Affiliations of authors: C. D. Harro, M. J. Reynolds, T. C. Mast, R. A. Karron (Center for Immunization Research, Department of International Medicine, School of Hygiene and Public Health), R. B. S. Roden (Department of Pathology, School of Medicine), The Johns Hopkins University, Baltimore, MD; Y.-Y. S. Pang, J. T. Schiller, D. R. Lowy (Laboratory of Cellular Oncology, Division of Basic Sciences), A. Hildesheim (Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics), National Cancer Institute, Bethesda, MD; Z. Wang, J. Dillner, Microbiology and Tumor Biology Centre, Karolinska Institute, Stockholm, Sweden; R. Robinson, Novavax, Inc., Columbia, MD; B. R. Murphy, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda.
Correspondence to: Douglas R. Lowy, M.D., National Institutes of Health, Bldg. 36, Rm. 1D-32, Bethesda, MD 20892 (e-mail: drl{at}helix.nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Clinical and molecular epidemiologic investigations have identified human papillomavirus (HPV) as the major cause of cervical cancer and cervical dysplasia (5,6). Virtually all cervical cancers contain the genes of high-risk HPVs (most commonly, types 16, 18, 31, and 45), and the relative frequency with which these types are found is remarkably similar in most regions of the world (6,7). HPV16 is found in approximately 50% of cervical cancers, and types 18, 31, and 45 account for an additional 25%30% of HPV-positive tumors.
Identification of HPV as a causal factor in virtually all cervical cancers implies that development of an effective vaccine against high-risk HPV could prevent the premalignant and malignant disease associated with HPV infection. Since prophylactic viral vaccines have a long record as a cost-effective approach to prevent infection or modify disease, such a vaccine might also lower the cost of screening and treating premalignant cervical disease. HPVs are DNA tumor viruses that contain oncogenes. There might be theoretic arguments against the presence of such genes, which can disrupt normal growth controls, in a vaccine destined for normal individuals. To develop a prophylactic vaccine against HPV infection, we and others (8) have, therefore, taken a subunit vaccine approach, analogous to that used successfully against hepatitis B-induced disease, including hepatocarcinoma (9).
Papillomaviruses encode a major capsid protein, L1, that has the intrinsic capacity to self-assemble into virus-like particles (VLPs) in the absence of other viral gene products (1013). Recombinant L1 VLPs are morphologically indistinguishable from authentic virions, contain the immunodominant conformationally dependent neutralization epitopes present in authentic virions, and have the ability to generate high titers of type-specific neutralizing antibodies (8).
Several trials of preventive papillomavirus vaccine candidates using L1 VLPs purified from insect cells have been conducted by use of the cutaneous cottontail rabbit papillomavirus (CRPV), the oral mucosal bovine papillomavirus 4 (BPV4), or the canine oral papillomavirus (COPV) disease model in its natural host. Three subcutaneous injections of CRPV L1 VLPs given without adjuvant, or combined with alum or Freund's adjuvant, protected rabbits against persistent infection and subsequent carcinoma after high-dose CRPV challenge (14,15). Protection lasted at least 1 year (15). Similarly, dogs or calves given two intramuscular injections of COPV L1 VLPs (without adjuvant) or BPV4 L1 VLPs (with alum), respectively, were protected from subsequent oral mucosal challenge (16,17). In the CRPV and COPV models, passive transfer of serum or immunoglobulin (Ig) G from animals immunized with the L1 VLPs protected naive animals challenged with the homologous virus, indicating that neutralizing antibodies were sufficient to confer protection (14,16).
Since papillomaviruses are species specific and HPVs do not induce disease in animals (4), further HPV vaccine development and evaluation require studies in humans. Therefore, as an initial step in demonstrating proof-of-principle, we developed a prophylactic recombinant HPV16 L1 VLP vaccine candidate, produced in insect cells with recombinant baculovirus, for testing in humans. This study was designed to evaluate the safety and immunogenicity in healthy young adults of two dose levels (10 and 50 µg) of the HPV16 L1 VLP vaccine given in aqueous solution without adjuvant or mixed with alum or MF59 adjuvant.
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SUBJECTS AND METHODS |
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Recombinant baculovirus expressing the full-length L1 capsid gene of HPV16 strain 114K (11) as VLPs was constructed by use of the bacmid system (18). A 1.5-kilobase BglII DNA fragment from pEVnod-KL1 (11) containing the HPV16 L1 gene was cloned in the BamHI site downstream of the polyhedrin promoter within the polh locus in the baculovirus donor plasmid pFASTBAC-1 (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) by site-specific recombination in Escherichia coli DH10Bac1. Recombinant baculovirus containing the HPV16 L1 DNA was isolated from Sf-9 insect cells transfected with the recombinant bacmid DNA by use of the cationic lipid Cellfectin (Life Technologies, Inc.). Recombinant baculovirus was plaque purified three times and screened for insert integrity by DNA sequencing and L1 capsid antigen expression in insect cells by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and western blot analyses. One recombinant baculovirus isolate that expressed high levels of L1 protein intracellularly and extracellularly was amplified in Sf-9 cells, tested for microbial contaminants and adventitious agents, and designated the master virus seed stock (bHPV16 L1 R-212). Working virus seed stocks of bHPV16 L1 virus were produced in Sf-9 cells infected for 3 days at a multiplicity of infection (moi) of 0.01 plaque-forming unit (pfu)/cell.
Production of clinical lots of recombinant HPV16 L1 VLP vaccines was performed in accordance to good manufacturing practice guidelines for well-characterized biologicals at the Vaccine Production Facility of Novavax, Inc. (formerly DynCorp) in Columbia, MD. Production lots of recombinant HPV16 L1 VLPs were manufactured in Sf-9 cells (23 x 106 cells/mL in 16.8-L-size batches) infected with bHPV-16 L1 virus at an moi of 35 pfu's/cell for 45 days at 28 °C as described previously (19). Sf-9 cells were cultivated as suspension cultures by use of Sf-900 II serum-free medium (Life Technologies, Inc.). Extracellular HPV16 L1 VLPs were recovered from infected cell suspension by low-speed centrifugation (1000g for 10 minutes at 4 °C), and supernatants were clarified by centrifugation (10 000g for 30 minutes at 4 °C). Clarified supernatants were concentrated 15-fold by ultrafiltration with the use of hollow fiber membranes (UFP-5-C-6A, MWCO 500 000) (A/G Technologies) and diafiltered against 20 volumes of phosphate-buffered saline (PBS) (pH 7.2). The dialysate was clarified by centrifugation as described above, the clarified supernatant was loaded onto 30% sucrose cushions in PBS, and the recombinant VLPs and baculovirus were pelleted through the sucrose cushions by ultracentrifugation (26 000g for 3 hours at 4 °C) by use of swinging bucket rotors. Pellets were solubilized in PBS and loaded onto 25%65% sucrose step gradients. VLPs were resolved as bluish bands on gradients by ultracentrifugation (25 000g for 1 hour at 4 °C) by use of swinging bucket rotors. Successive rounds of ultracentrifugation on sucrose gradients were used to obtain VLPs with a purity of more than 95%. VLPs were recovered from banded VLPs by ultracentrifugation (26 000g for 3 hours at 4 °C). Sucrose was removed from pelleted VLPs by dialysis against PBS. The VLPs were then diluted to 0.3 mg/mL and filtered aseptically through 0.22-µm membranes. The filtered VLPs were designated the final bulk product and stored at -20 °C. The final bulk product underwent both safety and analytic testing, and the batch records were audited by quality assurance before release of the final bulk product for formulation of final container vaccine product. The integrity of the VLPs was monitored by their morphology in the electron microscope, their ability to hemagglutinate mouse red blood cells efficiently (20), and their strong reactivity to HPV16 conformational and neutralizing monoclonal antibodies (H16.V5, H16.E70, and H16.U4) (21,22) and nonreactivity to an HPV11 neutralizing monoclonal antibody (H11.B2) (23).
The final bulk product of recombinant HPV16 L1 VLPs was formulated in a final volume of 0.5 mL as a nonadjuvanted vaccine at a 10- and 50-µg dose or in a final volume of 0.25 mL to be mixed with MF-59 adjuvant (Chiron Corporation, Emeryville, CA), which is a microfluidized oil-in-water emulsion consisting of 1.25 mg of sorbitan amonoleate (Tween 80®), 1.25 mg of sorbitan trioleate (Span 85®), and 10.75 mg of squalene per 0.5-mL dose. At the time of vaccine administration, 0.25 mL of MF59 emulsion was combined with 0.25 mL of vaccine to yield a vaccine dose of 10 and 50 µg. Alternatively, the final bulk product was formulated with alum (aluminum potassium sulfate, 10%; EM Merck, Darmstadt, Germany) at 120 mg of alum per mg of VLP antigen. The final volume of the VLPalum vaccine was 0.5 mL for 10- and 50-µg doses. Sterile saline served as the placebo vaccine. Formulated VLPs were dispensed aseptically into sterile vials (3.0-mL size, type 1 borosilicate glass, silanized, depyrogenated; Wheaton Glass, Wheaton, MD) as a single-unit dose and were designated final container vials. Vials containing only VLP antigen were stored at -20 °C before administration, whereas vials containing VLP antigen adsorbed to alum were stored at 4 °C because of stability concerns.
Study Design
This double-blind, randomized, placebo-controlled, phase I safety and immunogenicity trial was conducted at The Johns Hopkins University Center for Immunization Research (Baltimore, MD). Guidelines for human experimentation of the Joint Committee for Clinical Investigation of The Johns Hopkins University School of Medicine and its institutional review board were followed in the conduct of this study. Seventy-two healthy, human immunodeficiency virus (HIV)-1-seronegative, 18- to 29-year-old volunteers (58 females and 14 males) were recruited for this study. Subjects were determined by history to be at low risk for HPV16 exposure. Individuals were not eligible to participate if they had a history of more than four lifetime sexual partners or more than two sexual partners within the preceding 6 months. Additional exclusion criteria included history of abnormal cervical cytology, immunodeficiency, anaphylaxis to medicines or vaccines, receipt of blood products within 3 months of enrollment, current pregnancy or lactation, and any other condition that might interfere with the study objectives. All aspects of the protocol were explained to the subjects who met the eligibility criteria, and informed, witnessed, written consent was obtained. Preimmune HPV16 serostatus was not a criterion for eligibility, since it was expected that only about 10% of the volunteers would be seropositve (in this study, only six of 72 volunteers were found to be seropositive at study entry), its omission simplified recruitment, enrollment, and initiation of vaccination, and the presence of a few seropositive vaccine recipients would enable us to monitor the response of such individuals.
Before enrollment, a medical history was obtained from each subject, a physical examination was performed, and the following laboratory tests were done (Quest Diagnostics; Baltimore, MD): complete blood cell count (CBC), platelet count, alanine aminotransferase (ALT), serum creatinine, hepatitis B surface antigen, HIV antibody test, and urine dipstick for hemoglobin and protein. Volunteers were eligible to enroll if their medical history, physical examination, and laboratory tests were without clinically significant abnormalities. Individuals with clinically significant abnormalities were not enrolled but were counseled and referred for medical consultation as appropriate. To qualify for enrollment and subsequent vaccination, female subjects were required to have a negative urine pregnancy test on the day of each vaccination and to use an acceptable method of birth control until completion of the study.
To determine whether the dose of HPV16 L1 VLP vaccine and/or the addition of alum or MF59 adjuvant would influence the reactogenicity or immune response, the trial was conducted in a dose-escalation manner, starting with 10 µg of HPV16 L1 VLP vaccine given alone, with alum, or with MF59 adjuvants. When this dose was determined to be safe, we then evaluated a 50-µg dose of HPV16 L1 VLP vaccine given alone, with alum, or with MF59 adjuvant (Table 1). For each dose/adjuvant group, 10 subjects were randomly assigned to receive investigational vaccine, and two were randomly assigned to receive placebo (sterile physiologic saline). Subjects received vaccine or placebo as an intramuscular injection (0.5 mL) in the deltoid region at months 0, 1, and 4. To maintain blinding, the individual who prepared and administered the vaccine was not involved in data collection or clinical evaluation of volunteers.
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Serologic Assays
IgG-specific HPV16 L1 VLP-based enzyme-linked immunosorbent assays (ELISAs) were performed in a 96-well plate format as described previously (24), except that 200 ng of VLPs was used per well and end point dilution titers were determined. VLPs for the ELISAs were purified from the nuclei of HPV16 L1 recombinant baculovirus-infected Sf-9 cells as described previously (11). Fourfold dilutions of each serum were assayed, starting at a dilution of 10. Sera were designated ELISA positive at a given dilution if the absolute optical density (OD) was greater than or equal to 0.2 and was at least double the reactivity of the same serum dilution in a well containing blocking buffer but no VLPs. Seroconversion was defined as a fourfold or greater rise in titer. Vaccine recipients were considered to be HPV16 seropositive at enrollment if their prevaccination serum demonstrated an ELISA antibody titer that was greater than or equal to the reactivity of a standard pooled serum, which was assayed on the same plate (generally OD of 0.40.6 at 1 : 40 dilution). Reactivity to the standard serum had been validated as a cut point for seropositivity in a previous seroepidemiologic study (24).
The detailed procedure for IgM, IgA, and IgG isotyping and for determining IgG subclass with subclass-specific second antibodies is reported elsewhere (25). Sera from the individuals receiving vaccine without adjuvant or with alum were tested in triplicate at a dilution of 1 : 50 for IgG and IgG1 or at a dilution of 1 : 20 for IgG2, IgG3, and IgG4 by use of the corresponding isotype- or subclass-specific second antibody. A sample was considered to be positive if, after subtraction of the reactivity to denatured BPV1 VLPs, the OD was greater than the mean OD plus three standard deviations from the mean for a panel of sera from virgin women and was at least 0.100.
In vitro HPV16 pseudovirion neutralization assays were performed as described previously (26). Briefly, infectious virions composed of the HPV16 L1 and L2 capsid proteins and the BPV1 genome were generated by infection of BPHE-1 hamster cells (which contain autonomously replicating BPV genomes) with replication-defective Semliki Forest Virus vectors expressing HPV16 L1 and L2 genes. Individual infections by the pseudotype virions were detected as transformed foci in a monolayer of mouse C127 cells. Fourfold dilutions of the sera, starting with a dilution of 1 : 10, were mixed with approximately 50100 focus-forming units of pseudotype virions and assayed for inhibition of focal transformation. Neutralization was defined as at least a 50% reduction in the number of foci compared with the number obtained in the absence of human serum. The ELISA and neutralizing antibody titers are given as the reciprocal of the highest positive dilution for each assay.
Statistical Methods
The data for local and systemic reactions were grouped by vaccine dose and adjuvant. Data from placebo recipients were pooled for analysis. We compared proportions of reactions among different categories of volunteers by use of a continuity corrected test for proportions between two independent groups (27). Nonparametric statistics (KruskalWallis test) were used to make comparisons across groups at a given time. The KruskalWallis test was also used to compare the distribution of antibody titers in different groups over time (28). The Spearman correlation coefficient was used when comparing ELISA results against those obtained by using the neutralization test (28). Categorized ELISA and neutralization results were compared with the use of the Kappa statistic, and overall agreement percentages were also computed (29). All statistical tests were two-sided.
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RESULTS |
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Administration of three injections of HPV16 L1 VLP vaccine, given alone or with alum or MF59 adjuvant, was well tolerated at both dose levels (Table 3). For each group, the frequency and severity of reactions following each of the three doses were similar, and reactions for all three vaccinations have, therefore, been grouped together. Most of the local and systemic reactions were classified as mild. Clinical responses of the subjects who received 10 µg of vaccine without adjuvant were almost identical to those of the placebo group. Subjects who received 50 µg of vaccine without adjuvant reported local side effects about twice as frequently as the placebo recipients. Recipients of either 10 or 50 µg of vaccine with MF59 reported pain at the injection site more frequently than recipients of vaccine with alum or without adjuvant (10 µg24 (80%) of 30 versus 11 (36.7%) of 30 and seven (23.3%) of 30; P = .002 and P = .0001, respectively; 50 µg28 (93.3%) of 30 versus 14 (51.9%) of 27 and 17 (56.7%) of 30; P = .0005 and P = .003, respectively). However, pain was mild to moderate in intensity and resolved spontaneously within 4872 hours in all subjects. Similarly, recipients of 50 µg of vaccine with MF59 reported local induration and/or erythema more frequently than recipients of 50 µg of vaccine with alum or without adjuvant. Of note, one recipient of 50 µg of vaccine with MF59 reported erythema that peaked at 60 mm in diameter on the day of the third vaccination and resolved on day 2 after vaccination. A second recipient of 50 µg of vaccine with MF59 reported 45 mm of erythema and induration that began on the day of the second vaccination and resolved on day 4 after vaccination. Neither of these subjects required medication or other clinical intervention. There was no notable difference in the clinical responses of the five vaccine recipients who were seropositive at entry compared with the seronegative vaccine recipients in the same group nor was the frequency or severity of reactions greater after the third vaccination than after the initial vaccinations.
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HPV16-Specific Antibody Responses
The volunteers were evaluated for serum IgG responses to the vaccine by use of an HPV16 VLP ELISA (24) (Table 4). Overall, the sexual-history screening tool used in this study was an effective predictor of baseline HPV16 seronegativity by ELISA. A total of six of 72 subjects were IgG seropositive by ELISA at study entry. The geometric mean ELISA titer (GMT) of the prevaccination sera from these six individuals was 202 (range, 40640). Four of these subjects received 10 µg of HPV16 L1 VLP vaccine (one without adjuvant, two with alum, and one with MF59), one received 50 µg of HPV16 L1 VLP vaccine without adjuvant, and one received placebo. The final serum ELISA titers for each of the five vaccine recipients who were seropositive before vaccination were no more than one fourfold dilution above the final serum GMT of the prevaccination seronegative vaccinees in the same group.
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In each group that received vaccine (alone or with adjuvant), peak titers were observed at month 5 (1 month after the third injection). The month-5 antibody titers for the groups that received 10 µg of vaccine with alum and 10 µg of vaccine with MF59 were similar (P = .76) and were significantly higher (P =.007 and P = .01, respectively) than the month 5 antibody titers of the group that received 10 µg of HPV16 L1 VLP alone. Thus, within the low-dose group, the addition of alum or MF59 adjuvant enhanced the immune response.
Compared with the month-5 titers at the 10-µg dose, titers for the 50-µg groups were markedly higher in recipients of vaccine without adjuvant, modestly higher in recipients of vaccine with MF59, and slightly lower in recipients of vaccine with alum. In contrast to the results obtained with the 10-µg vaccine dose, the ELISA antibody titers achieved in the group that received 50 µg of vaccine without adjuvant were equivalent to those in the group that received 50 µg of vaccine with MF59 (P = .58). Month-5 antibody titers in these two groups were significantly higher (P = .002 and P = .001, respectively) than the antibody titers in the group that received vaccine with alum.
The sera from subjects vaccinated without adjuvant or with alum were also analyzed in HPV16 VLP ELISAs specific for IgM and IgA and for IgG subclasses (Table 5). The majority of the antibody response to VLP vaccination was of the IgG1 subclass. As expected, most vaccine recipients became transiently seropositive for HPV16 IgM antibodies, which were usually detected in the sample taken 1 month after the initial vaccination. Most vaccine recipients also became seropositive for serum IgA; however, relative to IgG responses, the responses varied considerably and were generally weaker. All vaccine recipients became strongly seropositive for IgG1, and the responses closely paralleled the response to total IgG. In contrast, only one subject became weakly, although consistently, seropositive in the IgG2 assay. Responses to IgG3 and IgG4 were weak and variable.
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The GMT neutralizing antibody titers in the group that received 50 µg of vaccine with MF59 were greater than the titers in recipients of 50 µg of vaccine without adjuvant, but this difference was not statistically significant (P = .08) (Table 4). However, the neutralizing antibody titers achieved in groups receiving VLPs with MF59 or with no adjuvant were significantly higher than the titers in the group receiving VLPs in alum (P = .0001 and P = .001, respectively). Thus, the relative hierarchy of the GMT neutralization resembled the GMT ELISA titers.
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DISCUSSION |
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The most commonly reported side effect in our study was pain at the site of injection. Most of the pain was mild and short-lived, consistent with other intramuscularly administered recombinant subunit vaccines, such as licensed hepatitis B vaccines (32,33). Side effects were similar in recipients of placebo and recipients of 10 µg of vaccine without adjuvant. However, recipients of the higher vaccine dose (50 µg) without adjuvant did report more side effects than the placebo recipients. As expected, a greater proportion of recipients of HPV16 L1 VLP with MF59 reported moderate pain at the injection site than recipients of HPV16 L1 VLP vaccine given alone or with alum. The proportion of subjects experiencing injection site reactogenicity after injection of HPV16 L1 VLP with MF59 was comparable to that reported with other investigational vaccine antigens given with MF59, such as hepatitis B surface antigen and herpes simplex type II gD glycoprotein (34,35).
In this study, the adjuvant effects of alum and MF59 were evident in recipients of the lower vaccine dose (10 µg). In addition, there was a dose-dependent response in the groups receiving vaccine without adjuvant or with MF59, although not in the groups receiving vaccine with alum. Specifically, as determined by GMT ELISA titers, there was a marked increase in the immune response to 50 µg without adjuvant compared with 10 µg without adjuvant (10 240 versus 640), a modest increase with MF59 (10 240 versus 3480), and no increase in the ELISA titers seen with alum (3040 versus 2190). Since at the 50-µg dose the ELISA titers in recipients of vaccine without adjuvant were comparable to the titers in the group that received vaccine with MF59, and even higher than the group that received vaccine with alum, there was no apparent benefit to using adjuvant at this dose when serum antibody titers were measured shortly after immunizations. It is unknown whether these properties would continue to be true following a longer interval after the third vaccine dose. Since the addition of MF59 adjuvant was associated with increased pain and induration at the injection site, the optimal immunogenicity and reactogenicity profile in the current study was obtained with 50 µg of HPV16 L1 VLP vaccine without adjuvant. It is encouraging to note that, with the higher dose without adjuvant or with MF59, the final serum titers were about 40 times higher than those detected systemically after natural infection in the subjects who were seropositive before vaccination.
The similar titers seen at the 50-µg dose with no adjuvant or with MF59, combined with the modest increase seen with MF59 at the higher dose, suggest that even higher doses of vaccine would probably not induce substantially higher antibody titers. For alum, the 10-µg vaccine dose induced maximum ELISA titers. A similar plateau effect was reported when alum was used for HPV11 L1 VLP vaccination of macaques (36). Although we do not understand the basis for this phenomenon, two factors that can have a substantial impact on the immune response of antigens delivered with alum are the degree of antigen adsorption onto alum and the dose of alum used (37). It is unlikely that the degree of antigen adsorption adversely affected immunogenicity because, in the formulation containing 50 µg of HPV16 L1 VLP with alum, more than 95% of the VLPs was complexed with the aluminum hydroxide. However, even with high levels of antigen adsorption, it is possible that the concentration of alum was insufficient to induce a maximal adjuvant effect. It is also possible that the VLPs in alum, which were stored at 4 °C, might have been less stable. Such instability would presumably have affected only the 50-µg dose, which was given after the 10-µg dose.
A predominantly IgG1 response was also not unexpected. We have found recently that IgG1 is also the IgG isotype that predominates after seroconversion to natural infection (25). Predominantly IgG1 responses are also commonly seen after other microbial infections (38). In C57BL/6 mice, HPV16 L1 VLPs induce a more varied IgG response, with substantial amounts of specific IgG1, IgG2b, and IgG3 detected (Heather Greenstone, Ph.D. Thesis, The Johns Hopkins University). Whether this reflects a stronger Th1 type response to HPV VLPs in humans than in mice remains to be determined. The inability to detect an IgG2 response in most vaccine recipients is unlikely to be due to poor sensitivity or specificity of the IgG2 assay. In fact, the sensitivity/specificity of the IgG2 assay was superior to that of the IgG1 assay for isotype-specific human Ig control subjects (25).
Neutralization assays are often considered to be the "gold standard" in assessing the immunogenicity of a prophylactic vaccine such as the one tested here. When the month-5 sera from the groups that received the 50-µg dose of vaccine were analyzed for HPV16 peudovirion neutralizing activity, there was an excellent quantitative correlation with the ELISA titers. This correlation held for individuals as well as for groups, further implying that the ELISA appears to represent an appropriate surrogate assay for the more cumbersome and expensive neutralization assay.
For both assays, there was a remarkable consistency of response within each vaccine group. The immunogenicity results seen here with the human volunteers parallel those obtained in animals, where consistently statistically significant immunogenicity and protection against experimental disease have been observed, even when adjuvant was not given. It is likely that the particulate nature and regular array of L1 in the VLPs contribute to their high immunogenicity. Perhaps efficient immune recognition is promoted by an interaction between the VLPs and cell surface pattern recognition receptors that bind the ordered external structure of icosahedral virions (39).
It is not possible to know from these studies whether systemic administration of vaccine will protect against cervical infection under natural conditions. However, the magnitude of the antibody responses in the human volunteers compares favorably with that seen in animal studies in which VLP vaccination, with alum or without adjuvant, induced protection from high-dose virus challenge (Table 6). Although some caution must be taken in comparing studies in which different vaccination protocols and ELISAs were used, our overall impression is that humans and experimental animals respond similarly to VLP vaccination.
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The safety and immunogenicity profile obtained in this study encourages further clinical investigation of HPV VLP-based immunoprophylactic vaccines. On the basis of these results, we have initiated a phase II trial of 50 µg of L1 VLP without adjuvant. The key question of whether systemic administration with a VLP vaccine can confer protection under natural conditions must await the outcome of controlled efficacy trials. If the vaccine were eventually shown to be effective, the type specificity of the neutralizing activity induced by the VLPs implies that protection would be type specific. Since multiple HPV types are implicated in cervical cancer, a multivalent vaccine would be needed. A vaccine composed of the four HPV types seen most frequently in cervical cancer (types 16, 18, 31, and 45) would theoretically be able to protect against approximately 80% of cervical cancers (6).
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NOTES |
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We thank Catherine Greer and Lori Hansen (Chiron, Emeryville, CA) for providing the MF59, Dr. Robert Chanock (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for a critical reading of the manuscript, and Dr. Richard Leapman and his colleagues (Division of Bioengineering and Physical Science, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health) for providing electron microscopic facilities and assistance. We also acknowledge the expert assistance of Gwen Hammer, Sabrina Drayton, Arlene Bloom, Nancy Frazier, Martin Blair, and Denos Harris, The Johns Hopkins University, Baltimore, MD.
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Manuscript received June 5, 2000; revised December 12, 2000; accepted December 21, 2000.
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