Abstract

Background.Cellular immunity is involved in spontaneous clearance of anogenital warts caused, most typically, by human papillomavirus (HPV) type 6 or 11, supporting the concept of therapeutic vaccination. A therapeutic vaccine composed of HPV-6 L2E7 fusion protein and AS02A adjuvant was evaluated in conjunction with conventional therapies in subjects with anogenital warts

Methods A total of 457 subjects with anogenital warts were screened, of which 320 with HPV-6 and/or HPV-11 infection were enrolled into 2 double-blind, placebo-controlled substudies. Three doses of vaccine or placebo were administered along with either ablative therapy or podophyllotoxin

Results Although a positive trend toward clearance was seen in patients infected with only HPV-6, in neither substudy did the vaccine significantly increase the efficacy of conventional therapies, despite induction of adequate immune responses. Extensive HPV typing by polymerase chain reaction demonstrated that a majority of screened subjects (73.7%) were infected with HPV-6 and/or HPV-11 and that a large proportion (40.1%) were infected with multiple HPV types. HPV types that put subjects at high risk of development of cervical cancer were detected in 39.8% of subjects

Conclusions Infection with multiple HPV types, including high-risk types, is common in anogenital wart disease. Therapeutic vaccination failed to increase the efficacy of conventional therapies

Anogenital human papillomavirus (HPV) infections represent one of the most prevalent sexually transmitted diseases (STDs) worldwide [1–3]. Mucosal HPV infections are subdivided into 2 groups according to the risk of malignant progression. Infection with high-risk HPV types (primarily 16, 18, 31, and 45) are strongly associated with the development of cervical cancer, whereas infection with low-risk HPV types (predominantly 6 and 11) cause benign mucosal tumors [4, 5]. Anogenital warts, or condyloma acuminata, are usually benign tumors of the genital mucosa and&rank as one of the most prevalent STDs, with an estimated incidence of 1–3 million new cases per year in the United States [1]. The incidence has increased by 2.5- to 4.5-fold during the past 3 decades [6]

The numerous therapies used to eliminate anogenital warts fall into 2 classes: ablative (physical destructive methods or cytotoxic or antiproliferative compounds) and immune based (predominantly imiquimod and interferon) [7, 8]. Although the cure rate is highly variable from study to study, the recurrence rate is generally similar regardless of the therapy used, with an average of 30% of subjects with recurrences within 3 months after therapy

Cell-mediated immune responses appear to play a major role in regression of anogenital warts [9] and are probably down-regulated or modified by HPVs [10–12]. A characteristic of all HPV lesions is their propensity to spontaneously regress. Spontaneously regressing anogenital warts are massively infiltrated with CD4+ T cells, and an increased level of interleukin-12 expression is observed [13, 14]. This phenomenon, together with results from animal studies, forms the main argument supporting the rationale for therapeutic vaccination as a potential therapy for HPV-induced diseases [15]

In the cottontail rabbit model, spontaneous regression of anogenital warts was associated with a high immune response to early (E) protein 2 [16], and vaccination with E1 or E2 prior to challenge induced a faster regression of anogenital warts [17]. In cattle, late (L) protein 2 from bovine papillomavirus (BPV) type 2 induces a rapid regression of anogenital warts and a massive immune cell infiltrate [18, 19]. Conversely, infection with BPV-4 can be prevented by vaccination with L2 from BPV-4, and vaccination with E7 from BPV-4 can limit the growth of anogenital warts and induce their regression [20]

Consequently, a therapeutic vaccine composed of a fusion protein of L2 and E7 from HPV-6 was developed [21]. The high homology between HPV-6 and HPV-11 and the observation that both types, but predominantly HPV-6, are almost exclusively detected as causative agents of anogenital warts supported the decision to test a vaccine that included only HPV-6 components. In an uncontrolled trial of 27 subjects who received 3 aluminum adjuvanted L2E7 vaccine doses [22], 5 had complete regression at week 8, and, after additional therapy, 8 more subjects did not experience recurrences. Subsequently, the L2E7 antigen was combined with GlaxoSmithKline Biologicals proprietary adjuvant AS02A, which contains 2 immunostimulants, monophosphoryl lipid A and QS21, and an oil-in-water emulsion. It has been shown to induce high antibody titers, a strong CD4+ cell response, and cytotoxic T lymphocyte activity in humans [23, 24] and has an acceptable safety profile. Two placebo-controlled substudies were conducted to evaluate the therapeutic efficacy of the L2E7-AS02A vaccine administered in addition to standard therapy

Subjects, Materials, and Methods

Subjects and proceduresMale and female subjects, 18–50 years old, with anogenital warts as defined by clinical aspect of condyloma acuminata (cauliflower) on external anogenital areas were recruited in 26 centers in 8 countries. At screening, HPV typing of anogenital warts was conducted, and, at each visit, the extent and location of the anogenital warts were mapped and photographed. The surface of each anogenital wart area was calculated by multiplying the 2 longest perpendicular diameters. Anogenital symptoms (pain, itching, and burning) and signs (ulcers, bleeding, crusts, scars, redness, and swelling) were recorded at each visit. Information about the current anogenital wart disease, sexual activity, the history of anogenital warts, previous sexually transmitted diseases, smoking habits, and general medical history were recorded at screening

Subjects were not eligible for the study if they presented with the following conditions: pregnancy or lactation, life-threatening or serious medical condition, history of autoimmune disease or of severe allergic reaction to vaccination, or current severe allergic disease at entry. If a febrile illness (temperature ⩾38°C) was present at the time of vaccination, vaccination was postponed until resolution of the fever

Subjects were stratified according to sex, country, and HPV type: HPV-6 with or without another HPV type excluding HPV-11, HPV-11 with or without another HPV type excluding HPV-6, or HPV-6 and HPV-11 with or without another HPV type. Subjects were randomized (1:1 ratio) to receive 3 doses of either the vaccine or the placebo at 2-week intervals. Local and general signs and symptoms were monitored on the day of vaccination and for the following 6 days. A urine pregnancy test was performed before each vaccination in female subjects. All subjects gave written, informed consent. The study was approved by local ethics committees and conformed with the ethical guidelines of the Declaration of Helsinki

Samples and data collectionAt screening, a cytobrush was used to obtain duplicate samples from each of the 2 largest areas with anogenital warts in each anatomic site. Another sample was obtained with a cytobrush on the occasion of the first confirmed recurrence after a period of complete remission. Anti–HPV-6 L2E7 antibodies were measured by ELISA at each visit

HPV DNA detectionIn each investigator’s laboratory, various HPV L1 consensus primer polymerase chain reaction (PCR) systems, including MY09/11, PGMY09/11, GP5+/6+, and SPF primers, were used, with detection of products and HPV typing by EIA, reverse line blot, and line probe assay systems. The samples were analyzed in the central laboratory, using a general primer GP5+/GP6+-mediated PCR-EIA method described elsewhere [25]. Briefly, this PCR-EIA method allows the detection of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk HPV types (6, 11, 40, 42, 43, and 44), after a general primer GP5+/biotinylated GP6+ (bio GP6+) PCR. For EIA analysis, GP5+/bio GP6+–generated PCR products were captured on streptavidin-coated microwells, denatured by alkaline treatment, hybridized to cocktails of digoxigenin-labeled internal oligonucleotide probes, and detected immunochemically by reading optical density values at different time intervals (1 h, 3 h, and overnight), as described elsewhere [26]. The samples were first assessed for the presence or absence of high-risk and low-risk HPV types, and when HPV was detected, type-specific PCR-EIA was performed

Vaccine and placeboThe L2E7 antigen is a fusion protein of a minor capsid protein (L2) and an early viral protein (E7) from HPV-6 [21]. The AS02A adjuvant contains 3 major components: monophosphoryl lipid A, QS21, and an oil-in-water emulsion [24]. The placebo was composed of purified lactose and of the same oil-in-water emulsion without immunostimulants (AS03). The vaccine or placebo was injected intramuscularly

Substudy-specific procedures and end pointsIn substudy A, vaccine or placebo in addition to ablative therapy was given to subjects. Eligible subjects had persistent or recurrent anogenital warts for a minimum of 3 months, with or without previous therapy. The last therapy was ended a minimum of 2 weeks before study entry. After randomization, subjects underwent removal of their anogenital warts by CO2 laser or by electrocautery at week 2 and, if necessary, at week 4, with follow-up for 6 months. The primary outcome measure was the time to recurrence of external anogenital warts from the day of administration of first ablative therapy up to month 6 after treatment

In substudy B, vaccine or placebo in addition to podophyllotoxin therapy was given to subjects. Eligible subjects had a first episode of anogenital warts or recently occurring anogenital warts and had never been treated. Subjects were given 0.5% podophyllotoxin solution to be applied twice daily for 3 days, followed by 4 days free of treatment, for 5 consecutive weeks. They were evaluated regularly for 2 months after the first vaccination. The primary outcome measure was the complete disappearance of all external anogenital warts, evaluated 2 months after the first vaccination

Statistical methodsIn substudy A, the time-to-recurrence censored data were estimated nonparametrically by the Kaplan-Meier method and were analyzed using the Cox procedure. In both substudies, binary data were compared between groups, using the Cochran-Mantel-Haenszel test controlling for country. The homogeneity of odds ratios across countries was assessed using the Breslow-Day test. Ordinal data were compared between groups by means of the extended Cochran-Mantel-Haenszel mean score test on the standardized midranks controlling for country. Continuous data were compared between groups, using an analysis of covariance. Primary analyses were performed on all subjects infected with HPV-6 and/or HPV-11 with or without another HPV type. Subanalyses of subjects infected with only HPV-6 were also performed

Results

Demographics and baseline dataA total of 457 subjects, 315 with persistent or recurrent anogenital warts and 142 with new or recent anogenital warts, were screened. There were 271 men and 182 women (sex was not recorded for 4 subjects). Enrollment started in June 1999 and ended in December 1999. Both substudies were completed in September 2000

The distribution of HPV types for all screened subjects is presented in figure 1. Overall, 32.6% of screened subjects were infection with only HPV-6, and 67.2% were infected with HPV-6 with or without another HPV type. Infection with only HPV-11 was detected in 4.8% of screened subjects, and HPV-11 was detected in 19.7% of screened subjects. A high-risk HPV type was present in 39.8% of screened subjects

Figure 1

Distribution of human papillomavirus (HPV) types among all screened subjects. HR, high risk of development of cervical cancer; LR, low risk of development of cervical cancer

Figure 1

Distribution of human papillomavirus (HPV) types among all screened subjects. HR, high risk of development of cervical cancer; LR, low risk of development of cervical cancer

In screened subjects with new or recent anogenital warts and in screened subjects with persistent or recurrent anogenital warts, HPV-6 was detected in 59.9% and 70.5%, respectively, and a high-risk HPV type was detected in 37.3% and 41.0%, respectively (figure 2). In male and female screened subjects, HPV-6 was detected in 66.0% and 68.0%, respectively, and a high-risk HPV type was detected in 35.4% and 46.7%, respectively (data not shown). There were no significant differences in the distribution of the HPV types between screened subjects with new or recent anogenital warts and screened subjects with persistent or recurrent anogenital warts or between male and female screened subjects. In total, 19.4% of screened subjects had a previous STD, including either Chlamydia trachomatis infection (10.8%), genital herpes (4.9%), or gonorrhea (2.0%)

Figure 2

Human papillomavirus (HPV) types in subjects with new or recent anogenital warts and in subjects with persistent or recurrent anogenital warts. HR, high risk of development of cervical cancer

Figure 2

Human papillomavirus (HPV) types in subjects with new or recent anogenital warts and in subjects with persistent or recurrent anogenital warts. HR, high risk of development of cervical cancer

Of the 457 screened subjects, 191 were enrolled in substudy A (ablation and vaccine or placebo). A total of 129 screened subjects (92 with new or recent anogenital warts and 37 with persistent or recurrent anogenital warts) were enrolled in substudy B (podophyllotoxin and vaccine or placebo)

In substudy A, there was no significant difference in the mean age and the male:female ratio the between groups (table 1). Most subjects were current or previous smokers (72.2% and 74.4% in the vaccine and placebo groups, respectively). The majority of subjects were infected with HPV-6, with or without another HPV type (91.7% and 92.5% in the vaccine and placebo groups, respectively), and a high-risk HPV type was present in 36.1% and 36.2% of subjects in the vaccine and placebo groups, respectively (table 2)

Table 1

Characteristics of the study population

Table 1

Characteristics of the study population

Table 2

Characteristics of anogenital wart disease in the study population

Table 2

Characteristics of anogenital wart disease in the study population

In substudy B, the mean age was 28.4 years and 25.8 years, and the male:female ratio was 1.6:1 and 1.2:1 in the vaccine and placebo groups, respectively (table 1). Overall, 70.5% of subjects were current or previous smokers. HPV-6 was detected in most subjects (92.2% and 87.7% in the vaccine and placebo groups, respectively), and a high-risk HPV type was present in 29.5% of subjects (table 2)

Analysis of efficacyIn substudy A, there was no significant difference between groups in the number of recurrences up to month 6 after treatment (P=.246) (table 3). Similarly, subjects in both groups underwent a similar number of ablative therapy sessions (P=.434). The distribution of recurrences was not significantly different between groups (P=.2097) (figure 3). A subanalysis of subjects infected with only HPV-6 yielded similar results

Table 3

Ablative therapy and vaccine or placebo (substudy A): efficacy at month 6 after treatment in patients with persistent or recurrent anogenital warts

Table 3

Ablative therapy and vaccine or placebo (substudy A): efficacy at month 6 after treatment in patients with persistent or recurrent anogenital warts

Figure 3

Kaplan-Meier estimates of survival in patients in substudy A with anogenital warts. Shown are recurrences during the 6-month study period in subjects receiving ablation and vaccine or placebo (solid line) or ablation and placebo (dashed line). The primary end point was time to first recurrence

Figure 3

Kaplan-Meier estimates of survival in patients in substudy A with anogenital warts. Shown are recurrences during the 6-month study period in subjects receiving ablation and vaccine or placebo (solid line) or ablation and placebo (dashed line). The primary end point was time to first recurrence

In substudy B, there was no significant difference between the vaccine and placebo groups at month 2 after treatment in the complete clearance of anogenital warts (48.4% vs. 46.2%; P=.673), the clearance of initial anogenital warts (57.8% vs. 64.6%; P=.459), or the partial clearance of anogenital warts (68.8% vs. 78.5%; P=.131) (table 4). However, a subanalysis of subjects infected with only HPV-6 showed that there was a positive trend toward complete clearance in the vaccine group (51.6% vs. 33.3%; P=.246), although the difference between the groups was not significant, perhaps because of the small number of subjects studied (33 in the vaccine group and 31 in the placebo group)

Table 4

Podophyllotoxin plus vaccine or placebo study (substudy B): efficacy at month 2 after treatment

Table 4

Podophyllotoxin plus vaccine or placebo study (substudy B): efficacy at month 2 after treatment

Serological analysisAll subjects in both studies mounted anti-L2E7 antibody responses after vaccination. In substudy A, subjects had a maximum geometric mean titer of 1150 pg/mL after 3 vaccinations, and, at that time, all subjects had antibody titers above the cutoff level (20 pg/mL). The antibody titers decreased rapidly, to a geometric mean of 249.2 pg/mL at month 6 after treatment. In substudy B, subjects had a maximum geometric mean titer of 1377.5 pg/mL after 2 injections, and, at that time, all subjects had antibody titers above the cutoff level. Again, the titers decreased to a geometric mean of 196 pg/mL at month 6 after treatment

SafetyIn substudy A, 97.6% of subjects in the vaccine group and 84.9% of subjects in the placebo group reported general and local symptoms. The most frequently reported local symptoms in the vaccine and placebo groups were pain at the injection site (83.6% and 63.3%), followed by redness (40.4% and 26.5%) and swelling (20.2% and 10.9%); grade 3 pain was reported by 2.2% and 0.7% of subjects, respectively. The most frequently reported general symptoms in the vaccine and placebo groups were fatigue (36.2% and 38.2%), myalgia (29.6% and 22.2%), and headache (22.6% and 11.6%). Grade 3 general symptoms were rarely reported. No vaccine-related serious adverse event was recorded. The safety profile was identical in substudy B

Discussion

Anogenital warts are benign HPV-induced anogenital lesions and are among the most prevalent STDs worldwide [1]. They are caused primarily by HPV-6 and, to a lesser extent, by HPV-11. The present study examined the largest cohort of subjects with external anogenital warts for whom full clinical and virological information was available. HPV-6 was the most frequently detected HPV type and was found in 67.2% of screened subjects. However, infection with only HPV-6, only HPV-11, or HPV-6 and HPV-11 was detected in 32.6%, 4.8%, and 6.8% of screened subjects, respectively. All other screened subjects, 55.8% of the total, were either infected with HPV-6 and/or HPV-11 and coinfected with other HPV types, predominantly high-risk HPV types, or were infected with HPV types other than HPV-6 and HPV-11. A high-risk HPV type was detected in 39.8% of screened subjects

Few other studies have examined infections with multiple HPV types by use of PCR techniques. Greer et al. [27] examined 37 subjects with anogenital warts: HPV-6 was detected in 94% of subjects, HPV-11 was detected in 8% of subjects, and other HPV types were detected in 30% of subjects. Eight subjects had >1 HPV type. Brown et al. [28] recruited 41 otherwise healthy and 24 immunosuppressed patients. Anogenital warts were removed by surgical excision and were analyzed by PCR. All but 2 anogenital warts (96.9%) contained either HPV-6 or HPV-11. Although HPV-6 was predominant in healthy subjects, HPV-11 was the most frequent type detected in immunosuppressed patients. In addition, coinfection with high-risk HPV types was more frequent in immunosuppressed (100%) than in healthy (44%) subjects

Our results are in accordance with those of Brown et al. [28], but our cohort was larger. The high rate of coinfection does not necessarily mean that there is a potential causal relationship between infection with non-6, non-11 HPV types and the development of anogenital warts; it could mean that subjects with anogenital warts have a high risk of coinfection with high-risk HPV types. Coinfection of anogenital warts could represent either double infection of individual cells [29] or regionally separate infections within individual anogenital warts [30]. Cervical infection with one HPV genotype is known to increase the risk of coinfection with another [31]. The ability of HPV to down-regulate local innate immune responses may facilitate the development of such HPV coinfections [10, 11], and such phenomena may play a role in HPV coinfection in anogenital warts. Several studies have shown a higher risk of cervical cancer in women who previously had anogenital warts [32–35]. In a large case-control study of 10,838 women in Denmark, women who previously had anogenital warts were 1.9 times more likely than other women to report an abnormal result of a cervical smear [36]. The presence of anogenital warts could, therefore, be a marker for subjects at high risk of anogenital and cervical infection with high-risk HPV types

Smoking has been reported to be associated with cervical neoplasia, and it has been suggested to act as a cofactor for HPV infection [37–40]. Smoking has also been associated with the risk of having anogenital warts [36, 38]. Similarly, in the present study, >70% of subjects were either current or previous smokers

Approximately 20% of subjects in the present study reported a history of other STDs, predominantly C. trachomatis infection. In the Danish cohort noted above, women who previously had C. trachomatis infection were 30% more likely than other women to have had anogenital warts [36]

In the present study, subjects with either new or recent anogenital warts were treated with a 0.5% podophyllotoxin solution. The cure rate at month 2 after treatment was 46.2% for complete clearance of all anogenital warts, including those appearing after the initiation of therapy. The clearance rate of anogenital warts that were present at study entry was 64.6%, and these rates are very similar to those in studies of podophyllotoxin monotherapy [7, 8]

The failure of therapeutic vaccination in the present study was disappointing. L2 antigen has been shown to induce either neutralizing antibodies or a cellular response, depending on the model [20, 41]. E7 antigen is recognized as a major target for therapeutic vaccination, mainly against high-risk HPV types [42–44]. Goldstone et al. [45] reported results from an open study of patients with high-grade squamous intraepithelial lesions who also had anogenital warts. They were vaccinated with HPV-16 E7 protein fused to heat-shock protein 65 (Hsp65) from Mycobacterium bovis Six months after vaccination, 3 (21%) of 14 patients had a complete regression of anogenital warts, and 10 patients had a reduction in anogenital wart size. These results are somewhat surprising in view of the absence of cross-reactivity between HPV-16 and the causative agents of benign anogenital warts—namely, HPV-6 and HPV-11. Because of the absence of a control group, a spontaneous regression—or, alternatively, a nonspecific effect induced by Hsp65—cannot be excluded

In the present study, the reason for the failure of therapeutic vaccination may be that mucocutaneous lesions are not accessible by a systemic immune response or that the immune response induced by the vaccine was not adequate. AS02A is a strong CD4+ T cell inducer [23], yet, although it also induces a cytotoxic response in humans [24], studies have not demonstrated an induction of CD8+ T cells. However, a CD8+ T cell response may not be needed, because the majority of the T cells infiltrating spontaneously regressing anogenital warts are CD4+ T cells [13]. Therefore, this adjuvant was probably the correct one to use. In addition, vaccination was administered with standard therapies in an attempt to cause a local inflammatory reaction and attract CD4+ T cells primed by vaccination

The failure of the vaccine could have resulted from choosing the incorrect antigens. L2 is a weaker inducer of neutralizing antibodies and cellular immunity than is the major capsid protein L1, which is the major candidate for a prophylactic vaccine. E7 may be expressed too late in the virus cycle, and early proteins such as E2 or E4 may be preferable choices. Finally, in the construct of the fusion protein L2E7, the E7 part is much smaller than the L2 moiety, and the T cell epitopes on E7 may have been hidden. To verify this hypothesis, we measured the cell-mediated immune response by stimulating peripheral-blood lymphocytes from vaccinated subjects with either L2E7 or E7 proteins or peptides. A strong response to L2E7 was detected but no response to E7 was found (data not shown). Therefore, this factor may have contributed to the failure of the vaccine. In addition, the high rate of coinfection may have played a role in the maintenance of anogenital warts. The positive trend toward clearance seen in patients with anogenital warts due to infection with only HPV-6 supports this hypothesis

In conclusion, our data suggest that coinfection with multiple HPV types is a common phenomenon in external anogenital warts. It cannot be ascertained whether multiple HPV types play an active role in the development of anogenital warts, but the presence of anogenital warts should be considered a risk factor for infection with high-risk HPV types. The failure of therapeutic vaccination deserves further evaluation, so that the immune mechanisms involved in the regression of HPV-induced anogenital warts can be clarified

Acknowledgments

The following investigators participated in the study. Canada: F. Aoki, University of Manitoba, Winnipeg; W. Gulliver, St. Johns; D. Haase, Victoria General Hospital, Halifax; M. Lassonde, Centre de Recherche–Centre Hospitalier Universitaire de Montréal, Montreal; A. Martel, Centre Medical Halles St. Foy, St. Foy; K. Papp, Probity Medical Research, Waterloo; B. Romanowski, Sexually Transmitted Disease Control, Edmonton; M. Roy-Fortier, Hôpital St. Sacrement, Quebec; J. Sellors, Master University Medical Center, Hamilton. Denmark: C. Sand-Petersen, Bisjebjerg Hospital, Copenhagen. Finland: A. M. Ranki, Helsinki University Hospital; A. Vaalasti, Tampere University Hospital, Tampere. France: R. Barrasso, Bichat and Salpetriere University Hospitals, Paris; M. H. Cayrol, Clinique St. Jean de Languedoc, Toulouse; Y. Drouault, Hôpital Tarnier, Paris; M. Janier, Hôpital St. Louis, Paris; J. P. Ortonne, Hôpital de l’Archet, Nice; F. Pouget, Hôpital Henri Mondor, Creteil. Germany: E. Petersen, Albert Ludwig Universität, Freiburg; T. Krieg, Universität zu Köln, Köln; U. Wagner, Universitätsklinikum Tubingen, Tubingen. Norway: H. Moy, Grensen Klinic for Sexuell Helse, Oslo. New Zealand: M. Reid, Auckland Sexual Health Center, Auckland. The Netherlands: I. Cairo, STD Clinic, Municipal Health Service, Amsterdam; W. Van der Meijden, Academish Ziekenhuis Rotterdam, Rotterdam; C. J. L. M. Meijer and J. Walboomers, University Hospital, Vrije Univesiteit, Amsterdam (central laboratory). Sweden: G. Johanisson, Sociale Huset, Göteborg; P. Lidbrink, Hudkliniken, Huddigne; A. Strand, Akademiska Sju Khuset, Uppsala. United Kingdom: C. J. N. Lacey, University of York, York

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Presented in part: 18th International Papillomavirus Conference, Barcelona, 21–28 July 2005 (abstract 128)
Financial support: GlaxoSmithKline Biologicals
Potential conflicts of interest: P.V. and M.W. are GlaxoSmithKline employees
Deceased