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A Phase 1 Study of a Recombinant Viruslike Particle Vaccine against Human Papillomavirus Type 11 in Healthy Adult Volunteers

  1. Thomas G. Evans1,a,
  2. William Bonnez1,
  3. Robert C. Rose1,2,
  4. Scott Koenig3,
  5. Lisa Demeter1,2,
  6. JoAnn A. Suzich3,
  7. Diane O’Brien1,
  8. Meredith Campbell1,
  9. Wendy I. White3,
  10. James Balsley3 and
  11. Richard C. Reichman1,2
  1. 1Infectious Diseases Unit, Department of Medicine, and
  2. 2Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York;
  3. 3MedImmune, Gaithersburg, Maryland
  1. Reprints or correspondence: Dr. Richard C. Reichman, Box 689, 601 Elmwood Ave., Rochester, NY 14642 (richard_reichman{at}urmc.rochester.edu)
 
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Abstract

Viruslike particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)–11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV-11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen–specific interferon–γ and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types

Human papillomaviruses (HPVs) infect epithelial tissues of skin and mucous membranes. More than 30 different HPV types infect the genital tract, producing anogenital warts and a variety of benign and malignant neoplasms, including cervical cancer [1, 2]. Treatment of these extraordinarily common sexually transmitted diseases (STDs) consists primarily of ablation or removal of infected tissues, although antiviral and immunomodulatory drugs have also been shown to have some therapeutic benefit [3, 4]. All modes of therapy are associated with significant rates of failure and relapse, and some have significant toxicity. Prevention of HPV infections consists primarily of avoiding contact with infectious lesions, although barrier methods of contraception may offer some protection [5, 6]. Widespread infection and serious disease, as well as inadequate modes of therapy and prevention, have led to interest in the development of HPV vaccines

Because HPVs are exquisitely species-specific and have not been propagated in tissue culture, significant amounts of well-characterized HPV antigens have not been widely available. Few immunologic studies have been performed with uniform antigen preparations, and immune correlates of disease resolution and prevention from infection have not been established. However, in recent years, several groups have recognized that viruslike particles (VLPs) closely approximate the antigenic characteristics of wild-type virions [7 –15]. HPV VLPs derived from expression in eucaryotic cells of the major capsid protein can be used to detect antibodies in the serum of infected patients and to induce the production of neutralizing antibodies in laboratory animals. In addition, studies conducted in animal models have demonstrated that VLP vaccines can induce an effective immune response. Studies in the cow, rabbit, and dog all have shown that vaccination with homologous VLPs can protect from live virus challenge [16 –18]

In the present study, we administered an HPV type 11 (HPV-11) VLP candidate vaccine to 65 healthy adults without serologic evidence of previous HPV-11 infection. The purpose of the study was to evaluate the tolerability, safety, and antigenicity of the vaccine. Antigenicity was assessed by measuring binding and neutralizing antibodies and by determining proliferative and cytokine responses of PBMC after in vitro stimulation, using L1 VLPs from the homologous HPV type (HPV-11) and from 3 heterologous types (HPV-6, -16, and -18)

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Subjects and Methods

Study Subjects

All subjects were enrolled at the University of Rochester Vaccine Center. Subjects were adults between the ages of 18 and 45 years and were healthy, as determined by medical history; physical examination, including genital examination and pelvic examination for women; electrocardiogram; and routine laboratory tests. Laboratory criteria for eligibility included hemoglobin level >11 g/dL, white blood cell count >4000 cells/mm3, platelet count >120,000 cells/mm3, normal urinalysis findings, and aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin, calcium, phosphorus, total protein, albumin, serum electrolytes, glucose, blood urea nitrogen, and creatinine values within the normal range

Criteria for exclusion included abnormal serum immunoglobulin G, A, or M level; positive urine pregnancy test or abnormal Pap smear for women; any acute or chronic condition limiting the volunteer’s ability to complete the study; a history or clinical manifestation of significant metabolic, pulmonary, cardiovascular, hematologic, gastrointestinal, dermatologic, or genitourinary disease; a history of cancer or chronic hepatitis; a history of alcohol or other drug abuse within 2 years of enrollment; current use of immunosuppressive medications or a history of immunodeficiency; receipt of immunoglobulin or blood products within 4 months of study entry; anogenital warts within the previous year; serum antibodies to hepatitis C, hepatitis B surface antigen, or human immunodeficiency virus (HIV) type 1; and positive urine test results for drugs of abuse or a positive blood alcohol level. Before enrollment, all subjects had negative HPV-11 ELISA tests, as described below

Study Design

This was a blinded, randomized, placebo-controlled dose escalation study. The VLPs in the vaccine preparation were formed by self-assembly of HPV L1 capsid protein produced by baculovirus vectors in Tricholusia ni [19 –21]. The HPV-11 L1 VLP vaccine is referred to as “MEDI-501” (MedImmune). Either MEDI-501 formulated in aluminum hydroxide or an alum adjuvant control containing no protein was administered to ∼12 vaccine recipients and 4 placebo recipients in each of the 4 dosing groups (2.8 μg, 9.4 μg, 31.3 μg, and 104.3 μg, referred to hereafter as the 3, 9, 30, and 100 μg doses). Sequential eligible volunteers were randomized within each treatment group by a computer-generated scheme to receive either vaccine or adjuvant control, in a 3:1 ratio. Immunizations were administered intramuscularly in the deltoid at 0, 4, and 16 weeks. Study subjects were seen and evaluated before and 3, 7, 14, and 28 days after each injection and at weeks 12, 24, and 48 after initial vaccination. Evaluations consisted of a medical history, physical examination, and performance of routine laboratory tests, as outlined above

Serologic and Virologic Studies

ELISA tests HPV-11 L1 VLPs were diluted in PBS and were bound to 96-well polystyrene plates by incubation at 37°C for 1 h. Plates were washed 4 times with PBS containing 5% nonfat dry milk. After a 1-h incubation at room temperature, the plates were washed 4 times with PBS-Tween. Human serum samples were diluted in PBS containing 1% nonfat dry milk and were added to the plates for a 2-h incubation at room temperature. After further washing, VLP-specific antibodies were identified, using peroxidase-labeled anti–human IgG. After a 1-h incubation at room temperature, the plates were washed, and peroxidase substrate was added to the wells. Optical density readings were taken 30 min after the addition of substrate

Neutralization testsAn in vitro reverse-transcriptase–polymerase chain reaction (RT-PCR)–based assay, originally described by Smith et al. [22], was used to detect neutralizing antibodies against HPV-11. HaCaT cells, an aneuploid human keratinocyte cell line [23], were grown to 85%–100% confluency in 24 well plates. HPV-11 particles obtained from xenograft propagation were preincubated with dilutions of serum samples at 35° C for 75 min and were added to the HaCaT cells [24]. Six days after infection, total RNA was harvested from the cells, and 10% of the total RNA was used for cDNA synthesis, using an oligo d-T primer. Ten percent of the cDNA then was used as a template for nested RT-PCR, using primers specific for the E1^E4 spliced message. RT-PCR products were separated on an agarose gel and were visualized by ethidium bromide staining. Absence of the E1^E4 band indicated that viral neutralization had occurred. Neutralization titer was defined as the highest serum dilution that inhibited the synthesis of the E1^E4 spliced message. As a control for RNA isolation, amplification of β-actin–specific DNA was performed on all cDNA samples found to be E1^E4 negative. To control for specificity and virus infection, amplification with E1^E4-specific primers was carried out with cDNA prepared from uninfected HaCaT cells, cells infected with HPV-11 that had not been preincubated with serum, and cells infected with HPV-11 that had been preincubated with a seronegative human control serum

Lymphoproliferative assays (LPAs)L1 VLPs corresponding to HPV-6, -11, -16, and -18 and to parvovirus B19 VP2 were produced at MedImmune, using the baculovirus expression system. Purification was accomplished by cesium chloride and sucrose gradient centrifugation and by phenyl hydrophobic interaction chromatography. The final bulk product was dialyzed and sterilized by filtration. Phytohemagglutinin was purchased from Sigma, and tetanus toxoid from Connaught Laboratories. LPAs were conducted by using PBMC that were drawn on days 0, 42 (2 weeks after the second vaccination), and 126 (4 weeks after the third and final vaccination). Separation, cryopreservation, and thawing of PBMC followed consensus techniques used in our laboratories that have been established by the National Institutes of Health AIDS Vaccine Evaluation Group and the AIDS Clinical Trials Group [25]. All assays were conducted after all PBMC were collected and were performed without knowledge of treatment assignment (vaccine or placebo). In brief, 105 PBMC per well were incubated in round-bottom plates for 6 days with medium or antigen in quadruplicate, in RPMI 10% heat-inactivated autologous serum (drawn before vaccination). For the 2 lower vaccine doses (3 and 9 μg, also referred to as the low-dose group), the HPV antigens were used in vitro at final concentrations of 2 and 10 μg/mL, respectively. For the 2 higher vaccine dose groups (30 and 100 μg, also referred to as the high-dose group), the HPV antigens were limited in supply and were used in vitro at 1 and 5 μg/mL, respectively. The concentrations of other antigens used were 5 lytic-forming units/mL for tetanus and 5 μg/mL for the B19 baculovirus control. The B19 control was available in sufficient quantity only to perform assays on the first 3 vaccine dose groups. On the sixth day of incubation, 50 μL of media was removed from each well, 50 μL of tritiated thymidine (1 μCu) was added, and the plates were harvested and were read the following morning on a Packard Instrument Top-Count. The stimulation index (SI) was calculated as the mean antigen stimulation/mean medium stimulation

Cytokine assayThe 50 μL samples from the 4 replicate wells for each condition, which were removed on the sixth day, were pooled and were stored at −70°C until use. At that time, the interleukin (IL)–5 and interferon (INF)–γ concentrations of the samples were determined by ELISA, using antibody pairs obtained from Pharmingen, as described elsewhere [26]. Assays were performed in duplicate. The lower limit of sensitivity for each assay was 50 pg/mL

Statistical Methods

This phase 1 trial was designed to obtain safety and immunogenicity information and not to test for efficacy differences among treatment groups. Summary and descriptive statistics and graphs were used to describe results across treatment groups. All volunteers who received study vaccine were included in analyses of safety. Adverse events that occurred or worsened within 8 weeks after the last scheduled administration of study medication were assessed blindly for severity and relationship to study drug. Volunteers were considered to be evaluable for immunogenicity if they received an immunization and were followed up for ⩾3 weeks. Neutralizing antibody studies were performed only for volunteers who received all 3 injections of study medication

In the LPA analysis, assays in which none of the control antigens or mitogens revealed an SI >3.0 were considered to have nonviable T cells and were removed from the analysis before unblinding. The assay results for the VLP recipients are presented in tables and figures as the overall responses in each vaccine dose group. However, for our formal statistical analysis, we retained only the data from those patients who had observations at all time points. Because of missing observations and the use of smaller amounts of VLPs in some assays with the 30 and 100 μg dosing groups, the formal statistical analysis was confined to the lower-dose groups (3 and 9 μg) and their matching control groups. Given the reduced data set, we sought to determine simply whether there were differences in lymphoproliferative response, or in the production of INF-γ or IL-5, before and after immunization and between the placebo and MEDI-501 recipients, regardless of dose. The data were analyzed by a 2-way analysis of variance (ANOVA), with vaccination (MEDI-501 vs. placebo) treated as a between-groups variable and time of PBMC collection (before vs. after immunization) treated as a within-subjects variable. A statistically significant interaction between these 2 variables meant that a response after vaccination noted only in the MEDI-501 recipients could be attributed to the vaccine. Two-sided P values ⩽.05 were considered to be statistically significant

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Results

Characteristics of Study Participants

A total of 65 volunteers were enrolled. The mean age of study participants was ∼30 years, with a range of 19–45 years. Twenty-four volunteers (37%) were men, and 41 (63%) were women. Sixty (92%) were white, 2 were African American, 1 was Latino, and 2 were members of other racial groups. In general, study subjects were at low risk for acquiring STDs. Fifty-three (82%) had one or no sex partner during the year before enrollment; 10 had 2 partners in the past 12 months, and 1 each had 3 and 5 partners during that period. Few of the participants had histories of previous STDs. A history of chlamydial infection was obtained from 8 patients, 3 had histories of genital herpes, and 1 each had had gonorrhea and anogenital warts

Sixteen subjects were enrolled in each of groups A, B, C, and D. An additional subject was enrolled in group D when a volunteer who was randomized to the vaccine arm became permanently unavailable for follow-up after moving out of the country after receipt of only the initial vaccination. Three subjects in group A (2 in the vaccine arm and 1 in the adjuvant control arm) received only 2 doses. One vaccine recipient was noncompliant, and another developed hives after the second injection, which were believed by the clinical staff to be possibly related to study medication. The study team decided not to administer a second booster injection to that individual. The adjuvant control recipient became pregnant and was not given the third injection. Two subjects in the highest-dose group (both in the vaccine arm) received only 2 injections. One volunteer was noncompliant, and the second moved out of town and refused further study participation. Overall, 59 (91%) of the 65 study participants received all 3 scheduled doses of study medication

Clinical and Laboratory Toxicity

Study medications were well tolerated. No changes in vital signs were associated with vaccine administration. Pain within 4 days after injection was the most frequently observed adverse event and occured after 72% (136/188) of immunizations (after 73% of active vaccine injections and after 70% of adjuvant control injections). Only 1 volunteer, an adjuvant control recipient, had an episode of severe pain. This episode occurred after the primary immunization, resolved within 72 h, and did not recur after the 2 booster vaccinations. Frequencies of other injection site adverse events (bruising, induration, inflammation, and edema) did not vary significantly among the treatment groups

Headache was reported more frequently within 4 days after an injection of vaccine than after an injection of adjuvant control (31% vs. 4%). Frequency of headaches did not vary among vaccine groups, and headaches were always of mild or moderate severity. Six volunteers reported rash and/or pruritis that was judged by the clinical investigator to be related to study vaccine. Five received MEDI-50l, and one received adjuvant control. In 4 of these volunteers, rashes or pruritis developed within 4 days of the study vaccine injection. In all but 1 volunteer, these manifestations were mild to moderate in severity and did not recur with subsequent injections. As described above, 1 volunteer developed hives and did not receive further injections. No significant changes in any of the treatment groups were observed in complete blood cell counts, serum chemistry studies, or urinalyses

Immunologic Studies

SerologyAdministration of each of the 4 doses of MEDI-501 vaccine produced dramatic increases in serum antibodies, as measured by ELISA (figure 1). Geometric mean titers (GMTs) 4 weeks after the initial vaccination ranged from a low of 34 in the 3 μg arm to 449 in the 100 μg arm. Four weeks after the first dose, GMT measurements ranged from 1324 to 2016. An additional increase was noted after the second booster, which produced GMT values 4 weeks later of 2786–6400. Thus, a clear dose response curve was generated, although differences among values measured in the 9, 30, and 100 μg groups were not readily apparent after 1 week following the first booster. ELISA titers decreased by about 0.5 log10 between weeks 20 and 48 of study

Figure 1
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Figure 1

Seroreactivities toward human papillomavirus (HPV)–11 L1 viruslike particle (VLP) vaccine of vaccinated subjects over time and according to vaccine dose. The placebo recipients tested negative before and after immunization (not shown)

Serum HPV-11 neutralizing antibody titers were measured in all patients before vaccination and among patients receiving all 3 injections at weeks 20 and 48 (table 1). No subject had serum neutralizing activity before vaccination, and GMT values at week 20 were similar among the 9, 30, and 100 μg groups. Among subjects who received all 3 injections, 7 of 10 subjects in the 3 μg arm had titers >1000, whereas 10 of 10 subjects in the 100 μg arm had neutralizing antibody titers of that magnitude. As was noted for antibodies measured by ELISA, neutralization antibody titers decreased by about 0.5log10 by week 48 (data not shown)

LymphoproliferationThe overall data for these LPA studies are summarized in table 2. The placebo patients from each group (n=4) are summed, to make a total of 16 control recipients. The responses of the others are shown by the vaccine dose group for days 0, 42, and 126. Proliferative responses to HPV antigens were found to be independent of the VLP vaccine dose. This is graphically illustrated by the response to high-dose in vitro HPV-11 antigen stimulation (figure 2). However, proliferation to the tetanus control was lower in the 100 μg MEDI-501 group, suggesting that the cells for the assays may not have been equivalently viable and responsive

Figure 2
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Figure 2

Stimulation indices (mean±SEM) for volunteers studied from each vaccine dose group after in vitro stimulation with human papillomavirus (HPV)–11 viruslike particle (VLP) vaccine. The 3 μg (n=7) and 9 μg (n=11) dose groups were stimulated with 10 μg/mL of VLP vaccine in vitro, and the higher-dose groups (n=12 for 30 μg and n=13 for 100 μg) were stimulated with 5 μg/mL of VLP vaccine in vitro. The placebo recipients from each group (4 per group) were stimulated with similar amounts of antigen

Due to the dose independence, data from all MEDI-501 recipients were combined to compare with those from placebo recipients (figure 3). There was a substantial increase due to vaccination after all HPV antigen stimulations, when results in the placebo group are compared with results in the vaccine group. No SI increases were observed in response to any antigen in the placebo group. The statistical analysis done on the matched data sets showed no statistically significant increase between baseline and the later time points in proliferation to either tetanus (P=.49) or parvovirus (P=.26). In contrast, such an interaction was present for the high-dose HPV-11 antigen (P=.04). A trend was also present for the 10 μg HPV-6 antigen stimulation (P=.09), which shares 93% amino acid homology in the L1 gene, and for the HPV-16 high-dose stimulation (P=.06), which shares 68% homology

Figure 3
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Figure 3

Stimulation indices (mean±SEM) for each human papillomavirus antigen stimulation are shown over time with placebo volunteer values pooled and vaccinees from all 4 dosing groups similarly pooled. Results are shown for the higher dose of the preparation for each antigen used for in vitro stimulations

Many individuals had baseline responses >3.0 (table 2). Whether these were due to prior exposure to HPV, to a cross-reacting antigen in the VLP preparation, or to proliferation due to baculovirus contamination was not ascertained. Similar background responses to baculovirus preparations have been seen in trials in which baculovirus-derived antigens were used for proliferation studies (data not shown)

Table 1
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Table 1

Neutralizing antibody titers observed after human papillomavirus immunization

Table 2
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Table 2

Mean lymphoproliferation stimulation indices (SIs) for each vaccine and dose group

When it became clear that T cell responses had developed not only to the VLP-11 antigen, but also possibly to VLPs of other HPV types (HPV-6 and -16), a decision was made to test more purified antigen preparations, to decrease the likelihood that the responses were due to baculovirus contamination. A new set of L1 VLP antigens for LPA was purified by using chromatography and filtration after isolation of VLPs by cesium and sucrose gradients. A comparison of proliferation between the original and more purified VLP preparations was performed, using cells from 4 volunteers in the 30 μg dose group, and showed no difference between the original and more purified antigens (data not shown)

Cytokine analysisINF-γ and IL-5, which were secreted during the lymphoproliferation cultures, were measured in supernatants obtained after 144 h of in vitro stimulation. The mean cytokine levels for each of the groups are presented in table 3. Once again, the data have been combined across all vaccine dosing groups, as no statistical difference was observed for any 1 group. There were substantial increases in cytokine production in response to most of the HPV antigen stimulations. By ANOVA, these increases in INF-γ production over time in the vaccine recipients were significant in comparison with the response of placebo recipients for the HPV-11 high-dose in vitro stimulation (P=.006), with a trend toward significance for high-dose HPV-16 (P=.08). No significant interaction effect was observed with the parvovirus B19 (P=.50) or tetanus antigen (P=.17) stimulation in the same volunteers

Table 3
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Table 3

Cytokine values in 6-day culture supernatants

The analysis of the IL-5 data also revealed HPV-specific stimulation induced by vaccination for this Th2-phenotypic cytokine (table 2). Again, no significant increase was seen in MEDI-501 recipients, compared with the placebo control recipients, when their PBMC were stimulated with either tetanus (P=.31) or parvovirus B19 VLPs (P=.55). However, increases were seen in response to the high-dose HPV-11 in vitro stimulation (P=.05)

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Discussion

This study presents the results of an HPV VLP vaccine preparation to be administered to humans. We observed that VLP vaccines induce robust B and T cell responses in humans and that such responses are partially conserved across clinically and serologically distinct HPV types. The results demonstrate clearly that this HPV 11 preparation, formulated in an aluminum hydroxide adjuvant, is safe, well tolerated, and highly immunogenic in healthy seronegative volunteers. At doses of ∼3, 9, 30, and 100 μg, all volunteers briskly produced antibody, as measured by ELISA. There was an obvious dose response relationship, although no significant differences were observed among the recipients of the 3 higher doses. In addition, high levels of serum neutralizing antibody were produced. All study volunteers who received 3 doses of MEDI-50l at a dose of ⩾9 μg achieved serum neutralizing antibody titers of ⩾1000. Both binding and neutralizing antibodies remained high at 48 weeks after initial vaccination

We also observed that the MEDI-501 vaccine induced T cell proliferation and cytokine production. Epidemiologic observations point to a role for cell-mediated immune mechanisms in the control of HPV infections, including anogenital warts (condylomata acuminata). A marked lymphocyte and macrophage infiltrate consistent with a delayed-type hypersensitivity reaction is present in naturally regressing condylomata acuminata and exhibits an increase in the CD4+:CD8+ ratio and expression of lymphocyte activation markers [27, 28]. Patients with HIV infection have a 2–7-fold increased prevalence of condylomata acuminata, compared with noninfected control subjects [29, 30]

T cell responses to natural HPV infection have been difficult to measure [31] (and present authors’ unpublished data). The SIs induced by the MEDI-501 vaccine were far higher than those reported in natural HPV infections and were especially noteworthy in view of the fact that these assays were conducted with cryopreserved rather than fresh PBMC. In past studies, there has been little difference in in vitro proliferation between patients with clinical warts or cervical dysplasia, or between infected and uninfected control subjects [32 –34]. It is possible that this may be due, in part, to the relatively protected location of productive HPV infections. In the most complete study analyzing T cells associated with genital warts, Hong et al. recovered wart-infiltrating lymphocytes (WIL) from patients with condyloma acuminatum [33]. The WILs of 10 of the 13 patients proliferated in vitro in response to several of a series of overlapping 20-mer peptides (offset of 10) covering the entire E7 and L1 proteins of HPV-6. Of note, the responses of these patients’ PBMC to an HPV-6 L1 VLP were low, with an overall geometric mean SI of only 3.3

To our knowledge, this is the first report concerning lymphoproliferative responses to an HPV-11 VLP vaccine in human subjects. Using an algorithm to predict T cell epitopes in HPV-16 L1, Strang et al. observed an in vitro lymphoproliferative response when selected peptides were presented by HLA-DR to the lymphocytes of healthy human donors [35, 36]. Most of the epitopes selected were HPV type-specific, since the homologous peptides synthesized from other HPVs failed to induce a proliferative response [36, 37]. In addition, an epitope able to induce cytotoxic T lymphocyte activity has been mapped in the HPV-16 L1 protein [38]

In addition, vaccinee PBMC responded to heterotypic VLP antigens, indicating that T cell epitopes are conserved across serologically distinct genotypes. Because epidemiologic evidence points to a T cell–mediated protection from disease, the responses noted here may be important either in prevention of infection or in protection from disease development in those who do become infected. Most of the studies to date that correlate peripheral T cell responses and disease status in natural HPV infection have been conducted by using E6, E7, and L1 proteins of HPV-16 [31, 36, 39]. Because of low proliferation indices, the most successful methods for conducting such studies requires the establishment of short-term T cell lines before assaying for proliferation [31]. Such lines were used to examine the T cell response to HPV-16 L1 synthetic peptides in patients with cervical intraepithelial neoplasia (CIN) or cancer and in healthy control subjects [40, 41]. More patients than control subjects responded to ⩾1 L1 peptides, and a higher grade of CIN appeared to be associated with a greater likelihood of response. In other studies, the T cell response to E6 and E7 peptides appeared to be associated with clearance of viral infection [42]. On the other hand, a different HPV-16 L1 peptide was used by Nakagawa et al. to measure the T cell proliferative response of CIN patients and control subjects [43], and no significant difference between the 2 groups was observed. Additionally, a prospective study of T cell responses to L1 found no association with either persistence or clearance [44]

MEDI-501 vaccination elicited high levels of neutralizing antibody, which likely required T cell help, and was capable of inducing both a Th1 (INF-γ) and a Th2-like cytokine (IL-5). Although we did not definitely characterize the Th1/Th2 pattern elicited by this vaccination, these results are indicative of a mixed pattern [45]. The available published literature is mostly based on animal models and is usually limited to HPV-16 antigens. Dupuy et al. immunized mice with HPV-16 L1 VLP [46]. A significant lymphoproliferative response developed that was associated with the secretion of both INF-γ and IL-2, a pattern consistent with a Th1 profile. In P. S. Shepherd’s studies, Th1 responses were not as predominant in cancer patients as those measured in control subjects, which suggests that the T cell response may have played a protective role [41]. HPV-specific T cell IL-2 production has likewise been observed to decrease with cervical disease grade [47]

Several study participants had baseline stimulations with our VLP antigens that were above a standard SI cutoff of 3.0. We did not establish whether this was due to small amounts of contaminating baculovirus components in our antigens or to prior HPV infection. Many adults have serological evidence of exposure to some HPV types [48, 49], and thus it could be that these responses may have been due to prior T cell activation. The fact that other studies have failed to demonstrate significant differences in responses between infected individuals and control subjects supports this hypothesis

We also observed that 3 MEDI-501 recipients had a notable increase in proliferation to B19 VLP control, which suggests that some of the response seen to the HPV antigens was not specific. However, in these individuals, an increase in response to tetanus antigen was also observed, and thus this increase was likely due to a nonspecific bystander or activation effect of the nonplacebo immunization itself [50, 51]. Overall, there was no statistically significant increase in either lymphoproliferation or cytokine response to either tetanus or parvovirus B19 antigens

This HPV-11 vaccine preparation was well tolerated and safe. Although almost three-quarters of vaccine injections were accompanied by some pain, a similar rate was noted among recipients of the adjuvant control, and all episodes of pain, except one that occurred in an adjuvant control recipient, were mild or moderate in nature and resolved within 72 h. This rate and severity of pain are typical of commonly used vaccine preparations administered in alum adjuvants. Of interest was the observation of headache more commonly among MEDI-50l recipients than among adjuvant control recipients. The headache was unrelated to vaccine dose, was of only mild to moderate severity, and resolved in all subjects within 72 h. We have no explanation for our observation of a higher rate of mild to moderate headaches among vaccine recipients

Rash and/or pruritis was observed in 6 study participants. In 1 individual, hives were noted, and this reaction was believed by the investigators to be potentially related to the vaccine. This individual received no additional vaccine injections. Other study subjects with complaints of rash and/or pruritis received additional injections without adverse consequences. Results of routine complete blood cell counts, serum chemistry studies, and urinalyses were unremarkable and did not demonstrate significant differences among treatment groups or significant changes over time in any treatment group

In addition to establishing the safety and immunogenicity of MEDI-501, this study provides information that will be useful for designing a variety of subsequent investigations. First, the magnitude of T cell responses in peripheral blood is large enough to be able to compare different adjuvant or antigen strategies. Second, the cells from these studies could serve as useful reagents for mapping epitopes to peptides responsible for proliferation and/or cytokine secretion. These epitopes are likely to be conserved across many of the L1 proteins from different HPV types and could lead to new understandings in HPV vaccine design. Third, the brisk T cell response may enable the assessment of whether CD8+ activity was also induced by the VLP vaccines, as has been observed in animal models [46, 52]. Last, the implication of cross-type HPV responses may indicate that a vaccine against low–oncogenic risk HPV genotypes may provide some protection against high-risk HPV genotypes, and vice versa. Limited epidemiologic data suggest that such cross-protection by HPV types may indeed exist [53 –55]

This study demonstrated that HPV L1 VLP vaccines are highly immunogenic and induce brisk B cell and T cell responses. Ultimately, the efficacy of these preparations in the prevention of HPV-associated disease must be determined by an appropriately conducted field trial

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Acknowledgments

We would like to acknowledge the laboratory assistance of Jolene Andrews, Vesta Broumand-Mizani, and Theresa Fitzgerald of the University of Rochester in these studies

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Footnotes

  • ↵Presented in part: 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, September 1998 (abstract H-50)

    Written informed consent, as approved by the institutional review board at the University of Rochester, was obtained from all study subjects

    Financial support: MedImmune, Gaithersburg, MD; National Center for Research Resources, National Institutes of Health (General Clinical Research Center grant [5 M01 RR00044])

    S.K., J.A.S., W.I.W., and J.B. are employees of MedImmune

  • ↵Present affiliation: Division of Infectious Diseases, University of California, Davis

  • Received March 17, 2000.
  • Revision received January 31, 2001.
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References

  1. ↵
    1. zur Hausen H
    . Papillomavirus infections: a major cause of human cancers. Biochim Biophys Acta 1996;1288:F55-78.
    Medline
  2. ↵
    1. Mandell GL,
    2. Bennet JE,
    3. Dolin R
    1. Bonnez W,
    2. Reichman RC
    . Papillomaviruses. In: Mandell GL, Bennet JE, Dolin R, editors. Principles and practice of infectious diseases. New York: Churchill Livingstone; 2000.
  3. ↵
    1. Baker GE,
    2. Tyring SK
    . Therapeutic approaches to papillomavirus infections. Dermatol Clin 1997;15:331-40.
    CrossRefMedlineWeb of Science
  4. ↵
    1. Dolin R,
    2. Masur H,
    3. Saag M
    1. Bonnez W
    . Diseases due to human papillomavirus (sexually transmitted). In: Dolin R, Masur H, Saag M, editors. AIDS Therapy. Philadelphia: WB Saunders; 1999. p. 530-64.
  5. ↵
    1. Evans BA,
    2. Kell PD,
    3. Bond RA,
    4. MacRae KD
    . Heterosexual relationships and condom-use in the spread of sexually transmitted diseases to women. Genitourin Med 1995;71:291-4.
    MedlineWeb of Science
  6. ↵
    1. Schiffman MH,
    2. Brinton LA
    . The epidemiology of cervical carcinogenesis. Cancer 1995;76:1888-901.
    CrossRefMedlineWeb of Science
  7. ↵
    1. Bonnez W,
    2. Kashima HK,
    3. Leventhal B,
    4. et al
    . Antibody response to human papillomavirus (HPV) type 11 in children with juvenile-onset recurrent respiratory papillomatosis (RRP). Virology 1992;188:384-7.
    CrossRefMedline
  8. ↵
    1. Bonnez W,
    2. Da Rin C,
    3. Rose RC,
    4. Tyring SK,
    5. Reichman RC
    . Evolution of the antibody response to human papillomavirus type 11 (HPV-11) in patients with condyloma acuminatum according to treatment response. J Med Virol 1993;39:340-4.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Rose RC,
    2. Reichman RC,
    3. Bonnez W
    . Human papillomavirus (HPV) type 11 recombinant virus–like particles induce the formation of neutralizing antibodies and detect HPV-specific antibodies in human sera. J Gen Virol 1994;75:2075-9.
    Abstract/FREE Full Text
  10. ↵
    1. Carter JJ,
    2. Wipf GC,
    3. Hagensee ME,
    4. et al
    . Use of human papillomavirus type 6 capsids to detect antibodies in people with genital warts. J Infect Dis 1995;172:11-8.
    Abstract/FREE Full Text
  11. ↵
    1. Heim K,
    2. Christensen ND,
    3. Hoepfl R,
    4. et al
    . Serum IgG, IgM, and IgA reactivity to human papillomavirus types 11 and 6 virus-like particles in different gynecologic patient groups. J Infect Dis 1995;172:395-402.
    Abstract/FREE Full Text
  12. ↵
    1. Schiller JT,
    2. Roden RBS
    . Papillomavirus-like particles. Papillomavirus Rep 1995;6:121-8.
  13. ↵
    1. Wikstrom A,
    2. van Doornum GJ,
    3. Quint WG,
    4. Schiller JT,
    5. Dillner J
    . Identification of human papillomavirus seroconversions. J Gen Virol 1995;76:529-39.
    Abstract/FREE Full Text
  14. ↵
    1. Hagensee ME,
    2. Kiviat N,
    3. Critchlow CW,
    4. et al
    . Seroprevalence of human papillomavirus types 6 and 16 capsid antibodies in homosexual men. J Infect Dis 1997;176:625-31.
    Abstract/FREE Full Text
  15. ↵
    1. van Doornum G,
    2. Prins M,
    3. Andersson-Ellstrom A,
    4. Dillner J
    . Immunoglobulin A, G, and M responses to L1 and L2 capsids of human papillomavirus types 6, 11, 16, 18, and 33 L1 after newly acquired infection. Sex Transm Infect 1998;74:354-60.
    Abstract
  16. ↵
    1. Christensen ND,
    2. Reed CA,
    3. Cladel NM,
    4. Han R,
    5. Kreider JW
    . Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus. J Virol 1996;70:960-5.
    Abstract/FREE Full Text
  17. ↵
    1. Suzich JA,
    2. Ghim SJ,
    3. Palmer-Hill FJ,
    4. et al
    . Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci USA 1995;92:11553-7.
    Abstract/FREE Full Text
  18. ↵
    1. Kirnbauer R,
    2. Chandrachud LM,
    3. O’Neil BW,
    4. Neil BW,
    5. et al
    . Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization. Virology 1996;219:37-44.
    CrossRefMedline
  19. ↵
    1. Kirnbauer R,
    2. Booy F,
    3. Cheng N,
    4. Lowy DR,
    5. Schiller JT
    . Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA 1992;89:12180-4.
    Abstract/FREE Full Text
  20. ↵
    1. Rose RC,
    2. Bonnez W,
    3. Da Rin C,
    4. McCance DJ,
    5. Reichman RC
    . Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus–like particles. J Gen Virol 1994;75:2445-9.
    Abstract/FREE Full Text
  21. ↵
    1. Rose RC,
    2. Bonnez W,
    3. Reichman RC,
    4. Garcea RL
    . Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles. J Virol 1993;67:1936-44.
    Abstract/FREE Full Text
  22. ↵
    1. Smith LH,
    2. Foster C,
    3. Hitchcock ME,
    4. Isseroff R
    . In vitro HPV-11 infection of human foreskin. J Invest Dermatol 1993;101:292-5.
    CrossRefMedlineWeb of Science
  23. ↵
    1. Boukamp P,
    2. Petrussevska RT,
    3. Breitkreutz D,
    4. Hornung J,
    5. Markham A
    . Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988;106:761-71.
    Abstract/FREE Full Text
  24. ↵
    1. Kreider JW,
    2. Howett MK,
    3. Lill NL,
    4. et al
    . In vivo transformation of human skin with human papillomavirus type 11 from condylomata acuminata. J Virol 1986;59:369-76.
    Abstract/FREE Full Text
  25. ↵
    1. Keefer MC,
    2. Bonnez W,
    3. Roberts NJ Jr,
    4. Dolin R,
    5. Reichman RC
    . Human immunodeficiency virus (HIV-1) gp160-specific lymphocyte proliferative responses of mononuclear leukocytes from HIV-1 recombinant gp160 vaccine recipients. J Infect Dis 1991;163:448-53.
    Abstract/FREE Full Text
  26. ↵
    1. Evans TG,
    2. Fitzgerald T,
    3. Gibbons DC,
    4. Keefer MC,
    5. Soucier H
    . Th1/Th2 cytokine responses following HIV-1 immunization in seronegative volunteers: the AIDS Vaccine Evaluation Group. Clin Exp Immunol 1998;111:243-50.
    CrossRefMedlineWeb of Science
  27. ↵
    1. Bishop PE,
    2. McMillan A,
    3. Fletcher S
    . Immunological study of condylomata acuminata in men infected with the human immunodeficiency virus. Int J STD AIDS 1990;1:28-31.
    Medline
  28. ↵
    1. Coleman N,
    2. Birley HD,
    3. Renton AM,
    4. et al
    . Immunological events in regressing genital warts. Am J Clin Pathol 1994;102:768-74.
    MedlineWeb of Science
  29. ↵
    1. Mellon RL,
    2. Liautaud B,
    3. Pape JW,
    4. Johnson WD Jr
    . Association of HIV and STDs in Haiti: implications for blood banks and HIV vaccine trials [letter]. J Acquir Immune Defic Syndr Hum Retrovirol 1995;8:214.
    MedlineWeb of Science
  30. ↵
    1. Chirgwin KD,
    2. Feldman J,
    3. Augenbraun M,
    4. Landesman S,
    5. Minkoff H
    . Incidence of venereal warts in human immunodeficiency virus–infected and uninfected women. J Infect Dis 1995;172:235-8.
    Abstract/FREE Full Text
  31. ↵
    1. Shepherd P,
    2. Luxton J
    . T-cell responses to human papillomavirs in cervical dysplasia. Papillomavirus Rep 1999;10:53-9.
  32. ↵
    1. Charleson FC,
    2. Norval M,
    3. Benton EC,
    4. Hunter JA
    . Lymphoproliferative responses to human papillomaviruses in patients with cutaneous warts. Br J Dermatol 1992;127:551-9.
    CrossRefMedlineWeb of Science
  33. ↵
    1. Hong K,
    2. Greer CE,
    3. Ketter N,
    4. Van Nest G,
    5. Paliard X
    . Isolation and characterization of human papillomavirus type 6–specific T cells infiltrating genital warts. J Virol 1997;71:6427-32.
    Abstract/FREE Full Text
  34. ↵
    1. Cubie HA,
    2. Norval M
    . Humoral and cellular immunity to papillomavirus in patients with cervical dysplasia. J Med Virol 1988;24:85-95.
    CrossRefMedlineWeb of Science
  35. ↵
    1. Rothbard JB,
    2. Taylor WR
    . A sequence pattern common to T cell epitopes. Embo J 1988;7:93-100.
    MedlineWeb of Science
  36. ↵
    1. Strang G,
    2. Hickling JK,
    3. McIndoe GA,
    4. et al
    . Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity. J Gen Virol 1990;71:423-31.
    Abstract/FREE Full Text
  37. ↵
    1. Davies DH,
    2. Hill CM,
    3. Rothbard JB,
    4. Chain BM
    . Definition of murine T helper cell determinants in the major capsid protein of human papillomavirus type 16. J Gen Virol 1990;71:2691-8.
    Abstract/FREE Full Text
  38. ↵
    1. Rudolf MP,
    2. Nieland JD,
    3. DaSilva DM,
    4. et al
    . Induction of HPV16 capsid protein–specific human T cell responses by virus-like particles. Biol Chem 1999;380:335-40.
    CrossRefMedlineWeb of Science
  39. ↵
    1. Sadovnikova E,
    2. Stauss HJ
    . T cell epitopes in human papilloma virus proteins. Behring Inst Mitt 1994: July (94):87-93.
  40. ↵
    1. Shepherd PS,
    2. Rowe AJ,
    3. Cridland JC,
    4. Coletart T,
    5. Wilson P
    . Proliferative T cell responses to human papillomavirus type 16 L1 peptides in patients with cervical dysplasia. J Gen Virol 1996;77:593-602.
    Abstract/FREE Full Text
  41. ↵
    1. Luxton JC,
    2. Rose RC,
    3. Coletart T,
    4. Wilson P,
    5. Shepherd PS
    . Serological and T-helper cell responses to human papillomavirus type 16 L1 in women with cervical dysplasia or cervical carcinoma and in healthy controls. J Gen Virol 1997;78:917-23.
    Abstract/FREE Full Text
  42. ↵
    1. Kadish AS,
    2. Ho GY,
    3. Burk RD,
    4. et al
    . Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia. J Natl Cancer Inst 1997;89:1285-93.
    Abstract/FREE Full Text
  43. ↵
    1. Nakagawa M,
    2. Stites DP,
    3. Farhat S,
    4. et al
    . T-cell proliferative response to human papillomavirus type 16 peptides: relationship to cervical intraepithelial neoplasia. Clin Diagn Lab Immunol 1996;3:205-10.
    Medline
  44. ↵
    1. de Gruijl TD,
    2. Bontkes HJ,
    3. Walboomers JM,
    4. et al
    . Differential T helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study. Cancer Res 1998;58:1700-6.
    Abstract/FREE Full Text
  45. ↵
    1. Romagnani S
    . Biology of human TH1 and TH2 cells. J Clin Immunol 1995;15:121-9.
    CrossRefMedlineWeb of Science
  46. ↵
    1. Dupuy C,
    2. Buzoni-Gatel D,
    3. Touze A,
    4. Le Cann P,
    5. Bout D
    . Cell mediated immunity induced in mice by HPV 16 L1 virus–like particles. Microb Pathog 1997;22:219-25.
    CrossRefMedlineWeb of Science
  47. ↵
    1. Tsukui T,
    2. Hildesheim A,
    3. Schiffman MH,
    4. et al
    . Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus–derived peptides: correlation with cervical pathology. Cancer Res 1996;56:3967-74.
    Abstract/FREE Full Text
  48. ↵
    1. af Geijersstam V,
    2. Eklund C,
    3. Wang Z,
    4. et al
    . A survey of seroprevalence of human papillomavirus types 16, 18 and 33 among children. Int J Cancer 1999;80:489-93.
    CrossRefMedlineWeb of Science
  49. ↵
    1. Cubie HA,
    2. Plumstead M,
    3. Zhang W,
    4. de Jesus O,
    5. Duncan LA
    . Presence of antibodies to human papillomavirus virus–like particles (VLPs) in 11–13-year-old schoolgirls. J Med Virol 1998;56:210-6.
    CrossRefMedlineWeb of Science
  50. ↵
    1. Tough DF,
    2. Borrow P,
    3. Sprent J
    . Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 1996;272:1947-50.
    Abstract/FREE Full Text
  51. ↵
    1. Tough DF,
    2. Sun S,
    3. Zhang X,
    4. Sprent J
    . Stimulation of naive and memory T cells by cytokines. Immunol Rev 1999;170:39-47.
    CrossRefMedlineWeb of Science
  52. ↵
    1. Schirmbeck R,
    2. Bohm W,
    3. Reimann J
    . Virus-like particles induce MHC class I–restricted T-cell responses: lessons learned from the hepatitis B small surface antigen. Intervirology 1996;39:111-9.
    MedlineWeb of Science
  53. ↵
    1. Silins I,
    2. Wang Z,
    3. Avall-Lundqvist E,
    4. et al
    . Serological evidence for protection by human papillomavirus (HPV) type 6 infection against HPV type 16 cervical carcinogenesis. J Gen Virol 1999;80:2931-6.
    Abstract/FREE Full Text
  54. ↵
    1. Sigurgeirsson B,
    2. Lindelof B,
    3. Eklund G
    . Condylomata acuminata and risk of cancer: an epidemiological study. BMJ 1991;303:341-4.
    Abstract/FREE Full Text
  55. ↵
    1. Luostarinen T,
    2. af Geijersstam V,
    3. Bjorge T,
    4. et al
    . No excess risk of cervical carcinoma among women seropositive for both HPV16 and HPV6/11. Int J Cancer 1999;80:818-22.
    CrossRefMedlineWeb of Science
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  1. J Infect Dis. (2001) 183 (10): 1485-1493. doi: 10.1086/320190
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