = 1.1 (0.4–3.7) p = 0.437 p = 0.275 p = 0.801 No 61 85.2 14.8 65.6 34.4 72.1 27.9 Colposcopy (%): Normal 2 100 0.0 100 0.0 0.0 100 Low-grade 27 96.3 3.74 p = 0.177 63.0 37.0 p = 0.668 85.2 14.8 p = 0.015 High-grade 28 77.8 22.2 72.2 27.8 61.1 28.9 1 2 NC, non-computable; Contraception used until past; Contraception in current use; *For linear trend; HPV-NCIN, HPV infection without CIN.... 0.5; 95%CI 0.2–0.9). The relationship between tobacco antibody response to L antigens could be an indication of smoking and CC is highly complex; however, the basic the deregulation of immune responses induced by smok- mechanisms must involve the host's inability to clear HR- ing, increasing the likelihood of persistent infection and HPV infection. The observed failure of smokers to mount the long-term risk for CC. This is in agreement with other Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 results showing current smoking to be associated with level of immunosuppression. With regard to the above persistent HR-HPV infection [43,44]. discussed failure of smokers to mount viral antibody response, we analysed the combined effect of current The relationship of contraception and HPV infection has smoking and HIV-status in this cohort. Based on the been incompletely studied and only in relation with HPV obtained results and our previously reported data [50,44], DNA detection [27,45,46], but not in relation to antibody it is tempting to speculate that HIV-positive women who detection. In the present analysis only the condom use, are current smokers comprise a special high-risk group, among the recorded epidemiological variables, was with extremely high probability of developing CIN3/CC, related to seroreactivity to L antigens; the lowest (61.1%) due to their failure to eradicate persistent HR-HPV infec- seropositivity (Table 1) was observed in those women tion. This failure could be attributed to their inability to who reported to always use condom in sexual intercourse. mount effective immunological responses against HPV antigens, of which deregulation the low seroreactivity is The relationship between HPV DNA detection and anti- an indication. body response to different HPV antigens is highly com- plex. Large sero-epidemiological studies seem indicate Two other observations merit some discussion. We that antibody response to viral antigens does not invaria- noticed that seroresponse to L proteins declined in paral- bly occur during natural HPV infection [14,15]. Similarly, lel with the increasing severity of abnormality in the refer- only approximately half of HPV DNA-positive women ral PAP smear, from 100% among ASCUS cases to 56.3% with normal cytology show antibody response to VLPs among those with HSIL (Table 2)(p = 0.031 for linear [18,19]. Moreover E6/E7 antibodies are detected in trend). This could substantiate the observations of Wiley patients with CC as well as in healthy controls. [14,15,47]. and collabotators [28] who reported that women with These observations are confirmed in the present cohort, ASCUS were twice more likely to test anti-HPV L1-positive where detection of HPV DNA, both LR+HR genotypes and as compared with those with normal PAP. In our cohort, LR- and HR-HPV separately, did not bear any significant we had no PAP-negative women to confirm this. relationship with seroreactivity to any viral antigens. However women who reported past HPV infection show As recently discussed [34], most of the HPV antibodies a significantly higher prevalence (89.5%) of E antibodies bound to these 5 antigens with an AI higher than 40%, as compared with that (63.6%) of women who did not even in the sera from women infected with HPV type dif- reported past infection (OR = 4.8; 95%CI 1.1–22.8) ferent from 16 type. When related to epidemiological data (Table 2). These results suggest that seropositivity to E and clinical observations, AI data yielded few significant antigens could be considered as a surrogate for past viral observations. No feasible explanation can be offered for infection [48]. the association of high AI of anti-E7 antibodies in 100% of women which did not use contraception in the past, as Into the HPV-PathogenISS cohort, HIV-positive women compared with the 80% of those using contraception (p = were also enrolled [39]. As generally accepted, these 0.044). Similarly, the significant difference (p = 0.011) women are at increased risk for progression to CIN3 and obtained in AI between the different colposcopy catego- CC [49], mainly because of their failure to eradicate per- ries does not show any linear trend with the grade of sistent HR-HPV infection, even if treated by HAART [50]. abnormality. HPV serology in HIV-positive women has been studied only recently. In these women we have observed a signifi- Conclusion cantly lower seroprevalance compared to the HIV-nega- In summary the results show that there is no meaningful tive ones; these data are in contrast with those reported in relationship between HPV DNA detection and seroreactiv- large cohort studies, where HIV-positive women usually ity to tested HPV antigens, which was not unexpected in have higher prevalence of HPV antibodies [51,25,29]. the light of published data [14,15,19]. However, two sig- Some reasons of this discrepancy can be the small number nificant observations are of particular interest. Current of HIV-positive women in our cohort leaving room for smokers show significantly (p = 0.009) less seroreactivity observations by chance and the fact that all patients had to HPV L antigens as compared with the non-smokers. been on HAART for several years and their HPV baseline The difficulty of smokers to mount antibody response to status was very similar to that of HIV-negative women. On L antigens could be an indication of a marked deregula- the other hand, this observation could indicate their lower tion of the immune system responses thus increasing the HPV exposure in the past. A feasible explanation could be long-term risk for CIN3/CC. Another interesting observa- the HIV-induced immunosuppression leading to per- tion is the significantly impaired seroreactivity to HPV L turbed early immune responses against HPV [50]; unfor- antigens among HIV positive women, further accentuated tunately, the CD4 data of these women are incomplete among women HIV positive and current smokers. It is and it is not possible to draw firm conclusions on their tempting to speculate that HIV positive women who are Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 current smokers comprise a special high-risk group, with specific hyper-immune sera obtained by injection in extremely high probability of developing CIN3/CC, due either mice or rabbits of the recombinant purified HPV16 to their failure to eradicate persistent HR-HPV infection. L1, L2, E4, E6 and E7 proteins were used [34]. ELISA and avidity test Methods Patients The presence in the sera of anti-HPV antibodies was eval- In the HPV-PathogenISS study a cohort of 286 women uated by an ELISA test based on the recombinant L1, L2, were enrolled during 2002–2003 by Gynaecologic outpa- E4, E6 and E7 HPV16 proteins [34]. A cut-off value for tient departments at 5 hospitals (S. Orsola Malpighi, each antigen was calculated as the arithmetic mean of the Bologna; Istituto Regina Elena, Roma; Università di Tor absorbance values of 40 control sera, plus two standard Vergata, Roma; Luigi Sacco, Milano; and Istituto San Gal- deviations (SD). After a first calculation the outlier values licano, Roma). Women were selected for having abnor- were excluded and the cut-off recalculated [52]. The repro- mal PAP test (ASC-US, LSIL or HSIL) [39]. The sera ducibility of ELISA results was assayed by testing each examined in this study are from a group of 96 women of serum in duplicate and repeating each test three times. this cohort that gave the informed consent for serum tak- ing for research use. All women were subjected to colpos- Avidity of the specific anti-HPV IgGs was evaluated and copy, PAP test (if not taken within the past 3 months), the AI, expressed as a percentage of the absorbance in biopsy (when indicated), as well as sampling for HPV ELISA with urea wash and the absorbance in ELISA with- DNA testing. This study protocol was separately approved out urea wash × 100 [35]. Because of the lack of data by the institutional Ethical Committees of all 5 hospitals. regarding the avidity antibodies in HPV infection, it was Epidemiological interview, Pap test, colposcopy, biopsy, difficult to establish an AI value discriminating between treatment by LLETZ are been previously described [39]. low and high avidity. We tentatively assumed the 40% value, a discriminating value reported for some infectious Sampling for HPV testing and HPV genotype identification diseases [40-42]. A careful sampling protocol for HPV testing was followed in all clinics. Both exo- and endocervical specimens were Statistical analyses Statistical analyses were performed using the SPSS soft- collected and stored at -70°C, until transported to the two virology laboratories (Tor Vergata and ISS) for HPV anal- ware package (SPSS for Windows, version 14.01). Fre- ysis. HPV genotyping was performed by sequence analy- quency tables were analysed using the Chi-square test, sis, as previously described [34]. When the genotype was with likelihood ratio (LR) or Fisher's exact test being used not resolved by sequencing the samples were examined to assess the correlation between categorical variables. OR for the presence of either HR- or LR- HPV by HC2 assay and 95% confidence intervals (95%CI) were calculated (Digene). Because the genotype determination was not where appropriate, using the exact method. Differences in possible for all samples the analysis reported in this paper the means of continuous variables were analysed using have been done by considering only the HR- and LR-HPV non-parametric tests (Mann-Whitney or Kruskal-Wallis) categories. or ANOVA (to obtain the mean values). Logistic regres- sion analysis was used to control for the confounding HIV testing from age to some observed significant univariate predic- The patient HIV-serostatus was determined at the entry tors of seroreactivity, e.g. HIV/smoking status. Confound- with ELISA test and confirmed by Western blot, with the ing was also controlled by calculating the common odds informed consent of the patients. The state of HIV disease ratio (OR) for the stratum-specific estimates, using the in sero-positive patients was assessed by CD4+ and CD8+ Mantel-Haenszel test. To evaluate the statistical power of lymphocytes counting and recording the opportunistic the study (e.g. for age differences), we used STATA/SE 9.2. infections or neoplasia. Unfortunately CD4 parameter power analysis option, with the default value (0.05) for was not available for all HIV-positive patients. All these alpha, and using the true sample sizes of the women in the patients were being treated with highly active anti-retrovi- two groups, e.g. seropositive and -negative women. In all ral treatment (HAART). HIV-positive patients in this study tests, the values p < 0.05 were regarded statistically signif- were only 12, of whom 9 current smokers. icant. HPV serology Authors' contributions Human sera and control sera CG conceived the study, participated in his design and The collection and treatment of sera of the 96 women coordination and in rafting the manuscript, PDB, FG, SM, have been previously described [34]. Control sera were MGD participated in acquisition analysis and interpreta- randomly taken among sera collected for studies unre- tion of serology data, LA participated in interpretation of lated with HPV infection. As positive controls, antigen data and critically revised the manuscript, MB, SC, LM, Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 11. Jochmus-Kudielka I, Schneider A, Braun R, Kimmig R, Koldovsky U, AA, MC participated in acquisition of serum samples and Schneweis KE, Seedorf K, Gissmann L: Antibodies against the epidemiological data, KS participated in design of the human papillomavirus type 16 early proteins in human sera: study, performed the statistical analysis and participated correlation of anti-E7 reactivity with cervical cancer. J Natl Cancer Inst 1989, 81:1698-1704. in drafting the manuscript. 12. Muller M, Viscidi RP, Sun Y, Guerrero E, Hill PM, Shah F, Bosch FX, Munoz N, Gissmann L, Shah KV: Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16- associated invasive cervical Acknowledgements cancer. Virology 1992, 187:508-514. This work was supported by grants from the Italian Ministry of Health (Min- 13. Dillner J, Lenner P, Lehtinen M, Eklund C, Heino P, Wiklund F, Hall- istero della Salute, Ricerca Corrente 1% 2002; Fasc.OG/C; Ministero della mans G, Stendahl U: A population-based seroepidemiological Salute, Ricerca Finalizzata 2003–2006, Proj. K07; the Italian AIDS Project study of cervical cancer. Cancer Res 1994, 54:134-141. 2005 Fasc 50F/F) and Ministero della Salute, Ricerca Finalizzata 2005–2008 14. Dillner J: The serological response to papillomaviruses. Semin Cancer Biol 1999, 9:423-430. Proj. N1D 15. Dillner J: Toward "serolomics": papillomavirus serology is taking a technologic lead in high-throughput multiplexed We are grateful to Mrs. Sabrina Tocchio for the editorial assistance in the antibody analysis. Clin Chem 2005, 51:1768-1769. preparation of the manuscript and to Mr. Armando Cesolini for animal han- 16. De Sanjose S, Hamsikova E, Munoz N, Bosch FX, Hofmannova V, Gili M, Izarzugaza I, Viladiu P, Tormo MJ, Moreo P, Munoz MT, Ascunce dling. N, Tafur L, Shah KV, Vonka V: Serological response to HPV16 in CIN-III and cervical- cancer patients. Case-control studies in HPV-PathogenISS Study Group: L Leoncini and M Alderisio, Unità Citois- Spain and Colombia. Int J Cancer 1996, 66:70-74. topatologia, Centro Nazionale di Epidemiologia, Sorveglianza e Promozi- 17. Viscidi RP, Schiffman M, Hildesheim A, Herrero R, Castle PE, Bratti one della Salute, Istituto Superiore di Sanità, Roma, Italy; S Costa, D Santini, MC, Rodriguez AC, Sherman ME, Wang S, Clayman B, Burk RD: Seroreactivity to human papillomavirus (HPV) types 16, 18, M De Nuzzo, Dipartimento di Ginecologia e Ostetricia, Ospedale S. Orsola or 31 and risk of subsequent HPV infection: results from a Malpighi, Bologna, Italy; L Di Bonito, D Bonifacio and F Zanconati, U.C.O. population-based study in Costa Rica. Cancer Epidemiol Biomar- Anatomia Patologica, Istopatologia e Citodiagnostica, Ospedale Maggiore, kers Prev 2004, 13:324-7. Trieste, Italy; M Galati, Dipartimento di Ginecologia e Ostetricia, IFO, Isti- 18. 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Branca M, Costa S, Mariani L, Sesti F, Agarossi A, di Carlo A, Galati M, Benedetto A, Ciotti M, Giorgi C, Criscuolo A, Valieri M, Favalli C, Paba P, Santini D, Piccione E, Alderisio M, De Nuzzo M, Di Bonito L, Syrjanen K: Assessment of risk factors and human papilloma- virus (HPV) related pathogenetic mechanisms of CIN in HIV-positive and HIV-negative women. Study design and baseline data of the HPV-PathogenISS study. Eur J Gynaecol Oncol. 2004, 25:689-698. 40. Kanno A, Kazuyama Y: Immunoglobulin G antibody avidity for serodiagnosis of hepatitis C virus infection. J Med Virol 2002, 68:229-233. 41. Eggers M, Bäder U, Enders G: Combination of microneutraliza- tion and avidity assays: improved diagnosis of recent primary human cytomegalovirus infection in single serum sample of Publish with Bio Med Central and every second trimester pregnancy. J Met Virol 2000, 60:324-330. scientist can read your work free of charge 42. 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Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as measured by a novel ELISA based on denatured recombinant HPV16 late (L) and early (E) antigens

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Copyright © 2008 by Giorgi et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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Abstract

Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV. Objectives: The objective was to assess the clinical and epidemiological correlates of HPV- seroreactivity in a cohort of HIV-negative and HIV-positive women. Methods: Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study. Results: No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women (OR = 0.158; 95%CI 0.036–0.695, p = 0.014; Fisher's exact test). Importantly, this association did not loose its significance when controlled for Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 confounding from age (continuous variable) in multivariate analysis or using Mantel-Haenszel test for age-groups. Conclusion: It is tempting to speculate that HIV-positive current smokers comprise a special high- risk group, with highly impaired immunological response that could prevent eradication of persistent HPV infections and thus contribute to development of CIN3/CC. evaluation of the new HPV vaccines the WHO recom- Background Human Papillomavirus (HPV) infection, known to be mends the standardisation of the different in-house L1- associated with both benign and malignant tumours at based serological methods by the use of reference sera and different mucosal sites, is widespread among female and promoted an international collaborative study with this male populations [1,2]. The oncogenic high-risk HPV proposal [21]. types (HR-HPVs) are the single most important etiological agents of cervical cancer (CC) and its precursor (CIN) To improve the understanding of correlation between lesions [3,4]. Despite global differences in the prevalence infection and antibody response in patients with and of individual HR-HPV types, HPV16 remains the most fre- without cervical lesions, prospective cohort studies using quent HR-HPV type in all geographic regions and the cur- a widespread panel of viral antigens would be needed rent HPV research is mostly focused on it [3,4]. Diagnosis [14,15,26]. Similarly, it would be necessary to study the of HPV infection is based on detection of viral DNA using dynamics of HPV-E and -L antigen-specific antibodies in different PCR-based methods or commercially available relation to their clinical and epidemiological correlates hybrid capture (HC2) test [5,6]. Until now, serological [27,28], including immune-suppression due to HIV infec- studies have had little to offer in diagnosis of HPV infec- tion [25,29]. There are recent growing evidences regarding tions [7]. The first serological assays for HPV antibody the induction of antibodies neutralising across papilloma- detection were introduced in the early 1970's [8], but the virus types after vaccination with VLP displaying L2 more widespread use of HPV serology had to await the epitopes or with fusion protein containing L2E7E6 viral development of more refined assays based on virus-like proteins or synthetic peptides of the HPV16 L2 protein particles (VLP) in the early 1990's [9,10]. A growing body [30-32]. Very recently Rizk and collaborators [33] have of serology data has being obtained using different viral shown that L1 fused to GST protein displays a broad vari- protein-based ELISA to analyse HPV antibodies in differ- ety of epitopes, both conformational and linear; the neu- ent settings, most notably in large-scale seroepidemilogi- tralising ones are conformational and, mostly, type cal- and case-control studies to assess the risk factors of specific while the cross-reacting ones are associated with cervical carcinoma (CC) in HPV exposed subjects [11-17]. linear epitopes. The main conclusion from the plethora of sero-epidemio- logical studies is that antibody response to viral proteins We have recently reported the development of an in- does not invariably occur during a natural HPV infection house ELISA test based on five recombinant HPV16 pro- [14,15]; only half of HPV DNA-positive women with nor- teins expressed in E. coli: L1 and L2 capsid proteins, E6 mal cytology shows antibody response to VLPs [18,19]. and E7 oncoproteins and the non-structural E4 protein, HPV E6/E7 antibodies are predominantly found in all used in unfolded form [34]. The idea of using dena- patients with CC, but they are also detected in healthy tured antigens comes from the observation of a high controls, precluding the use of their detection as predictor amino acid identity, ranging from 36% for E6 to 74% for of CC [14,15,20]. L1, among the same proteins belonging to different HPV genotypes and then the possibility to detect cross-reacting VLP-based assays have been recognized as reliable geno- antibodies directed against linear epitopes. By this assay type-specific assays [14,15,21] and potentially will con- we were able to detect an antibody response in women tribute to the understanding of the natural history of HPV infected with HPV genotypes different from that of the ref- infections [18,19,22]. New L1VLP-based vaccines have erence antigen. In this study we also determined the Avid- been shown to induce levels of HPV type specific antibod- ity Index (AI) [35] of the positive samples. Antibody ies higher than that found in natural infected women response to linear epitopes has been reported in the past [23]. Correlation between anti-L1VLPs antibodies and [36,37] and was correlated to the specific stage of HPV- protection from reinfection in natural infection is still a associated cervical disease [38]; nevertheless this antibody controversial issue [24,25]. An important problem with response can not be related to protection level against serological studies resides in the fact that the results from HPV infection, that is associated to the neutralising com- different laboratories are not comparable for the lack of ponent of humoral response. Our system, revealing anti- reference specific sera [26]. For the serological response bodies only against linear epitopes, could be utilized to Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 evaluate the immunological responsiveness of subjects to Results HPV infection and contribute to estimate the risk of infec- Correlation of epidemiological characteristics to serological response tion progression but it could not provide information about protection level from reinfection in both natural The recorded epidemiological characteristics of the infection and after vaccination. enrolled women have been correlated to their serological response to either all HPV16 antigens, or separately E and In the present study we have correlated the antibody L proteins (Table 1). The statistical analysis shows that response, evaluated with our system, to the baseline epi- antibody response is not related to patient's age or their demiological, clinical and HPV DNA data of women ethnic origin (data not shown); however, the power of the enrolled in a multi-centre cohort study designed to study to detect true differences in the age between the two explore pathogenetic mechanisms and prognostic factors groups is limited, due to the small sample size. Interest- of CIN lesions in HIV-positive and HIV-negative women, ingly, current smokers have significantly (p = 0.009) less known as HPV-PathogenISS study [39]. seroreactivity to L antigens as compared with the non- Table 1: Key epidemiological data of patients related to their serological response to HPV16 antigens Variables Anti-E and -L Anti-E -E and E Anti L and -L 4 6 7 1 2 + - Sign. + - Sign. + - Sign. Mean Age (yrs) 34.9 36.9 p = 0.460 34.8 36.0 p = 0.533 36.1 35.6 p = 0.805 Smoking status % Non-smoker (40) 92.5 7.5 OR = 2.8 (0.7–11.3); 72.5 17.5 OR = 1.4 (0.6–3.6); 85.0 15.0 OR = 3.7 (1.3–10.5); p = 0.211 p = 0.495 p = 0.009 Current smoker (48) 81.3 18.7 64.6 35.4 60.4 39.6 N. cigarette/d 13.4 10.1 p = 0.285 13.8 10.9 p = 0.225 13.3 12.0 p = 0.603 Age start smoking (yrs) 18.9 19.6 p = 0.776 18.2 20.6 p = 0.122 19.5 18.1 p = 0.379 Years smoked 15.2 13.3 p = 0.592 16.5 12.0 p = 0.089 14.0 16.8 p = 0.296 Contraception (%): Yes (53) 92.5 7.5 OR = 1.4 (0.3–6.8); 73.6 26.4 OR = 1.3 (0.5–3.4): 73.6 26.4 OR = 0.7 (0.2–2.1): p = 0.694 p = 0.658 p = 0.560 No (29) 89.7 10.3 69.0 31.0 79.3 20.7 ^New partner (%): Yes (5) 100 - OR = NC; p = 0.508 60.0 40.0 OR = 0.7 (0.2–2.2); 60.0 40.0 OR = 0.7 (0.2–2.1); p = 0.630 p = 0.619 No (76) 86.8 13.2 71.1 28.9 72.4 27.6 Sexually active (%): Yes (79) 89.9 10.1 OR = 5.9 (0.9–40.9); 70.9 29.1 OR = 1.6 (0.3–10.4); 74.7 25.3 OR = 4.4 (0.7–28.4); p = 0.105 p = 0.631 p = 0.124 No (5) 60.0 40.0 60.0 40.0 40.0 60.0 Partner HIV+ (%): Yes (8) 87.5 12.5 OR = 0.9 (0.1–8.7); 87.5 12.5 OR = 3.3 (0.4–28.3); 62.5 37.5 OR = 0.6 (0.1–2.8); p = 1.000 p = 0.679 No (75) 88.0 12.0 68.0 32.0 73.3 26.7 Actual contraception % Yes (37) 86.5 13.5 OR = 0.7 (0.2–2.7); 67.6 32.4 OR = 0.8 (0.3–2.1); 64.9 35.1 OR = 0.4 (0.1–1.1); p = 0.726 p = 0.660 p = 0.093 No (43) 90.7 9.3 72.1 27.9 81.4 18.6 Condom Use % Never (43) 90.7 9.3 72.1 27.9 72.1 27.9 Often (14) 100 - p = 0.138 78.6 21.4 p = 0.334 100 - p = 0.022 Always (18) 77.8 22.2 55.6 44.4 61.1 38.9 STD since last visit % Yes (6) 100 - OR = NC; 100 - OR = NC; 83.3 16.7 OR = 1.7 (0.2–15.7) p = 1.000 p = 0.175 No (74) 89.2 10.8 68.9 31.1 74.3 25.7 1 2 NC, non-computable; Contraception used until past; Contraception in current use; *For linear trend; HPV-NCIN, HPV infection without CIN.... ^New partner since last visit Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 smokers (OR = 0.26; 95%CI 0.095–0.764), whereas the HPV negative women had high-avidity antibodies to E6, difference in reactivity to E and E+L antigens is not signif- as compared with 29.4% of HR-HPV positive women (p = icant. No differences in seroreactivity could be attributed 0.059). Interestingly, women with normal colposcopy to the other smoking variables (age at onset, number of had the highest AI for E7, followed by those with high- cigarettes/day, smoking years) (data not shown), or to any grade colposcopy (77.1%) and low-grade abnormality of the indicators of obstetric history or sexual behaviour, (54.1%) (p = 0.011). AI was not related to HIV/smoking including contraception used in the past. Women's sero- status, being practically identical between the two groups reactivity was not influenced by their being sexually for both L and E antigens. active, their having a new partner since the last visit in the clinic, or their having a HIV-positive sex partner. Interest- Correlation of HIV status to serological response ingly, the actual mode of contraception itself was not sig- When the HIV status was taken into account (Table 2), nificant, but the different practice of condom use was HIV-positive women showed less seroreactivity to E+L or significantly (p = 0.022) related to seroreactivity to L anti- E and L separately than the HIV-negative ones. Interest- gens, being the least frequent among women using always ingly only the difference of seroreactivity to L antigens condom in sexual intercourse. reached statistical significance (Fisher's exact test; p = 0.030). Correlation of clinical data to serological response The key clinical data were then related to HPV seroreactiv- HIV-positive and current smoker women, showed the ity; the results are shown in Table 2. Seroreactivity to E, L lowest L seroprevalence (33.3%), as compared with or E & L antigens seems to be not associated with HPV 75.9% among all other women (p = 0.014; Fisher's exact DNA positivity, either for all HPVs or for LR-HPV and HR- test), with OR = 0.158 (95%CI 0.036–0.695). This associ- HPV genotypes separately. Other baseline clinical data, as ation was not confounded by age, when tested either the reported past HPV infection, abnormality in the refer- using Mantel-Haenszel test for age groups (common OR = ral Pap smear and grade of CIN, were analysed regarding 0.178; 95%CI 0.038–0.832) (p = 0.028) or logistic regres- the sero-status. Only a reported past HPV infection was sion analysis for age as continuous variable (adjusted OR associated with significantly (p = 0.046) higher probabil- = 0.167; 95%CI 0.037–0.736) (p = 0.020). On the other ity of having antibodies to E antigens (OR = 4.8; 95%CI way round, women who are HIV-negative and not smok- 1.1–22.8). No statistical correlation was found between ers are significantly more likely to mount antibody the sero-status and the other considered clinical data, as response (OR = 6.31; 95%CI 1.43–27.71). the presence of external genital warts (Condylomata acu- minata) or their number (data not shown). Mild abnor- Discussion mality on colposcopy was found associated with In a previous study we have reported the baseline data of significantly increased seropositivity to L antigens, as a cohort comprising HIV-positive and HIV-negative compared with normal or high-grade colposcopy (p = women [39]. The present report provides data on HPV 0.015), no such difference was found in reactivity to E or exposure status of a group of these women that gave their to E+ L antigens. blood. The analysis regards the serological response to recombinant HPV16 L1, L2, E4, E6 and E7 proteins eval- The key epidemiological and clinical data were also uated by a new ELISA technique [34]. related to the AI values of L and E antigens determined in a previous study [34]. This is a useful parameter used in a We have correlated the women's epidemiological charac- large number of viral diseases to discriminate between teristics to their serological response to HPV16 antigens, low and high antibody avidity; 40% is considered the dis- using three possible outcomes; i) seroreactivity to either E crimination AI value [40-42]. In our system AI has been or L (= HPV seropositive or not), ii) reactivity to E, and iii) considered as an indirect measure of the specific interac- reactivity to L proteins. Only a few of the recorded epide- tion between antigen and cross-reacting antibodies. Most miological variables proved to be significantly related to reactions with L1, L2, E4 and E7 antigens showed an AI serostatus of the women in this cohort. Interestingly cur- higher than 40%, but not those reacting with E6 antigen. rent smokers showed significantly less seroreactivity to L Few significant associations were observed between high antigens as compared with the non-smokers (OR = 3.7; AI and the different epidemiological and clinical varia- 95%CI 1.3–10.5) (p = 0.009), while the difference in reac- bles. High AI of E7 antibodies was associated with 100% tivity to E and E+L antigens was not significant. This is in of women who did not use contraception in the past, as full agreement with a recent report, where almost identi- compared with 80% among women who used regular cal observations were reported [28]. In this study, smokers contraception (p = 0.044). Similarly, regular use of con- were twice as likely as non-smokers to test negative for dom was marginally (p = 0.076) associated with lower anti-HPV L1 antibodies, even when the effects of other probability of high-avidity antibodies to L1. None of HR- covariates were considered in the analysis (adjusted OR = Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 Table 2: Key clinical characteristics of the patients related to their serological response to HPV16 antigens Variables Anti-E and -L Anti-E -E and E Anti L and -L 4 6 7 1 2 + - Sign. + - Sign. + - Sign. HPV DNA (%): Positive 76 89.5 10.5 OR = 2.8 (0.8–9.9) 71.1 28.9 OR = 1.3 (0.4–3.7) 71.1 28.9 OR = 1.6 (0.6–4.6) p = 0.136 p = 0.604 p = 0.350 Negative 20 75.0 25.0 65.0 35.0 60.0 40.0 HR-HPV (%) Positive 66 87.9 12.1 OR = 1.4 (0.4–4.8) 68.2 31.8 OR = 0.8 (0.3–2.0) 69.7 30.3 OR = 1.1 (0.5–2.9) p = 0.536 p = 0.608 p = 0.767 Negative 30 83.3 16.7 73.3 26.7 66.7 33.3 LR-HPV (%): Positive 10 100 0.0 OR = NC 90.0 10.0 OR = 4.3 (0.5–30.0) 80.0 20.0 OR = 1.9 (0.4–9.7): p = 0.348 p = 0.273 p = 0.720 Negative 86 84.9 15.1 67.4 32.6 67.4 32.6 Previous: HPV (%): Yes 19 94.7 5.3 OR = 2.8 (0.3–23.9); 89.5 10.5 OR = 4.8 (1.1–22.8) 73.7 26.3 OR = 0.9 (0.3–3.1); p = 0.445 p = 0.046 p = 0.961 No 66 86.4 13.6 63.6 36.4 74.2 25.8 HIV-Status (%): Negative 84 89.3 10.7 OR = 4.2(1.1–16.5) 71.4 28.6 OR = 1.8 (0.5–6.2) 72.6 27.4 OR = 3.7 (1.1–12.9) p = 0.032 p = 0.355 p = 0.030 Positive 12 66.7 33.3 58.3 41.7 41.7 58.3 Smokers 9 66.6 33.3 55.6 44.4 33.3 66.7 Previous: PAP (%): Never 10 90.0 10.0 p = 0.793 70.0 30.0 p = 0.111 80.0 20.0 p = 0.445 Irregular 26 92.3 7.7 84.6 15.4 61.5 38.5 Regular (1–3 yrs) 49 85.7 14.3 61.2 38.8 75.5 24.5 Referral PAP (%): ASC-US 7 100 0.0 p = 0.431 85.7 14.3 p = 0.623 100 0.0 p = 0.094 LSIL 48 91.7 8.3 64.6 35.4 75.0 25.0 p = 0.031* HSIL 16 81.3 18.7 68.8 31.2 56.3 43.7 Biopsy Result (%): Normal 1 100 0.0 0.0 100 100 0.0 HPV-NCIN 9 100 0.0 p = 0.262 66.7 33.3 p = 0.526 77.8 22.2 p = 0.564 CIN1 11 90.9 9.1 54.5 45.5 63.6 36.4 CIN2 14 92.9 7.1 71.4 28.6 85.7 14.3 CIN3 15 93.3 6.7 73.3 16.7 73.3 26.7 SCC 2 50.0 50.0 50.0 50.0 50.0 50.0 Genital Warts (%): Yes 20 95.0 5.0 OR = 3.3(0.4–27.7) 80.0 20.0 OR = 2.1 (0.6–7.0) 75.0 25.0 OR = 1.1 (0.4–3.7) p = 0.437 p = 0.275 p = 0.801 No 61 85.2 14.8 65.6 34.4 72.1 27.9 Colposcopy (%): Normal 2 100 0.0 100 0.0 0.0 100 Low-grade 27 96.3 3.74 p = 0.177 63.0 37.0 p = 0.668 85.2 14.8 p = 0.015 High-grade 28 77.8 22.2 72.2 27.8 61.1 28.9 1 2 NC, non-computable; Contraception used until past; Contraception in current use; *For linear trend; HPV-NCIN, HPV infection without CIN.... 0.5; 95%CI 0.2–0.9). The relationship between tobacco antibody response to L antigens could be an indication of smoking and CC is highly complex; however, the basic the deregulation of immune responses induced by smok- mechanisms must involve the host's inability to clear HR- ing, increasing the likelihood of persistent infection and HPV infection. The observed failure of smokers to mount the long-term risk for CC. This is in agreement with other Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 results showing current smoking to be associated with level of immunosuppression. With regard to the above persistent HR-HPV infection [43,44]. discussed failure of smokers to mount viral antibody response, we analysed the combined effect of current The relationship of contraception and HPV infection has smoking and HIV-status in this cohort. Based on the been incompletely studied and only in relation with HPV obtained results and our previously reported data [50,44], DNA detection [27,45,46], but not in relation to antibody it is tempting to speculate that HIV-positive women who detection. In the present analysis only the condom use, are current smokers comprise a special high-risk group, among the recorded epidemiological variables, was with extremely high probability of developing CIN3/CC, related to seroreactivity to L antigens; the lowest (61.1%) due to their failure to eradicate persistent HR-HPV infec- seropositivity (Table 1) was observed in those women tion. This failure could be attributed to their inability to who reported to always use condom in sexual intercourse. mount effective immunological responses against HPV antigens, of which deregulation the low seroreactivity is The relationship between HPV DNA detection and anti- an indication. body response to different HPV antigens is highly com- plex. Large sero-epidemiological studies seem indicate Two other observations merit some discussion. We that antibody response to viral antigens does not invaria- noticed that seroresponse to L proteins declined in paral- bly occur during natural HPV infection [14,15]. Similarly, lel with the increasing severity of abnormality in the refer- only approximately half of HPV DNA-positive women ral PAP smear, from 100% among ASCUS cases to 56.3% with normal cytology show antibody response to VLPs among those with HSIL (Table 2)(p = 0.031 for linear [18,19]. Moreover E6/E7 antibodies are detected in trend). This could substantiate the observations of Wiley patients with CC as well as in healthy controls. [14,15,47]. and collabotators [28] who reported that women with These observations are confirmed in the present cohort, ASCUS were twice more likely to test anti-HPV L1-positive where detection of HPV DNA, both LR+HR genotypes and as compared with those with normal PAP. In our cohort, LR- and HR-HPV separately, did not bear any significant we had no PAP-negative women to confirm this. relationship with seroreactivity to any viral antigens. However women who reported past HPV infection show As recently discussed [34], most of the HPV antibodies a significantly higher prevalence (89.5%) of E antibodies bound to these 5 antigens with an AI higher than 40%, as compared with that (63.6%) of women who did not even in the sera from women infected with HPV type dif- reported past infection (OR = 4.8; 95%CI 1.1–22.8) ferent from 16 type. When related to epidemiological data (Table 2). These results suggest that seropositivity to E and clinical observations, AI data yielded few significant antigens could be considered as a surrogate for past viral observations. No feasible explanation can be offered for infection [48]. the association of high AI of anti-E7 antibodies in 100% of women which did not use contraception in the past, as Into the HPV-PathogenISS cohort, HIV-positive women compared with the 80% of those using contraception (p = were also enrolled [39]. As generally accepted, these 0.044). Similarly, the significant difference (p = 0.011) women are at increased risk for progression to CIN3 and obtained in AI between the different colposcopy catego- CC [49], mainly because of their failure to eradicate per- ries does not show any linear trend with the grade of sistent HR-HPV infection, even if treated by HAART [50]. abnormality. HPV serology in HIV-positive women has been studied only recently. In these women we have observed a signifi- Conclusion cantly lower seroprevalance compared to the HIV-nega- In summary the results show that there is no meaningful tive ones; these data are in contrast with those reported in relationship between HPV DNA detection and seroreactiv- large cohort studies, where HIV-positive women usually ity to tested HPV antigens, which was not unexpected in have higher prevalence of HPV antibodies [51,25,29]. the light of published data [14,15,19]. However, two sig- Some reasons of this discrepancy can be the small number nificant observations are of particular interest. Current of HIV-positive women in our cohort leaving room for smokers show significantly (p = 0.009) less seroreactivity observations by chance and the fact that all patients had to HPV L antigens as compared with the non-smokers. been on HAART for several years and their HPV baseline The difficulty of smokers to mount antibody response to status was very similar to that of HIV-negative women. On L antigens could be an indication of a marked deregula- the other hand, this observation could indicate their lower tion of the immune system responses thus increasing the HPV exposure in the past. A feasible explanation could be long-term risk for CIN3/CC. Another interesting observa- the HIV-induced immunosuppression leading to per- tion is the significantly impaired seroreactivity to HPV L turbed early immune responses against HPV [50]; unfor- antigens among HIV positive women, further accentuated tunately, the CD4 data of these women are incomplete among women HIV positive and current smokers. It is and it is not possible to draw firm conclusions on their tempting to speculate that HIV positive women who are Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 current smokers comprise a special high-risk group, with specific hyper-immune sera obtained by injection in extremely high probability of developing CIN3/CC, due either mice or rabbits of the recombinant purified HPV16 to their failure to eradicate persistent HR-HPV infection. L1, L2, E4, E6 and E7 proteins were used [34]. ELISA and avidity test Methods Patients The presence in the sera of anti-HPV antibodies was eval- In the HPV-PathogenISS study a cohort of 286 women uated by an ELISA test based on the recombinant L1, L2, were enrolled during 2002–2003 by Gynaecologic outpa- E4, E6 and E7 HPV16 proteins [34]. A cut-off value for tient departments at 5 hospitals (S. Orsola Malpighi, each antigen was calculated as the arithmetic mean of the Bologna; Istituto Regina Elena, Roma; Università di Tor absorbance values of 40 control sera, plus two standard Vergata, Roma; Luigi Sacco, Milano; and Istituto San Gal- deviations (SD). After a first calculation the outlier values licano, Roma). Women were selected for having abnor- were excluded and the cut-off recalculated [52]. The repro- mal PAP test (ASC-US, LSIL or HSIL) [39]. The sera ducibility of ELISA results was assayed by testing each examined in this study are from a group of 96 women of serum in duplicate and repeating each test three times. this cohort that gave the informed consent for serum tak- ing for research use. All women were subjected to colpos- Avidity of the specific anti-HPV IgGs was evaluated and copy, PAP test (if not taken within the past 3 months), the AI, expressed as a percentage of the absorbance in biopsy (when indicated), as well as sampling for HPV ELISA with urea wash and the absorbance in ELISA with- DNA testing. This study protocol was separately approved out urea wash × 100 [35]. Because of the lack of data by the institutional Ethical Committees of all 5 hospitals. regarding the avidity antibodies in HPV infection, it was Epidemiological interview, Pap test, colposcopy, biopsy, difficult to establish an AI value discriminating between treatment by LLETZ are been previously described [39]. low and high avidity. We tentatively assumed the 40% value, a discriminating value reported for some infectious Sampling for HPV testing and HPV genotype identification diseases [40-42]. A careful sampling protocol for HPV testing was followed in all clinics. Both exo- and endocervical specimens were Statistical analyses Statistical analyses were performed using the SPSS soft- collected and stored at -70°C, until transported to the two virology laboratories (Tor Vergata and ISS) for HPV anal- ware package (SPSS for Windows, version 14.01). Fre- ysis. HPV genotyping was performed by sequence analy- quency tables were analysed using the Chi-square test, sis, as previously described [34]. When the genotype was with likelihood ratio (LR) or Fisher's exact test being used not resolved by sequencing the samples were examined to assess the correlation between categorical variables. OR for the presence of either HR- or LR- HPV by HC2 assay and 95% confidence intervals (95%CI) were calculated (Digene). Because the genotype determination was not where appropriate, using the exact method. Differences in possible for all samples the analysis reported in this paper the means of continuous variables were analysed using have been done by considering only the HR- and LR-HPV non-parametric tests (Mann-Whitney or Kruskal-Wallis) categories. or ANOVA (to obtain the mean values). Logistic regres- sion analysis was used to control for the confounding HIV testing from age to some observed significant univariate predic- The patient HIV-serostatus was determined at the entry tors of seroreactivity, e.g. HIV/smoking status. Confound- with ELISA test and confirmed by Western blot, with the ing was also controlled by calculating the common odds informed consent of the patients. The state of HIV disease ratio (OR) for the stratum-specific estimates, using the in sero-positive patients was assessed by CD4+ and CD8+ Mantel-Haenszel test. To evaluate the statistical power of lymphocytes counting and recording the opportunistic the study (e.g. for age differences), we used STATA/SE 9.2. infections or neoplasia. Unfortunately CD4 parameter power analysis option, with the default value (0.05) for was not available for all HIV-positive patients. All these alpha, and using the true sample sizes of the women in the patients were being treated with highly active anti-retrovi- two groups, e.g. seropositive and -negative women. In all ral treatment (HAART). HIV-positive patients in this study tests, the values p < 0.05 were regarded statistically signif- were only 12, of whom 9 current smokers. icant. HPV serology Authors' contributions Human sera and control sera CG conceived the study, participated in his design and The collection and treatment of sera of the 96 women coordination and in rafting the manuscript, PDB, FG, SM, have been previously described [34]. Control sera were MGD participated in acquisition analysis and interpreta- randomly taken among sera collected for studies unre- tion of serology data, LA participated in interpretation of lated with HPV infection. As positive controls, antigen data and critically revised the manuscript, MB, SC, LM, Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:9 http://www.infectagentscancer.com/content/3/1/9 11. Jochmus-Kudielka I, Schneider A, Braun R, Kimmig R, Koldovsky U, AA, MC participated in acquisition of serum samples and Schneweis KE, Seedorf K, Gissmann L: Antibodies against the epidemiological data, KS participated in design of the human papillomavirus type 16 early proteins in human sera: study, performed the statistical analysis and participated correlation of anti-E7 reactivity with cervical cancer. J Natl Cancer Inst 1989, 81:1698-1704. in drafting the manuscript. 12. 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