Demographic, Clinic characteristics and risk factors of studied subjects
All the women enrolled in this study were from Tunisia. The age range across the studied population was 18 to 73 years with a mean age of 37 years. The majority of women from the general population or with cervical lesion (HGSIL and LGSIL) were married (85%) and monogamous. The median age at first intercourse was found to be 19 years (ranges l2 to 27 years). This population was homogenous in terms of social habits. None of the women had history of using contraceptive or condoms systematically, smoking or drinking alcohol. Out of 43 women with cervical lesions, 16 were diagnosed as LGSIL and 27 as HGSIL. All the sex workers enrolled in the study were legal, relatively homogenous in terms of sexual activity and high exposure risk factors for HPV infection. Fourteen (27.4%) sex workers had experienced first intercourse before the age of 20. The mean of years of sex work was four years (ranging 0 to 16 years). Majority of sex workers reported smoking (n=45, 88.2%) and drinking alcohol (n=25, 49%). The condom use was reported as follow: 18 women (35%) never, 14 women (27%) sometimes and 19 (37%) all of the time.
Binding of the peptides to the N-MAbs
To demonstrate if the linear and the cyclic peptides are recognized by neutralizing antibodies we tested their antigenicity by using three specific monoclonal antibodies (MAbs) raised against the L1 capsid protein of the HPV16 (Fig. 1) [19-21]. The H16.H5 antibody, is a murine monoclonal antibody with no neutralizing activity or type specificity. H16.H5 is reactive to a linear epitope including residues from 174 to 185, in the EF loop [19,20]. The H16.V5 and the H16.E70 are both neutralizing antibodies that recognize conformational epitopes on the surface of HPV16 VLPs [21]. H16.V5 and H16.E70 recognize different epitopes on the surface of HPV16 VLPs, although there is some overlap in the residues recognized by the two antibodies localized at the FG loop. Both the FG and DE loops were necessary for binding of the H16.E70. While, the FG loop is the predominant epitope recognized by H16.V5 [21]. Compared to the H16.H5 and H16.E70 antibodies, the H16.V5 was the most reactive against both the linear and the cyclic peptides. Both peptides likely have comparable antigenicity against the H16.V5 antibody. The reactivity curves of the H16.H5 and H16.E70 antibodies were overlapped for each form of the peptides, but lower than H16.V5. However, the cyclic peptide was more reactive than the linear one with H16.H5 and H16.E70 antibodies (Fig. 1).
Linear and cyclic peptide correlations
To show if the tow shapes of the peptide (linear and cyclic) have the same antigenicity to human sera, we tested the two peptides against sera samples from the several group of the study population. Pearson correlation analysis of the ELISA optical density results (405nm) for IgG detection using linear or cyclic peptide showed no significant correlation (r=-0.036; p=0.830) (Fig. 2). Unlike what we observed with mouse Mab, the two shapes of the peptides seem to react differently when binding to human polyclonal antibodies. The cyclic peptide is more reactive with human sera. This difference in reactivity, may have several reasons. First, antibodies in human sera are directed against a larger panel of epitopes. Secondly, cyclic peptides are known to be more stable than linear peptides with identical amino acid sequence. They are especially resistant to hydrolysis by exopeptidase as they lack amino and carboxyl ends. Besides, the rigidity of cyclic peptides increases binding affinity which can extend their biological activity and improve recognition of epitope mimics [32,33]. For all these reasons, only results obtained with the cyclic peptide were presented below.
Detection of systemic and cervical anti-L1FG/HPV16 antibodies
The distribution of positive women to anti-L1FG/HPV16 antibodies is summarized in Table 2. For statistical analysis, the reference group was the healthy women from the general population. The systemic IgG antibody prevalence was significantly higher among women with LGSIL and sex workers (44% and 25%, respectively). However, comparable frequencies were observed among women with HGSIL and those from the general population (15% and 12%, respectively). The systemic IgA and the cervical IgG antibody prevalence’s were significantly more elevated among the sex workers, suggesting the influence of the lifestyle on antibody production (23% and 16%, respectively).
Compared to healthy women from the general population, the HPV DNA prevalence was significantly higher among sex workers and women with LGSIL or HGSIL (39%, 62%, and 81%, respectively) (Table 2). As antibodies are a marker of past as well as present infection, we examined the relationship between HPV16 capsid FG loop sero-reactivity and the status of HPV16 infection (Table 3). The overall frequency of HPV16 DNA positivity was 13% (23/179), interestingly none of the HPV16 positive women showed positive systemic or local IgG anti-peptide antibodies. However, among HPV16 DNA negative women but infected by HPV types other than the HPV16 (HPV18, 31, 33, 45, 56, 58, 68, 82, 53, 66, 6, 11, 61, 70, 81, 83 and 84) [28], we detected a higher antibody prevalence in both sera and cervical secretions. These results suggesting that detection of anti-L1FG/HPV16 IgG antibodies is unrelated to a current infection with HPV16.
To identify a prognostic signification of the anti-LlFG/HPV16 antibodies, we extended our analysis and compared results of LGSIL and HGSIL patients. The proportion of local IgG and IgA was very low in cervical samples and could not be compared. However, in sera, the frequency of the antibodies was significantly more elevated among women with LGSIL compared to HGSIL (44% versus 15%; p=0.04), suggesting that women with anti-peptide antibodies appear to have a better prognosis than those without antibodies.
We have previously shown that HPV infection decreased with age, among healthy women [27,28]. To assess if we observed the same pattern with the antibody reactivity to the L1FG/HPV16, we overlapped HPV DNA and IgG prevalence’s curves according to age (Fig. 3). Among healthy women from the general population, the HPV DNA and IgG prevalence’s were less than 20% and did not change significantly with age. Among the sex workers, the prevalence of systemic IgG increases (21% to 66%), and conversely HPV DNA prevalence decreased (54% to 25%) from the age of 31 years old. Among the women with cervical lesions, HPV DNA prevalence remained elevated, but prevalence of systemic IgG markedly decreased from the age of 31 years old. Altogether, when we compare the pattern among sex workers and women with cervical lesions, we observed inverted and positive progress, suggesting that anti-L1FG/HPV16 antibodies may have efficient effect on HPV clearance.