Human Papillomavirus (HPV) infection, known to be associated with both benign and malignant tumours at different mucosal sites, is widespread among female and male populations 12. The oncogenic high-risk HPV types (HR-HPVs) are the single most important etiological agents of cervical cancer (CC) and its precursor (CIN) lesions 34. Despite global differences in the prevalence of individual HR-HPV types, HPV16 remains the most frequent HR-HPV type in all geographic regions and the current HPV research is mostly focused on it 34. Diagnosis of HPV infection is based on detection of viral DNA using different PCR-based methods or commercially available hybrid capture (HC2) test 56. Until now, serological studies have had little to offer in diagnosis of HPV infections 7. The first serological assays for HPV antibody detection were introduced in the early 1970's 8, but the more widespread use of HPV serology had to await the development of more refined assays based on virus-like particles (VLP) in the early 1990's 910. A growing body of serology data has being obtained using different viral protein-based ELISA to analyse HPV antibodies in different settings, most notably in large-scale seroepidemilogical- and case-control studies to assess the risk factors of cervical carcinoma (CC) in HPV exposed subjects 11121314151617. The main conclusion from the plethora of sero-epidemiological studies is that antibody response to viral proteins does not invariably occur during a natural HPV infection 1415; only half of HPV DNA-positive women with normal cytology shows antibody response to VLPs 1819. HPV E6/E7 antibodies are predominantly found in patients with CC, but they are also detected in healthy controls, precluding the use of their detection as predictor of CC 141520.

VLP-based assays have been recognized as reliable genotype-specific assays 141521 and potentially will contribute to the understanding of the natural history of HPV infections 181922. New L1VLP-based vaccines have been shown to induce levels of HPV type specific antibodies higher than that found in natural infected women 23. Correlation between anti-L1VLPs antibodies and protection from reinfection in natural infection is still a controversial issue 2425. An important problem with serological studies resides in the fact that the results from different laboratories are not comparable for the lack of reference specific sera 26. For the serological response evaluation of the new HPV vaccines the WHO recommends the standardisation of the different in-house L1-based serological methods by the use of reference sera and promoted an international collaborative study with this proposal 21.

To improve the understanding of correlation between infection and antibody response in patients with and without cervical lesions, prospective cohort studies using a widespread panel of viral antigens would be needed 141526. Similarly, it would be necessary to study the dynamics of HPV-E and -L antigen-specific antibodies in relation to their clinical and epidemiological correlates 2728, including immune-suppression due to HIV infection 2529. There are recent growing evidences regarding the induction of antibodies neutralising across papillomavirus types after vaccination with VLP displaying L2 epitopes or with fusion protein containing L2E7E6 viral proteins or synthetic peptides of the HPV16 L2 protein 303132. Very recently Rizk and collaborators 33 have shown that L1 fused to GST protein displays a broad variety of epitopes, both conformational and linear; the neutralising ones are conformational and, mostly, type specific while the cross-reacting ones are associated with linear epitopes.

We have recently reported the development of an in-house ELISA test based on five recombinant HPV16 proteins expressed in E. coli: L1 and L2 capsid proteins, E6 and E7 oncoproteins and the non-structural E4 protein, all used in unfolded form 34. The idea of using denatured antigens comes from the observation of a high amino acid identity, ranging from 36% for E6 to 74% for L1, among the same proteins belonging to different HPV genotypes and then the possibility to detect cross-reacting antibodies directed against linear epitopes. By this assay we were able to detect an antibody response in women infected with HPV genotypes different from that of the reference antigen. In this study we also determined the Avidity Index (AI) 35 of the positive samples. Antibody response to linear epitopes has been reported in the past 3637 and was correlated to the specific stage of HPV-associated cervical disease 38; nevertheless this antibody response can not be related to protection level against HPV infection, that is associated to the neutralising component of humoral response. Our system, revealing antibodies only against linear epitopes, could be utilized to evaluate the immunological responsiveness of subjects to HPV infection and contribute to estimate the risk of infection progression but it could not provide information about protection level from reinfection in both natural infection and after vaccination.

In the present study we have correlated the antibody response, evaluated with our system, to the baseline epidemiological, clinical and HPV DNA data of women enrolled in a multi-centre cohort study designed to explore pathogenetic mechanisms and prognostic factors of CIN lesions in HIV-positive and HIV-negative women, known as HPV-PathogenISS study 39.

In a previous study we have reported the baseline data of a cohort comprising HIV-positive and HIV-negative women 39. The present report provides data on HPV exposure status of a group of these women that gave their blood. The analysis regards the serological response to recombinant HPV16 L1, L2, E4, E6 and E7 proteins evaluated by a new ELISA technique 34.

We have correlated the women's epidemiological characteristics to their serological response to HPV16 antigens, using three possible outcomes; i) seroreactivity to either E or L (= HPV seropositive or not), ii) reactivity to E, and iii) reactivity to L proteins. Only a few of the recorded epidemiological variables proved to be significantly related to serostatus of the women in this cohort. Interestingly current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers (OR = 3.7; 95%CI 1.3–10.5) (p = 0.009), while the difference in reactivity to E and E+L antigens was not significant. This is in full agreement with a recent report, where almost identical observations were reported 28. In this study, smokers were twice as likely as non-smokers to test negative for anti-HPV L1 antibodies, even when the effects of other covariates were considered in the analysis (adjusted OR = 0.5; 95%CI 0.2–0.9). The relationship between tobacco smoking and CC is highly complex; however, the basic mechanisms must involve the host's inability to clear HR-HPV infection. The observed failure of smokers to mount antibody response to L antigens could be an indication of the deregulation of immune responses induced by smoking, increasing the likelihood of persistent infection and the long-term risk for CC. This is in agreement with other results showing current smoking to be associated with persistent HR-HPV infection 4344.

The relationship of contraception and HPV infection has been incompletely studied and only in relation with HPV DNA detection 274546, but not in relation to antibody detection. In the present analysis only the condom use, among the recorded epidemiological variables, was related to seroreactivity to L antigens; the lowest (61.1%) seropositivity (Table 1) was observed in those women who reported to always use condom in sexual intercourse.

The relationship between HPV DNA detection and antibody response to different HPV antigens is highly complex. Large sero-epidemiological studies seem indicate that antibody response to viral antigens does not invariably occur during natural HPV infection 1415. Similarly, only approximately half of HPV DNA-positive women with normal cytology show antibody response to VLPs 1819. Moreover E6/E7 antibodies are detected in patients with CC as well as in healthy controls. 141547. These observations are confirmed in the present cohort, where detection of HPV DNA, both LR+HR genotypes and LR- and HR-HPV separately, did not bear any significant relationship with seroreactivity to any viral antigens. However women who reported past HPV infection show a significantly higher prevalence (89.5%) of E antibodies as compared with that (63.6%) of women who did not reported past infection (OR = 4.8; 95%CI 1.1–22.8) (Table 2). These results suggest that seropositivity to E antigens could be considered as a surrogate for past viral infection 48.

Into the HPV-PathogenISS cohort, HIV-positive women were also enrolled 39. As generally accepted, these women are at increased risk for progression to CIN3 and CC 49, mainly because of their failure to eradicate persistent HR-HPV infection, even if treated by HAART 50. HPV serology in HIV-positive women has been studied only recently. In these women we have observed a significantly lower seroprevalance compared to the HIV-negative ones; these data are in contrast with those reported in large cohort studies, where HIV-positive women usually have higher prevalence of HPV antibodies 512529. Some reasons of this discrepancy can be the small number of HIV-positive women in our cohort leaving room for observations by chance and the fact that all patients had been on HAART for several years and their HPV baseline status was very similar to that of HIV-negative women. On the other hand, this observation could indicate their lower HPV exposure in the past. A feasible explanation could be the HIV-induced immunosuppression leading to perturbed early immune responses against HPV 50; unfortunately, the CD4 data of these women are incomplete and it is not possible to draw firm conclusions on their level of immunosuppression. With regard to the above discussed failure of smokers to mount viral antibody response, we analysed the combined effect of current smoking and HIV-status in this cohort. Based on the obtained results and our previously reported data 5044, it is tempting to speculate that HIV-positive women who are current smokers comprise a special high-risk group, with extremely high probability of developing CIN3/CC, due to their failure to eradicate persistent HR-HPV infection. This failure could be attributed to their inability to mount effective immunological responses against HPV antigens, of which deregulation the low seroreactivity is an indication.

Two other observations merit some discussion. We noticed that seroresponse to L proteins declined in parallel with the increasing severity of abnormality in the referral PAP smear, from 100% among ASCUS cases to 56.3% among those with HSIL (Table 2)(p = 0.031 for linear trend). This could substantiate the observations of Wiley and collabotators 28 who reported that women with ASCUS were twice more likely to test anti-HPV L1-positive as compared with those with normal PAP. In our cohort, we had no PAP-negative women to confirm this.

As recently discussed 34, most of the HPV antibodies bound to these 5 antigens with an AI higher than 40%, even in the sera from women infected with HPV type different from 16 type. When related to epidemiological data and clinical observations, AI data yielded few significant observations. No feasible explanation can be offered for the association of high AI of anti-E7 antibodies in 100% of women which did not use contraception in the past, as compared with the 80% of those using contraception (p = 0.044). Similarly, the significant difference (p = 0.011) obtained in AI between the different colposcopy categories does not show any linear trend with the grade of abnormality.

In summary the results show that there is no meaningful relationship between HPV DNA detection and seroreactivity to tested HPV antigens, which was not unexpected in the light of published data 141519. However, two significant observations are of particular interest. Current smokers show significantly (p = 0.009) less seroreactivity to HPV L antigens as compared with the non-smokers. The difficulty of smokers to mount antibody response to L antigens could be an indication of a marked deregulation of the immune system responses thus increasing the long-term risk for CIN3/CC. Another interesting observation is the significantly impaired seroreactivity to HPV L antigens among HIV positive women, further accentuated among women HIV positive and current smokers. It is tempting to speculate that HIV positive women who are current smokers comprise a special high-risk group, with extremely high probability of developing CIN3/CC, due to their failure to eradicate persistent HR-HPV infection.

CG conceived the study, participated in his design and coordination and in rafting the manuscript, PDB, FG, SM, MGD participated in acquisition analysis and interpretation of serology data, LA participated in interpretation of data and critically revised the manuscript, MB, SC, LM, AA, MC participated in acquisition of serum samples and epidemiological data, KS participated in design of the study, performed the statistical analysis and participated in drafting the manuscript.