Key points are not available for this paper at this time.
Background: Infection with cancer-linked human papilloma-virus (HPV) types such as HPV type 16 (HPV16) is the most important risk factor in the development of cervical cancer. It has been shown that immunoglobulin G (IgG) antibody responses against HPV16 virus-like particles (VLPs) are specifically associated with genital HPV16 infection. Purpose: The aim of this study was to determine the temporal relationships between the presence of HPV16 VLP-specific IgGs, HPV16 infection patterns, and the course of premalignant cervical disease. Methods: Plasma samples from 133 women who had been diagnosed originally with mild to moderate cervical dyskaryosis and enrolled in a prospective nonintervention cohort study conducted in Amsterdam, The Netherlands, from 1991 through 1996 were analyzed for the presence of HPV16 VLP-specific IgGs by use of an enzyme-linked immunosorbent assay. A detailed analysis was performed on 43 women with different HPV16 infection patterns during a follow-up period of 10-34 months. Progression or regression of cervical intraepithelial neoplasia (CIN) lesions was monitored by cytologic and colposcopic testing at intervals of 3-4 months. HPV typing in cervical smears was performed by use of a polymerase chain reaction-based assay. Statistical analysis of the serologic data was performed by use of the Mann-Whitney U test or2×2 table analyses. Results: The presence of HPV16 VLP-specific IgGs in the plasma of the patients was found to be associated with the presence of HPV16 DNA in the cervical smear. Significantly higher proportions of patients with persistent HPV16 infections (i.e., who were polymerase chain reaction positive in three to 11 consecutive tests) than of patients with cleared HPV16 infections were found to be positive for the presence of HPV16 VLP-specific IgGs (18 69.2% of 26 versus nine 28.1% of 32, respectively; P = .003). HPV16 VLP-specific IgGs were consistently detected in all women (n = 11) who were persistently HPV16 DNA positive during follow-up and whose disease ultimately progressed to CIN III (histologically diagnosed severe dysplasia or carcinoma in situ). Conclusion: HPV16 VLP-specific IgG responses are present in the plasma of a majority of patients with persistent HPV16 infections and histologically confirmed high-grade lesions but only in a smaller subset of patients with cleared HPV16 infections and either normal cervical histology or low-grade CIN lesions. Implications: These results suggest that HPV16 VLP-specific antibodies are not responsible for the clearance of virally induced CIN lesions but that they might, in patients with persistent HPV16 infections, be indicative of an increased cervical cancer risk. J Natl Cancer Inst 1997;89:630-8 Compelling evidence has accumulated during recent years that shows infection by high-risk human papillomavirus (HPV) types such as HPV type 16 (HPV16) to be the most important risk factor in the development of carcinoma of the uterine cervix ( 1-3 ) . A lack of HPV-specific immunity may contribute substantially to the persistence of HPV in the cervix and to the subsequent development of premalignant or malignant lesions. The observation of increased incidences of HPV-associated lesions in immunocompromised individuals argues in favor of this hypothesis ( 4-6 ) . Our group is currently evaluating HPV16-specific immune responses in a nonintervention cohort study of patients originally diagnosed with initial mild to moderate cervical dyskaryosis ( 7 ) . This study design allows us to study the relationship between immune responses with HPV infection patterns and disease development over time. It complements several recent studies (810) designed to investigate seroconversion in relation to the initial acquisition of HPV infection. Immune responses against HPV during early stages of infection may be important in blocking reinfection, inhibiting viral spread, and preventing viral persistence. Virus-neutralizing antibodies at the mucosal surface, directed against native virion epitopes, may play an important role in this regard. However, the temporal relationship between virion antibody response and the course of cervical disease has not been examined critically. Information about naturally occurring immune responses against HPV16 capsids might have important implications for possible vaccine strategies. The lack of an in vitro culture system for high-risk HPV types has hindered the production of sufficient amounts of virions to test for specific antibody responses. Recently, HPV16 virus-like particles (VLPs), consisting of the major capsid protein L1 alone or L1 in combination with the minor capsid protein L2, were obtained through self-assembly in a baculovirus expression system and were used to detect serologic responses against epitopes on the capsid ( 11-14 ) . Serum immunoglobulin G (IgG) responses against conformational epitopes on HPV16 VLPs, as determined in an enzyme-linked immunosorbent assay (ELISA) format, were shown to be associated with HPV16 infection in patients with cervical intraepithelial neoplasia (CIN) ( 15 ) . HPV16 VLPs are also excellent candidates for use in prophylactic vaccination. Indeed, vaccination with papillomavirus capsid-like structures was recently shown to confer protection from a subsequent experimental viral challenge and to prevent the outgrowth of papillomavirus-induced lesions in several animal models ( 16-18 ) . In this prospective study, we determined the relationship between HPV16 VLP IgG reactivity, viral clearance or persistence, and the course of disease in patients with premalignant cervical lesions. Our study was nested within a larger prospective nonintervention cohort study of patients with CIN that was designed and conducted to examine the relationship between HPV infection patterns and the course of CIN disease (7 ) . Women (n 352) presenting with abnormal cervical cytology (mild to moderate dyskaryosis) were referred to the gynecological outpatient clinic of the Free University Hospital in Amsterdam, The Netherlands; after giving written informed consent to participate, they were enrolled in the study. The study design was approved by the ethics committee of the hospital. Fig. 1. Schematic representation of the cervical intraepithelial neoplasia (CIN) study design. After 1056 months of clinical follow-up (varying among patients), plasma was available from 187 patients. After the exclusion of 54 patients with human papillomavirus (HPV) type-X infections (i.e., infections with unidentified HPV types), 133 patients with CIN (HPV negative HPV-neg., n 29; HPV non-16 HPV type 16 negative but positive for other HPV types, n 32; and HPV16, n 72) were tested for immunoglobulin G (IgG) reactivity to HPV type 16 (HPV16) virus-like particles (VLPs) in a cross-sectional analysis at the start of plasma collection. HPV16-positive patients, from whom multiple plasma samples were available (n 43), were subsequently tested for VLP-specific IgG reactivity during follow-up (length of follow-up time being 10-34 months, varying among patients). At the end of the study, histologic data were available for 37 patients tested for VLP-specific IgG reactivity during follow-up and for 17 HPV16-positive patients tested only once in the cross-sectional analysis. Clinical disease course, histologic data from biopsy specimens taken at the end of clinical follow-up, and HPV infection patterns over time were related to the observed IgG antibody responses to HPV16 VLPs. Clinical follow-up consisted of cytologic and colposcopic examinations that were performed every 3-4 months. Biopsy specimens were not taken during the follow-up period to avoid interfering with the natural course of the disease. Results from the cytomorphologic tests were classified according to a modified Papanicolaou (Pap) system as employed in The Netherlands (7 ) ; i.e., Pap 1 no cytomorphologic abnormalities, Pap 2 inflammation, Pap 3a mild to moderate dyskaryosis, Pap 3b severe dyskaryosis, Pap 4 carcinoma in situ, and Pap 5 microinvasive carcinoma. Colposcopy was used to indicate the predicted grade of dysplasia (termed “ CINc” ) and to determine the extent of the observed lesions in cervical quadrants. At the end of follow-up, biopsy specimens were taken and a histologic diagnosis was made (no CIN normal or metaplastic epithelium, CIN I mild dysplasia, CIN II moderate dysplasia, and CIN III severe dysplasia or carcinoma in situ). A detailed description of the tight clinical surveillance of the patients and the evaluation of clinical follow-up were reported by Remmink et al. (7 ) . The design of this study is presented in . Of the 352 patients with CIN who participated in the study, 227 were still in clinical follow-up when plasma collection was started. Plasma samples were collected from 187 patients who consented to one or more blood donations. Patients infected by unidentified HPV types (HPV-X; n 54) were excluded. Plasma samples from the remaining 133 patients (including all HPV16-positive patients; n 72) were tested for IgG reactivity against HPV16 VLPs in a cross-sectional analysis after 10-56 months of clinical follow-up (mean length of follow-up, 31.2 months). HPV16-positive patients from whom multiple plasma samples were available were studied serologically and clinically over a follow-up period of 10-34 months (mean length of follow-up, 20.7 months; n 43; ). Of the total of 72 tested HPV16positive patients, 54 had reached the end of clinical follow-up this study was patients, histologic data were available that be related to the IgG determined The 133 CIN patients in the cross-sectional analysis in from to years (mean of the These patients were to different test on the of HPV typing and infection patterns over time to the of serologic A group of women with HPV infections and normal cervical cytology 1 or Pap was in the cross-sectional analysis. These participated in a follow-up study of women with normal cervical the CIN patients, they were from the at a of years These women had the HPV (n HPV16 (n (n and (n Plasma samples from a group of women were samples had been at the of Clinical of the Free University These women the during the period from 1991 through 1996 with to cervical disease and were found to have no or other clinical were in the study as with HPV and cytologic and were to the of the test (mean of the years were no in the of the patients with the women with normal cervical and the with CIN versus women with normal P patients with CIN versus P versus women with normal P HPV and were performed on from cervical as ( 7 ) . obtained from the cervical smears used for cytology were collected in in 1 and at the polymerase chain reaction were These were for on and at for 1 at being used in an HPV samples were tested in an HPV for the genital HPV types and of samples that to be negative in the were and after to DNA from genital HPV types 32, and ( ) . HPV smears that not be in this were The 133 CIN patients in this study were different test on the of HPV infection patterns as determined by during clinical HPV16 DNA was found to be present in the cervical of 72 patients at or plasma collection. These patients were to the HPV16 was the three The group patients who were found consistently to be negative for HPV16 DNA for at consecutive months after been HPV16 DNA positive (mean time the patients were determined to be positive for HPV16 months of the years The group patients with HPV16 DNA (mean of the years The group 26 patients who were consistently positive three to 11 consecutive tests) for HPV16 DNA over a period of at months serologic testing (mean of the years Patients who were negative for HPV during the follow-up period and for at months (mean length of months to serologic testing the Plasma from such patients was available for this study (mean of the years Patients with a or infection of HPV types other than HPV16 the HPV non-16 group (n 32; of the years This group consisted of patients with infections of types (HPV types and and patients with types (HPV types and were no in the of the total HPV16-positive group with the group or the HPV non-16 group or between the of the and Plasma from blood was in and was at the plasma samples were at for at to protein HPV16 were in and by as ( ) . These VLPs were used in an to determine specific plasma IgG reactivity, a of the assay reported by et al. ( 15 ) . of VLPs in was to and at 37 for the were three with for 2 at 37 with The and and three with The were for 2 at with human plasma in This plasma consistently in the of the when plasma samples from different patients were tested in a from to After with human IgG in was The were for 1 at and with in was The reaction was by the of the at was by use of an tests were performed for all plasma the obtained for the test of plasma the reactivity found in the negative with and with was IgG were as the of the Plasma samples were when results were found between for we in relation to obtained for a of plasma samples positive and negative IgG that were in A were was on the of found in an of women from the as the women in the test This was reached by three the to the after exclusion of one analysis between was performed with the or test 2 table and intervals were with from 2 2 table analyses. IgG between were with the Mann-Whitney U tests were and results were when P At the start of serologic follow-up, a cross-sectional analysis was In this test were by HPV infection These patterns were determined during clinical follow-up a period of 10-56 IgG IgG found for the the women with normal cervical and the the HPV and the HPV16positive patients with CIN are shown in Significantly higher VLP-specific IgG were found in the HPV16positive CIN group than in the HPV non-16 group or the and the HPV non-16 CIN test when the HPV16-positive CIN group was patients with a a or a persistent HPV16 an between reactivity and HPV16 persistence HPV16 VLP-specific IgG in the group of women were than in the group of patients with the the and the HPV patients ( P A was also found with the group of women with normal cervical cytology and HPV this that IgG responses against the HPV16 capsid protein be and in the of cytologic ( P on the of IgG within the group of a was one IgG this were 1 shows the of positive for all the different test of 133 patients who had been tested were positive for HPV16 VLP-specific The observed in the HPV16-positive CIN group was higher than that in the other of patients with CIN (i.e., or HPV three patients in the HPV non-16 CIN group positive responses had cleared infections of HPV types not related to type 16 (i.e., types and of the patients with or infections of types a positive IgG reactivity The of women with positive IgG responses against HPV16 VLPs was found in the group this was higher than the observed in the group alone P or in the group with the group P the a in IgG was observed between patients who were found to be HPV16 positive once during follow-up of and patients who were positive more than once during follow-up of P A of positive was found in the group with normal cervical cytology and HPV infections of HPV of this from that found in the group of women P 1. G (IgG) against human papillomavirus (HPV) type 16 (HPV16) virus-like particles in patients with cervical intraepithelial neoplasia (CIN) and in normal cross-sectional analysis Fig. analysis of immunoglobulin G (IgG) reactivity against human papillomavirus (HPV) type 16 (HPV16) virus-like particles (VLPs) as the detected tested are IgG reactivity from one for all are between are by the P Mann-Whitney U Women were to the different by disease and HPV DNA women HPV and cytologic women with normal cervical intraepithelial neoplasia (CIN) polymerase chain reaction negative for HPV during clinical follow-up to HPV infected by HPV type other than type patients with or HPV16 HPV16-positive patients with CIN by HPV16 infection HPV16 HPV16 persistence. IgG reactivity against HPV16 VLPs was determined in 43 patients with CIN who had different HPV16 infection patterns and from whom multiple plasma samples were available over a period of 10-34 months (mean serologic months). Plasma samples were available from to different time Results from 17 and patients are shown in . IgG in the persistence and are shown to the time that the start of clinical for the clearance group are shown to the time that the positive HPV16 DNA In the clearance of patients had HPV16 VLP-specific IgG over time. patients, plasma samples were available the time of Of patients, one was IgG positive ( of 17 patients were found to be positive for the presence of HPV16 VLP-specific 11 of patients were consistently positive during follow-up, the other patients had IgG the three of patients with HPV16 DNA over time were consistently positive for HPV16 VLP-specific IgGs during 2 cytologic and colposcopic data in relation to the of HPV16 VLP-specific IgG for all HPV16-positive patients at the start of plasma collection. The of VLP-specific IgG were also determined for the different histologic CIN ultimately found for 54 HPV16-positive patients at the end of clinical shows a higher of IgG in patients whose disease had ultimately progressed to CIN III than in HPV16-positive patients with CIN I or II or CIN in the total HPV16-positive group and ( P and in the of patients with HPV16 persistence ( P results were found in 37 patients who were tested for HPV16 VLP-specific IgG reactivity over time and for whom histologic data were available by the end of serologic patients whose disease had progressed to CIN III over the course of follow-up had a persistent HPV16 infection and IgG against HPV16 VLPs (n In only of 26 patients lesions or with CIN were positive ( P within the a was found between the patients whose disease had progressed to CIN III of 11) and whose disease not of P Fig. papillomavirus (HPV) type 16 (HPV16) virus-like immunoglobulin G (IgG) reactivity over time in 43 HPV16positive patients with cervical intraepithelial were by HPV16 infection HPV16 clearance (n results shown to the time that the positive HPV16 DNA HPV16 persistence (n and HPV16 (n results shown to the start of clinical the at by HPV16 DNA by HPV16 DNA are from papillomavirus type 16 (HPV16) virus-like immunoglobulin G (IgG) in relation to cervical intraepithelial neoplasia (CIN) disease in HPV16 cross-sectional analysis shows follow-up data obtained from patients who had persistent HPV16 infections and who different clinical IgG over time are shown in relation to disease development and colposcopic over At the end of follow-up, the histologic is (no CIN CIN I CIN II or CIN III In the VLP-specific IgG during follow-up were observed in one of patients histologic CIN in of one with CIN in one of patients with CIN and in 11 of 11 patients with CIN IgG reactivity in of 11 patients who disease to CIN III in In this study, we have shown that HPV16 VLP-specific IgG reactivity is type specific and is associated with and HPV16 infections in patients with These results are in with that responses more in HPV16-positive patients with CIN and that IgGs against VLPs type . Fig. of patients with cervical intraepithelial neoplasia (CIN) who were positive for the presence of human papillomavirus type 16 (HPV16) virus-like G in the cross-sectional in relation to histologic at the end of clinical from test are the total HPV16positive group (n P the group (n CIN are no CIN no dysplasia CIN I mild dysplasia CIN II moderate dysplasia CIN III severe dysplasia The total of patients in is at the of the we observed IgG responses against HPV16 VLPs in a of patients whose HPV16 infections cleared with time 28.1% of was a between antibody responses and HPV16 persistence (18 69.2% of 26 patients who were IgG in the cohort of patients. 11 patients with whose disease over a follow-up period of months progressed to histologically confirmed CIN III dysplasia or carcinoma in had persistent HPV16 infections and consistently positive IgG to HPV16 VLPs at to different time over the course of In a recently reported study of patients with CIN III or cervical cancer in and et al. ( ) of HPV16 VLP-specific IgG in the CIN III from and This is to the that we found in the cross-sectional analysis for patients whose disease ultimately progressed to CIN III It is that the from higher of HPV16 VLP-specific IgG than the from to an higher of cervical cancer in These data an between the presence of antibodies in the and increased cervical cancer risk. recent study by et al. ( ) IgG reactivity against HPV16 VLPs to have for the development of cervical cancer. These results are in with of an between IgG reactivity against HPV16 VLPs and to CIN III and the VLP IgG reactivity might be in to in the of cervical cancer risk. as IgG reactivity against HPV16 VLPs to a subset of patients in the HPV16 group with an increased risk of disease to CIN III might to be a to the HPV DNA of for and present HPV et al. ( ) found of positive IgG in patients with cervical carcinoma than in patients with CIN also observed a in a of tested HPV16-positive patients with cervical carcinoma of being positive for HPV16 VLP-specific data not This observation may be by a expression of the L1 and capsid the from CIN to cervical carcinoma ( 26 ) . The L1 and have been reported to be present in low-grade CIN lesions L1 and expression was observed in high-grade cervical lesions ( 26 ) . These are by that of 11 patients with disease to CIN III during follow-up a in HPV16 VLP-specific IgG reactivity Fig. papillomavirus (HPV) type 16 (HPV16) virus-like immunoglobulin G (IgG) reactivity related to clinical follow-up in patients with cervical intraepithelial neoplasia (CIN) and HPV16 persistence. IgG as the at are shown in relation to cytologic and colposcopic data for time The histologic is shown at the end of clinical and serologic the Results are shown for the between IgG and the to CIN the of HPV16 infection may be an important Progression of cervical lesions in a subset of patients with a persistent infection of high-risk HPV types ( 7 ) . found a between IgG and HPV16 persistence. patients with a cleared HPV16 infection were more to be IgG positive when they were positive for HPV16 more than once These are in with a recent by patients . months months months and months . Pap no cytomorphologic Pap Pap mild to moderate predicted grade of dysplasia and extent of the observed lesions in cervical no no CIN mild CIN moderate CIN severe for are from et al. ( ) who found that patients who were positive for the presence of HPV16 DNA by at consecutive time a higher of positive IgG responses than patients who were positive at only one time persistent infections may the of between HPV capsid and the immune system and the subsequent of specific immune responses. as persistence is associated with disease IgG reactivity may also be associated with the development of CIN III lesions. low-grade CIN lesions with HPV infections may still be present in patients with cervical lesions that had progressed to CIN Indeed, of 11 patients with CIN who were ultimately diagnosed with CIN III lesions during the period of follow-up by serologic testing also had CIN I or II lesions as determined by histologic These lesions may still capsids and to the detected responses. lesions to with IgG may start of L1 in cervical biopsy specimens from women may this viral may also be a factor in the of responses against HPV This is by the of higher of IgG in normal women found to be HPV16 positive by the assay and the more than in women found to be positive by alone ( 15 ) . of IgG were also higher among patients with CIN who had been found to be HPV16 DNA positive by than among patients found to be positive by ( ) . women with a higher viral are more to higher amounts of capsids to antibody and to cervical lesions. examine this we are in the of HPV16 DNA in cervical smears from the patients tested in this study in a in ( ) . in viral after initial infection may also the of IgG responses in a of CIN patients with or HPV16 data on HPV16-positive patients with normal cervical cytology of the 17 women were also positive for the presence of HPV16 VLP-specific be that the presence of lesions is not a for IgG responses against HPV16 capsid This is in with studies that HPV16 VLP-specific within months after a HPV16 infection in normal women with multiple ( ) . A recent follow-up study of in the ( ) that such positive IgG for years and have a for the subsequent development of premalignant cervical lesions. one of patients who HPV16 clearance HPV16 VLP-specific IgG at that time This that other immune may be in lesions. that antibodies are detected with the test employed in this study, implications have for possible vaccination studies animal models the of VLPs for prophylactic vaccination. with papillomavirus VLP from experimental challenge with this and the development of ( 16 ) . vaccination with papillomavirus and papillomavirus type 4 the development of mucosal after experimental challenge ( 17 ) . VLP-specific detected after were with native virions and were to be and responsible for of protection by or IgG this ( 16 17 ) . However, VLPs were not found to be in vaccination. not regression of lesions ( ) . This to be in with HPV16 VLP-specific IgG responses are present in patients with viral clearance and may have virions in normal or epithelium, but they are in patients with persistent In in the they are associated with CIN lesions. These data suggest that HPV16 VLPs may be as a prophylactic but not for vaccination. be by of It has been that a of IgG mucosal by mucosal such as the of the cervix The of was by a from the and all at the of Clinical of the Free University Hospital for excellent also and for of the and and for in the evaluation of the clinical
Gruijl et al. (1997) studied this question.