Нарушена схема вакцинации ГепатитВ

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Vaccine 26 (2008) 6844–6851 HPV antibody levels and clinical efficacy following administration of a prophylactic quadrivalent HPV vaccine E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 6845 1. Introduction The lifetime risk of infection with the Human Papillomavirus (HPV) exceeds 50% [1,2]. HPV infection can cause epithelial dyspla-sia and cancer of the cervix, a significant proportion of cancers of the genitalia (both genders), anal canal, and the oropharynx, as well as benign tumors of the genitalia (condylomata acuminata) and the larynx (recurrent respiratory papillomatosis [RRP]) [3–8]. HPV types are defined by sequence variation in the gene encod¬ing the L1 protein, the major constituent of the viral capsid. Over 40 different HPV types are known to infect cervical, anogenital, and oropharyngeal epithelia. These types are divided into two groups: (a) high-risk HPV types that can cause cancer; and (b) low-risk HPV types that rarely cause cancer, but commonly cause dysplas-tic lesions. Among the high-risk HPV types, HPV 16 and HPV 18 cause approximately 70% of cervical and anal cancer cases [9], and over 80% of HPV-related external genital and oropharyngeal cancer cases. HPV 6 and HPV 11 are low-risk HPV types that cause approx¬imately 90% of all genital wart cases [10] and virtually all RRP cases [11]. A prophylactic vaccine targeting HPV types 6, 11, 16, and 18 has been developed, and is currently available in many countries. This vaccine contains L1 proteins of the 4 vaccine HPV types arranged as 4 separate species of virus-like particles (VLPs) adsorbed onto amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant. In studies, prophylactic administration of this vaccine to 16–26-year-old women was 96–100% effective in preventing HPV 16- and HPV 18-related cervical squamous cell cancer, cervical adenocarci-noma, vulvar cancer, and vaginal cancer (based on a demonstration of efficacy against HPV 16- and HPV 18-related cervical intraep-ithelial neoplasia [CIN] grade 3, cervical adenocarcinoma in situ [AIS], vulvar intraepithelial neoplasia [VIN] grades 2/3, and vaginal intraepithelial neoplasia [VaIN] grades 2/3, respectively) [12–15]. The vaccine was 98–100% effective in preventing HPV 6- and HPV 11-related genital warts and CIN. Because HPV infection is sexually transmitted, men and women remain at risk of infection as long as they are sexually active. Thus, tobemaximally effective, prophylactic HPV vaccines should induce long-lived protective efficacy (i.e., at least 10 years, preferably life¬long). In clinical trials, sustained protective efficacy was observed through at least 5 years following vaccination onset [16]. Ongoing studies are evaluating the longer-term effectiveness of the vaccine. Todate,an immune markerthat can identify vaccinatedsubjects who are protected from acquisition of infection with types targeted by the vaccine has not been identified. Suchamarker wouldbe use¬ful in defining the duration of vaccine-induced protective efficacy (and the timing of administration of a booster dose of vaccine, if needed). An immune marker would also simplify the bridging of protective efficacy of the quadrivalent HPV vaccine to new popu¬lations and to new formulations. Additionally, an immune marker would aid in the evaluation of follow-on multivalent vaccines. Preclinical studies have suggested that the protective efficacy of the quadrivalent HPV vaccine is mediated by anti-HPV L1 humoral responses [17–19]. Administration of L1 VLP vaccines tar¬geting animal papillomaviruses prevents infection and disease and is accompanied by induction of anti-L1 neutralizing antibodies. Transferofserum fromvaccinated animalstounvaccinatedanimals protected the unvaccinated animals from acquisition of infection and disease following a virus challenge. On the basis of these find¬ings, Phase II and Phase III clinical trials of the quadrivalent HPV vaccine in young–adult women have focused on measurement of serumanti-HPV L1responsesshortlyaftercompletionof the3-dose vaccination regimen and for up to 4.5 years thereafter. To define a candidate immune correlate of vaccine efficacy, an evaluation of the correlation between vaccine-induced serum anti-HPV responses and the vaccine’s protective efficacy was conducted. We evalu¬ated this correlation among 17,622 young adult women enrolled in efficacy studies of the quadrivalent HPV vaccine. 2. Materials and methods 2.1. Design of the phase III clinical trials Protocols 013 (NCT00092521) and 015 (NCT00092534) (termed FUTURE I and FUTURE II, respectively) were phase III, randomized, double-blind, placebo-controlled clinical trials designed to inves¬tigate the prophylactic efficacy of the quadrivalent (types 6, 11, 16, 18) HPV L1 VLP vaccine (GARDASILTM/SILGARDTM, Merck and Co., Inc., Whitehouse Station, NJ) on HPV 6/11/16/18-related CIN, AIS, or cervical cancer (protocol 013 co-primary endpoint); HPV 6/11/16/18-related condylomata acuminata, VIN, VaIN, vulvar can¬cer, or vaginal cancer (protocol 013 co-primary endpoint), and HPV 16/18-related CIN 2/3, AIS, or cervical cancer (protocol 015 primary endpoint) [12,15]. Between December 2001 and May 2003,17,622 15-26-year-old women were enrolled in the two trials (17,599 received at least 1 dose of vaccine or placebo). The trials enrolled women who reported 0-4 lifetime sexual partners at day 1. Enrolled subjects with clinical evidence of genital HPV disease at day 1 were discon¬tinued from the study prior to randomization. Subjects received intramuscular injections of quadrivalent HPV vaccine or visually indistinguishable placebo at enrollment (day 1), month 2, and month 6. Each protocol was approved by the institutional review boards (ethical review committees) at participating centers and informed consent was received from all subjects enrolled. The designs of protocols 013 and 015 are described elsewhere [15,20]. 2.2. Study vaccine The quadrivalent vaccine consisted of a mixture of four recom-binant HPV type-specific VLPs composed of full-length L1 major capsid proteins of HPV types 6, 11, 16 and 18 synthesized in Sac-charomyces cerevisiae [21-23]. The vaccine is comprised of 20 \±g of HPV 6 VLP, 40 (jig of HPV 11 VLP, 40 (jig of HPV 16 VLP and 20 (jig of HPV 18 VLP, formulated with 225 u,g of amorphous alu¬minum hydroxyphosphate sulfate adjuvant. The placebo contained the same adjuvant and was visually indistinguishable from vaccine. 2.3. Clinical follow-up and laboratory testing Examination for the presence of genital warts and vulvar and vaginal lesions was performed at enrolment (day 1), month 3 (pro¬tocol 013 only), and months 7,12, 24,36, and 48 (also at months 18 and 30 for protocol 013). ThinPrepTM (Cytyc, Boxborough MA, USA) cytology specimens for Pap testing were collected at enrollment (day 1), month 7, and at 6-12-month intervals thereafter. Cytology specimens were classified using The Bethesda System-2001 [24]. Procedures for algorithm-based cytology, colposcopy and biopsy referral have been described previously [12,15]. Biopsy material was first read for clinical management by pathologists at a cen¬tral laboratory (Diagnostic Cytology Laboratories, Indianapolis, IN), and then read for endpoint determination by a blinded panel of four pathologists as described previously. Blood samples were obtained at enrollment (day 1) for anti-HPV serology testing for HPV types 6, 11, 16, and 18 using competitive Luminex-basedimmunoassays(cLIA; developed by Merck Research Laboratories, West Point, PA, using technology from the Luminex Corporation, Austin, TX) [25]. Dilution-corrected serostatus cutoffs were 20 mMU/mL for HPV 6, 16 mMU/mL for HPV 11, 20 mMU/mL 6846 E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 for HPV 16, and 24mMU/mL for HPV 18 (epitopes H6.M48, K11.B2, H16.V5, and H18.J4; for HPV types 6, 11, 16, and 18, respectively). Ascertainment of HPV infection involved HPV PCR analysis performed on samples obtained at enrollment (day 1), month 3 (protocol 013 only), and month 7. For each subject, genital swab specimens were tested for the presence of HPV 6, 11, 16, and 18 DNA. For PCR analyses, swabs, biopsy samples and thin tissue sec¬tions cut adjacent to sections used for histopathological analysis were used to detect HPV DNA with primers specific for HPV 6, 11, 16, or 18. 2.4. Statistical analyses Analyses were conducted in a per-protocol population. Subjects included in this population received all three doses of vaccine or placebo within 1 year, were seronegative at day 1 and PCR negative from day 1 through month 7to relevantvaccineHPVtypes (subjects could be positive for one type and be counted in the per-protocol population of another vaccine HPV type if they were naпve to that type). Subjects did not deviate from the protocol; follow-up began 1 month post-dose 3 (month 7). Vaccine efficacy (defined as [1-relative risk]Ч100%) and the corresponding 95% confidence intervals were estimated using an exact procedure which accounted for the amount of follow-up (i.e., person-time at risk) in the vaccine and placebo groups. Subjects were pooled across the studies by vaccination group (vaccine or placebo) for analysis. Some efficacy endpoints in this report are composite endpoints, including more than one lesion type and/or more than one HPV type. If a subject met the criteria for one or more of the components of a composite endpoint, she was counted as a case for the composite endpoint once and only once. 3. Results Of the 17,622 women who were randomized in protocols 013 and 015, 17,599 were allocated and received either vaccine or placebo(8799wereallocatedtovaccine,and8800were allocatedto placebo) (Fig.1). Approximately, 97% of those subjects who received eithervaccineorplaceboreceivedallthreedosesand completedthe vaccination phase. At the time of this report 93% of vaccine recipi¬ents and 84% of placebo recipients had completed follow-up in the study in which they were enrolled. Data represent a mean follow-up time of 44 months (protocols 013 and 015 were closed early in order to vaccinate the placebo population). Quadrivalent HPV vaccine elicited a strong immunologic response against all vaccine HPV types in subjects who were included in the per-protocol immunogenicity population (Table 1). Geometric mean titers (GMT) reached a peak at month 7, and declinedthereafterasexpected.Over 99%ofsubjects seroconverted for vaccine HPV types by month 7. Variability was seen in the per¬centage of subjects who were seropositive for vaccine HPV types at end-of-study; a lower percentage of subjects were seropositive for HPV18 at end-of-study, when compared to other vaccine HPV types. At the end-of-study for protocols 013 and 015, efficacy of the quadrivalent HPV vaccine against any grade CIN or AIS related to HPV 6, 11, 16, or 18 in the per-protocol efficacy population was 96.0% (95% CI: 92.2–98.2) (Table 2). This efficacy is illustrated in a significant difference in the time to any grade CIN or AIS as seen in Fig. 2. Nine cases of any grade CIN were seen among subjects who received quadrivalent HPV vaccine versus 222 cases in those subjects receiving placebo. Vaccine efficacy against CIN 2 or worse related to HPV 6, 11, 16, or 18 in the per-protocol population was АЕ = adverse experience; PPE = per-protocol efficacy. Fig. 1. Study populations. E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 6847 Table 1 Anti-HPV 6, 11, 16 and 18 serologic responses by study time point among 16–26-year-old women who received quadrivalent HPV vaccine.a. Quadrivalent HPV vaccine (N=8787) n GMT (mMU/mL) Seroconversion GMT 95% CI n m Percent 95% CI Anti-HPV 6 Day 1 2632 <8 <8 to <8 2632 0 0.0 0.0-0.1 Month 7 2635 542.7 526.3-559.6 2635 2630 99.8 99.6-99.9 Month 24 2438 113.4 109.1-117.9 2438 2329 95.5 94.6-96.3 End-of-studyb 2375 74.6 71.6-77.7 2375 2131 89.7 88.4-90.9 Anti-HPV 11 Day 1 2656 <8 <8, <8 2656 0 0.0 0.0-0.1 Month 7 2659 761.6 735.4-788.6 2659 2652 99.7 99.5-99.9 Month 24 2469 144.5 138.9-150.3 2469 2414 97.8 97.1-98.3 End-of-studyb 2399 90.3 86.6-94.1 2399 2267 94.5 93.5-95.4 Anti-HPV 16 Day 1 2570 <12 <12 to <12 2570 0 0.0 0.0-0.1 Month 7 2573 2294.0 2185.2-2408.1 2573 2568 99.8 99.5-99.9 Month 24 2381 460.3 440.9-480.5 2381 2366 99.4 99.0-99.6 End-of-studyb 2330 334.6 319.4-350.5 2330 2293 98.4 97.8-98.9 Anti-HPV 18 Day 1 2796 <8 <8 to <8 2796 0 0.0 0.0-0.1 Month 7 2800 461.6 443.9-479.9 2800 2785 99.5 99.1-99.7 Month 24 2603 52.2 49.4-55.2 2603 1865 71.6 69.9-73.4 End-of-studyb 2536 33.8 32.0-35.7 2536 1529 60.3 58.4-62.2 N = number of subjects that were randomized to the respective vaccination group who received at least 1 injection and had non-missing data; n= number of evaluable subjects with serology data at relevant time point; m= number of seropositive subjects (subject is defined as seropositive to HPV 6, 11, 16 or 18 if her corresponding anti-HPV cLIA was ≥20mMU/mL, 16mMU/mL, 20mMU/mL, or 24 mMU/mL, respectively); CI=confidence interval; GMT =geometric mean titer; mMU =milli Merck units. a The per-protocol immunogenicity population includes all subjects who were not general protocol violators, received all three vaccinations within acceptable day ranges, were seronegative at day 1 and PCR negative day 1 through month 7 for the relevant HPV type(s), and had a month 7 serum sample collected within an acceptable day range. b End-of-study visits were generally scheduled earlier than month 48. This time point includes all visits occurring within 6 months of the approximate mean interval of 44 months. 98.2% (95% CI: 93.2–99.8). Two cases of CIN 3 were seen in sub¬jects receiving quadrivalent HPV vaccine versus 63 in those subjects receiving placebo. Efficacy against any grade CIN or AIS related to HPV 16 and HPV 18 was 94.3% (95% CI: 88.4–97.6) and 98.4% (95% CI: 90.5–100.0), respectively. Of the nine cases of HPV 6, 11, 16, or 18-related any grade CIN or AIS in subjects receiving vaccine, eight were related to HPV 16 (six cases of CIN 1 and two cases of CIN 3), and one was related to HPV 18 (CIN 1) (Table 3). Many subjects were positive for non-vaccine HPV types at enrollment into the studies. Sub¬jects diagnosed with CIN 1 or worse had vaccine-type HPV DNA found in biopsy/biopsies taken after cytologic abnormalities were seen on Pap test. However, if definitive therapy was performed (after at least a diagnosis of ASC-US with high-risk HPV positiv-ity), the specimens collected did not always contain vaccine type HPV DNA. Month 7 serum antibody titers against the vaccine HPV type detected in the index lesion were available for three subjects who became casesofCIN1orworse (cases 7–9; all HPV 16-related). There was variability seen in the immune response to HPV 16 fol¬lowing vaccination in each of these three subjects; antibody titers against the offending HPV type were 205 mMU/mL, 2011mMU/mL, and 7415mMU/mL. Table 2 Efficacy against HPV 6/11/16/18-related CIN (any grade) or AIS at end-of-study, by severity. Per-protocol efficacy population of protocols 013 and 015 at end-of-study. Quadrivalent HPV vaccine (N=8799) Placebo (N=8800) Observed efficacy (%) 95% CI n Cases Rate n Cases Rate 222 9 96.0 CIN (any grade) or AIS By lesion severity CIN 1 CIN 2 or worse CIN 2 CIN 3 or worse CIN 3 AIS By HPV type HPV 6 HPV 11 HPV 16 HPV 18 7629 7629 7 7629 2 7629 0 7629 2 7629 2 7629 0 6688 0 6688 0 6448 8 7158 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 7632 7632 168 0.8 95.9 7632 109 0.5 98.2 7632 70 0.3 100.0 7632 66 0.3 97.0 7632 63 0.3 96.8 7632 7 0.0 100.0 6619 46 0.2 100.0 6619 12 0.1 100.0 6257 135 0.7 94.3 7092 60 0.3 98.4 92.2-98.2 91.3-98.4 93.2-99.8 94.6-100.0 88.7-99.6 88.1-99.6 30.9-100.0 91.8-100.0 64.5-100.0 88.4-97.6 90.5-100.0 Subjects are counted once in each applicable endpoint category; a subject may appear in more than one category. N = number of subjects randomized to the respective vaccination group who received at least 1 injection; n= number of subjects evaluable, i.e., number of subjects in the given population who also have at least one follow-up visit; CI =confidence interval; CIN =cervical intraepithelial neoplasia; AIS =adenocarcinoma in situ; rate= incidence rate per 100 person years at risk. 6848 E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 Fig. 2. Time to HPV 6 (A), HPV 11 (B), HPV 16 (C), HPV 18 (D) and HPV 6/11/16/18-related (E) CIN (any grade) or AIS in the per-protocol efficacy population of protocol 013 and protocol 015 at end-of-study. *Case counting started at month 7. Vaccine lines along the horizontal axis are not visible. 4. Discussion The hallmark of prophylactic vaccine efficacy is disease preven¬tion. Accordingly, the WHO’s recommended clinical endpoints for determining the efficacy of prophylactic HPV vaccines are disease endpoints [26]. However, the significanceofvaccine immunogenic-ity becomes more apparent after efficacy against clinical disease endpoints has been established. Analyzing the immune response to E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 6849 Table 3 Description of cases of HPV 6, 11, 16, or 18-related CIN observed among subjects who received quadrivalent HPV vaccine. Casea Age Time of detection (month) HPV type Histology Narrative 17 22 22 23 18 2 3 4 5 6 7 8 9 23 50.37 13.11 36.86 46.19 32.00 45.44 22 14.52 23 12.88 20 12.42 16 16 18 16 16 16 16 16 16 CIN3 CIN 1 CIN 1 CIN 1 CIN3 CIN 1 CIN 1 CIN 1 CIN 1 PCR positive for HPV 51, and negative for other tested types at enrollment. Colposcopy and a single biopsy at month 49 revealed CIN 3, which was positive for HPV 16 only. Definitive therapy was performed at month 52, and revealed a negative pathology panel diagnosis, which was positive for HPV 56 and negative for HPV 16 and all other tested types. Negative to all 4 vaccine HPV types and 10 other high-risk HPV types at enrollment. At month 13, the subject underwent colposcopy and two biopsies revealed CIN 1 positive for HPV 58 DNA. One biopsy was positive for HPV 16. PCR positive for HPV 56 and negative for HPV 18 at enrollment. Biopsy at month 35 revealed CIN 1 (by Path panel; central lab diagnosis was CIN 2) which was HPV 56 positive and HPV 18 negative. Definitive therapy was performed at month 37. Therapy biopsy and 2 of 4 definitive therapy specimens were given a diagnosis of CIN 1, all were positive for HPV 56. One specimen was also positive for HPV 18. PCR positive for HPV 56 at enrollment, and negative for vaccine types. Colposcopy and three cervical biopsies at month 19 all revealed CIN 1. All biopsies were negative for HPV16 and 56, as well as all other tested HPV types. Colposcopy and two cervical biopsies were performed at month 46, and revealed HPV 16 only in one biopsy specimen, which was positive for CIN 1. Both biopsies were negative for HPV 56. PCR positive for HPV 52, and negative for vaccine HPV types at enrollment. Colposcopic biopsy at month 32.5 read as CIN 3. The specimen was positive for both HPV 52 and HPV 16. All specimens collected at definitive therapy were HPV 52 positive and HPV 16 negative. PCR negative at enrollment for all tested HPV types. Colposcopy and cervical biopsies were performed at month 42, and demonstrated CIN 1, which was positive for HPV types 16, 35, 52, and 58. Definitive therapy was performed, and the specimen was positive for CIN 1 and CIN 2, which tested positive for HPV types 35, 52, and 58, but not HPV 16. A vaginal lesion was also identified and biopsied, and demonstrated VaIN1, which was also PCR positive for HPV types 35, 52, and 58, but negative for type 16. PCR positive at enrollment to HPV 18, HPV 31, HPV 33, and HPV 39, but not to HPV 16. Colposcopic biopsy at month 11 was read as CIN 3. HPV 18, HPV 31, HPV 33, and HPV 39 (but not HPV 16) were detected in the lesion. Definitive therapy conducted at month 15 to treat this CIN 3 lesion generated seven specimens. Of these, two were negative for HPV (they were also read as being normal). A total of five of the seven specimens were positive for at least one of the four types for which the subject was positive at day 1. Only one of seven biopsies was positive for HPV 16. This biopsy was also positive for HPV 18, HPV 33, and HPV 39, and was read as CIN 1. At month 7, the subjects’ anti-HPV 16 titer was 2011 mMU/mL, similar to the average HPV 16 GMT in subjects that received vaccine. PCR positive for HPV 39 at enrollment. Colposcopy performed 28 days following the month 12 visit yielded two biopsies, one of which was given a diagnosis of CIN 1. The biopsy that was positive for CIN 1 was also positive for HPV 16 and negative to the remaining 13 tested HPV types. The second biopsy was negative both by pathology panel review and HPV testing. At month 7, the subject’s anti-HPV 16 titer was 7415mMU/mL, significantly higher than the average HPV 16 GMT in subjects that received vaccine. PCR negative for all 14 tested HPV types. Colposcopy at month 13 yielded two biopsies. One of them was read as CIN 1 and was HPV 16 DNA positive. At month 7, this subject had an anti-HPV 16 level of 205mMU/mL, lower than the average HPV 16 GMT in subjects that received vaccine. PCR=polymerase chain reaction; CIN=cervical intraepithelial neoplasia; GMT=geometric mean titer. a Month 7 GMT’s for the offending HPV type were not available for cases 1–6. vaccination with the quadrivalent HPV vaccine was useful in bridg¬ing the response from girls and women 16–23 years oldtothose too young to be ethically followed for disease endpoints [27]. However, no immune correlate of protection for prevention of HPV-related disease has been identified to date in any HPV vaccine or natural history study. Immunological correlates of protection from HPV-related disease could be valuable in defining the antibody levels needed for protection from vaccine HPV types, as well as the length of protection, and whether a booster dose is needed. Taking into account the trials reported herein, and although data are not available for all subjects, we found little correlation between month 7 antibody levels and the chances of becoming a case of vaccine HPV type-related disease. This is in part due to few cases of vaccine HPV type-related disease among vaccinated sub¬jects, which are needed to accurately determine an immunological correlate of protection. In addition, not all month 7 antibody titers were available for subjects that did become a case of vaccine-type related disease. However, the data presented show no evidence of vaccine HPV type-related breakthrough disease duetowaning anti¬body levels or seropositivity (particularly important in the case of HPV 18 immunogenicity). A robust neutralizing antibody response to all four vaccine HPV types was seen at month 7; ≥99% of previously naпve subjects seroconverted for all four HPV types. Twenty-four months after vac¬cination, measurable neutralizing antibodies against HPV 6, 11, and 16 were observed in more than 95% of vaccinated subjects. In con¬trast, neutralizing antibodies against the H18J4 epitope of HPV 18 weremeasurablein 71.6% ofvaccinatedsubjects at month 24, and in 60% of subjects by end-of-study (average follow-up of 44 months). Whereas seropositivity percentages seem to be lower for HPV 18 at month 24 and at end-of-study than for the other three vaccine HPV types, analysis of the cases of HPV 6/11/16/18-related disease among vaccinated subjects during the trials provides important insight. Only one of these cases of CIN was related to HPV 18, and this case was confounded by the presence of HPV 56. While HPV 18 neutralizing antibody levels are observed to be diminishing, it is clear that these lower levels are not leading to breakthrough dis¬ease. This suggests that even in the HPV 18 seronegative subjects (39.7% of all subjects at end-of-study), vaccine efficacy remains near 100% through 44 months, possibly due to immune memory. We have measured and presented data concerning vaccine HPV type-specific neutralizing antibody titers; those which are able to 6850 E.A. Joura et al. / Vaccine 26 (2008) 6844–6851 neutralize HPV 6, 11, 16, and 18 inhibit infection of basal epithe¬lial cells [28,29]. While vaccination with the quadrivalent HPV vaccine invariably results in the production of a plethora of HPV-specific antibodies, not all of these antibodies are capable of virus neutralization. The measurement of all HPV-specific antibodies produced in response to vaccination (both neutralizing and non-neutralizing) while a surrogate of total (potentially non-specific) antibody response, is therefore less informative about the direct inhibitory effect of vaccination on vaccine HPV types. However, the interpretation of specific HPV-related neutralizing antibody titers must be viewed against the background of the cLIA assay. While post-vaccination sera are analyzed for antibodies directed against the most dominant known neutralizing epitopes (H6.M48, K11.B2, H16.V5, and H18.J4; for HPV types 6,11,16, and 18, respectively), it is possible that antibodies specific for other neutralizing epitopes exist that will not be accounted for by the current cLIA assay. This could lead to an underestimation of neutralizing antibody titers directed against vaccine HPV types. Efficacy against HPV 18-related disease is of crucial importance. HPV 18 not only causes some 20% of cervical cancer and HPV-related vulvar cancer, it is also the predominant HPV type leading to adenocarcinoma of the cervix, a disease with increasing incidence and an aggressive course, especially in younger women. Cytologic screening often fails to detect its precursors due to atypical cellu¬lar patterns and being in the endocervical canal and out of range of colposcopy. The mechanism for protection among subjects who become nominally anti-HPV 18 seronegative several years after vac¬cination remains to be determined. Olsson et al. have demonstrated that subjects vaccinated with the quadrivalent HPV vaccine are able to generate an anamnestic response upon further exposure to HPV vaccine (HPV VLPs), even among subjects who had become HPV seronegative before antigen challenge [30]. Support is lent by data demonstrating heightened antibody responses among women receiving quadrivalent HPV vaccine who were seropositive for vac¬cine HPV types at enrollment, as recently published by Giuliano et al. [31]. Immune memory is likely central to the efficacy against HPV 18-related disease among recipients of the quadrivalent HPV vaccine, despite antibody titers which decline after vaccination. Protection through anamnestic responses has also been observed in the case of Hepatitis B virus (HBV) vaccine. Like HPV, HBV is a sexually transmitted infection (as well as a blood-borne virus in contradistinction to HPV) that can persist and may lead to hep-atocellular cancer. Among recipients of the HBV vaccine, efficacy persists in the absence of detectable antibody titers, indicating the importance of immune memory. Moreover, on exposure to nat¬ural virus (e.g. needlestick accidents) an anamnestic response is seen, with no disease breakthrough. Data suggest that the quadri¬valent HPV vaccine is able to largely prevent disease related to vaccine HPV types (e.g. nascent infection) through the generation of a timely anamnestic response after exposure to a fourth vaccine dose. Whether natural infection in the genital tract or other mucosal surfaces result in an anamnestic response is yet to be demonstrated. This supposition is plausible given that papillomaviruses will attach but not penetrate host cells for a significant period of time, and that virus bound in this way appears susceptible to inactivation by neutralizing antibodies [32]. In summary, we have shown continued immunogenicity and efficacy for vaccine HPV types 44 months after vaccination. While no immune correlate of protection from HPV 6/11/16/18-related disease can yet be determined, it is unlikely that waning of antibody titers is responsible for disease breakthrough in subjr who have received quadrivalent HPV vaccine. Moreover, it is pro^rie that the anamnestic response plays a key role in protection from infection and disease in those subjects who have low or no detectable HPV 6/11/16/18 antibody levels years after primary vaccination. Acknowledgements The authors wishtothank MargaretJames,Carolyn Maass, Kath¬leen McCarroll, Kathy Harkins, and MaryAnne Rutkowski for help with statistical programming and analysis. Conflict of interest information: NM has received lecture fees, advisory board fees, and consultancy fees from Merck and Sanofi Pasteur MSD. SEO has received lecture fees from Merck. MHA has received lecture fees and grant support from Merck. OEI has received lecture fees from Merck and GlaxoSmithKline. CMW has received funding through her institution to conduct HPV vac¬cine studies for GlaxoSmithKline. KA has received consultancy and advisory board fees. XB has received lecture fees from Merck and GlaxoSmithKline, and has received funding through his insti¬tution to conduct HPV vaccine studies GlaxoSmithKline. JP has received consultancy fees, advisory board fees, and lecture fees from Merck. JD has received consultancy fees, lecture fees, and research grants from Merck andSanofiPasteurMSD. SL has received lecture fees from Merck and Sanofi Pasteur MSD. EJ has received lecture fees from Merck, Sanofi Pasteur MSD and GlaxoSmithK-line. SKK has received consultancy fees, and has received funding through her institution to conduct HPV vaccine studies for Sanofi Pasteur MSD and Digene. SMG has received advisory board fees and grant support from Commonwealth Serum Laboratories (CSL) and GlaxoSmithKline,lecture feesfromMerck, andfunding through her institution to conduct HPV vaccine studies for GSK. DGF has received consultancy fees and funding through his institution to conduct HPV vaccine studies for GlaxoSmithKline, and lecture fees and consultancy fees from Merck. KS has received consultancy fees from Merck. SM has received lecture fees and advisory board fees from Merck. GP has received lecture fees and consultancy fees from Merck and Sanofi Pasteur MSD. DRB has received lecture fees, advi¬sory board fees, and intellectual property fees. MS has received lecture fees and grant support from Merck. Additionally,SEO, CMW, MHA, OEI, GWKT, XB, JP, JD, EHT, SL, EJ, SKK, GP, SMG, DGF, KS, MS, LK,andDRBhavereceivedfundingthroughtheirinstitutionstocon-duct HPV vaccine studies for Merck. FJT, CR, AT, JB, LCL, KEDG, SV, SL, TMH, RH, and EB are employees ofMerck and potentially own stock and/or stock options in the company. 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