CC BY-NC-ND
0Contents lists available at SciVerse ScienceDirect
Trials in Vaccinology
journal homepage: www.elsevier.com/locate/trivac
Clinical evaluation to confirm the manufacturing consistency of three lots of an adjuvanted glycoprotein D genital herpes vaccine in healthy seronegative pre-teen and adolescent girls: A phase III multi-center double-blind randomized trial
Geert Leroux-Roels a'*, Simon Dobsonb, David I. Bernsteinc, Sandra Fowlerd, Barbara Romanowskie, Isabel Leroux-Roels a, Brigitte Cheuvartf, Thomas Heineman g, Gary Dubin g
a Center for Vaccinology - CEVAC, Ghent University and Hospital, Ghent, Belgium b BC Children's Hospital and University of British Columbia, Vancouver, BC, Canada
c Cincinnati Children's Hospital Medical Center, and the University of Cincinnati College of Medicine, Cincinnati, OH, USA d Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA e Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada fGlaxoSmithKline Vaccines, Wavre, Belgium g GlaxoSmithKline Vaccines, King of Prussia, PA, USA
ARTICLE INFO ABSTRACT
Objective: To evaluate the consistency of three commercial scale lots of candidate herpes simplex virus (HSV) type 2 (HSV-2) vaccine in adolescent girls.
Methods: A total of 554 healthy girls aged 10-17 years, from Belgium, Canada, and United States, were enrolled and randomized to receive one of the three manufacturing lots of the candidate glycoprotein D2 vaccine (gD2-AS04, GlaxoSmithKline Vaccines) according to a 0,1 and 6-month schedule. Consistency was based on anti-gD geometric mean titers (GMTs) 1 month post-dose 3 among HSV seronegative subjects complying to study procedures (N = 312): two-sided 90% confidence interval (CI) for the GMT ratio between each pair of vaccine lots had to be within the [0.67; 1.5] consistency interval. Results: Pre-specified consistency criteria were reached. At month 2 (1 month after the second vaccine dose) anti-gD antibodies were detected in all study participants, while the proportion of subjects with HSV-2 neutralizing antibodies ranged from 93% to 96.2%, remaining >90% throughout the study (between 93.7% and 96.1% for the three vaccine lot groups at month 12). The three vaccine lots had similar reac-togenicity profiles. The incidence of grade 3 solicited or unsolicited adverse events (AEs) ranged from 17.9% to 22.2% of subjects.
Conclusions: This study demonstrated the lot-to-lot consistency of three commercial scale production lots of herpes simplex candidate vaccine. The vaccine was immunogenic and had a clinically acceptable safety profile when administered in HSV type 1 and HSV-2 seronegative girls aged 10-17 years. The study was registered at clinicaltrials.gov (identifier NCT00224471).
© 2013 Elsevier Ltd. All rights reserved.
CrossMark
Article history: Received 31 October 2012 Revised 18 March 2013 Accepted 18 March 2013
Keywords:
Herpes simplex virus Subunit vaccine Lot consistency Safety
Immunogenicity
1. Introduction
Herpes virus type 2 (HSV-2) infection is one of the most common sexually transmitted diseases, with a reported seropreva-lence varying considerably among different populations and age groups [1,2]. A systematic review of the literature estimated that in 2003 there were 536 million persons 15-49 years old who
* Corresponding author. Address: Ghent University, Center for Vaccinology -CEVAC, Ghent University Hospital, Blok A - 1st Floor, De Pintelaan 185, 9000 Gent, Belgium. Tel.: +32 9 332 36 54; fax: +32 9 332 63 11.
E-mail address: geert.lerouxroels@ugent.be (G. Leroux-Roels).
1879-4378/$ - see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.trivac.2013.03.001
were HSV-2 seropositive, accounting for 16% of the world's population in this age group [1]. Estimated prevalence varied by geographical region: in Western Europe, HSV-2 infection was reported in 18% of women and 13% of men [1,3], while in countries from Sub-Saharan Africa up to 70% of women and 55% of men were infected [1,4]. In the US, the National Health and Nutrition Examination survey reported a 30% increase in HSV-2 seropreva-lence between the periods 1976-1980 and 1988-1994 [5]; HSV-2 seroprevalence then decreased to an estimated 17% during the period 1999-2004 [6], and remained relatively stable between 2005 and 2008 [7].
Recent studies have focused on the interaction between human immunodeficiency virus (HIV) and HSV-2 infection [8,9]. Although initially considered to be an opportunistic infection, it is now accepted that HSV-2 potentiates the risk of HIV acquisition and transmission [10]. The results of a meta-analysis of 19 longitudinal studies showed that HSV-2 seropositivity increased the risk of acquiring HIV infection 3-fold [10].
HSV infection in women of reproductive age is associated with the riskofviral transmission from motherto the neonate [11], with potential consequences of spontaneous abortion, intrauterine growth retardation, preterm delivery, intrauterine or postnatal death or severe neurodevelopmental disabilities [11,12]. HSV transmission can occur in utero (ascending infection or transplacental due to maternal viremia HSV infection), during delivery (85-90% of neonatal HSV infections) or postnatally [11,12]. The risk of transmission is higher in women with primary infection during pregnancy than in women with recurrent infection [13].
High prevalence and the potential social and psychological impact of genital herpes infection have stimulated interest in the development of a vaccine to prevent infection or disease. With the incidence of HSV-2 doubling between the age groups 12-19 and 20-29 years [14], female adolescents represent a target population for potential HSV-2 vaccines.
The prophylactic candidate vaccine developed by GlaxoSmithK-line Vaccines is an aluminum hydroxide and 3-O-deacylated monophosphoryl HSV-2 gD-based subunit vaccine formulated in adjuvant system (AS04), lipid A (MPL). HSV-2 gD is obtained from Chinese hamster ovary cells transfected with a gene encoding a truncated form of gD from the HSV-2 strain G [15]. The immunoge-nicity and safety of this candidate vaccine have been previously assessed in clinical trials [15,16]. However, most of these studies were conducted in adults.
Clinical evaluations to confirm the manufacturing consistency of consecutively produced lots of vaccine intended for commercial use are prerequisites for licensure [17,18]. The aim of these trials is to confirm the reliability of the manufacturing process following transition from small-scale to full-scale production. The study reported here was designed to evaluate the consistency of three commercial scale lots of the candidate HSV-2 vaccine in HSV type 1 (HSV-1) and HSV-2 seronegative girls aged 10-17 years.
2. Methods
2.1. Study objectives
The primary objective of this trial was to demonstrate, 1 month following the third dose, the lot-to-lot consistency of three different commercial scale production lots of herpes simplex candidate vaccine gD2-AS04 as determined by GMTs of antibodies against HSV gD (anti-gD) 1 month post dose 3.
Secondary objectives were to evaluate the immune response rate based on anti-gD and anti-HSV neutralizing antibody responses and to describe safety and reactogenicity of the HSV candidate vaccine in all treatment groups.
2.2. Study design and participants
This was a phase III, multicenter, double-blind, randomized study, with three parallel treatment groups (registered at www.ClinicalTrials.gov: NCT00224471).
It was conducted at 13 study centers in three countries (Belgium, Canada, and United States) and aimed to enroll 522 healthy HSV-1 and HSV-2 seronegative girls at initial screening who were 10-17 years of age at the time of first vaccination. Exclusion criteria included: pregnancy (urine pregnancy test or
lactation; any previous history, or current clinical signs or symptoms of oro-labial, genital or non-genital HSV disease; previous vaccination against herpes; previous administration of MPL adjuvant; any confirmed or suspected immunosuppressive or immunodeficient condition; history of allergic disease or reactions likely to be exacerbated by any component of the study vaccines. If the participant was of childbearing potential, she was asked to be abstinent or use an effective method of birth control for 30 days prior to vaccination and agreed to continue such precautions for 2 months after completion of the vaccination series.
Enrollment began on February 3, 2004. Participants were stratified into two age groups, 10-15 years and 16-17 years, ensuring that no more than 50% and no fewer than 25% of subjects were in the 16-17 years age stratum. Enrolled subjects were randomized using a central randomization system on the Internet according to a balanced allocation (1:1:1) to receive one of three lots of the candidate gD2-AS04 vaccine. The randomization algorithm was based on minimization accounting for center and age stratum with equal weight. Each participant was followed for 12 months with visits at months 0,1, 2, 6, 7 and 12. At each visit, participants were provided individual, age-appropriate counseling on pregnancy prevention and methods to reduce risk of sexually transmitted infections.
The study protocol and all study-related documents were approved by the national, regional, or investigational center Independent Ethics Committee or Institutional Review Board. Written informed consent or assent was provided by all participants prior to the performance of any study-specific procedures.
2.3. Study vaccines
The vaccine used in this study was developed and manufactured by GlaxoSmithKline [15,16,19]. It contained 20 ig of truncated glycoprotein D from HSV-2 strain G and the AS04 adjuvant system comprising 50 ig of MPL, and 0.5 mg of aluminum hydroxide.
Participants were randomly allocated to receive one of three manufacturing lots of the vaccine as follows: AHS1020A2 (group A), AHS1021A2 (group B) and AHS1022A2 (group C). The study vaccine was administered intramuscularly in the deltoid region of the non-dominant arm according to a 0, 1 and 6 months schedule.
2.4. ¡mmunogenicity evaluation
Initial screening for HSV serostatus was performed to determine eligibility for entry into the study, using both the HSV non-type specific Enzygnost® anti-HSV IgG enzyme-linked immunosorbent assay (ELISA) (Behring) and the HSV-2 type specific HerpeSelect-2™ ELISA (Focus Technologies). All participants determined to be seronegative by these two ELISA assays were considered eligible and were included in the total vaccinated cohort (TVC). Participants' serostatus was later confirmed by Western blot (University of Washington, Seattle, WA) [20] on all serum samples collected at study entry and by an anti-gD in-house ELISA (described below) on serum samples collected at time of randomization (month 0) for all participants included in the TVC. A participant was considered HSV seropositive by Western blot if two of three bands (for glycoprotein B, virion polypeptide 5, and infected cell protein-35) were visualized. The confirmed results of participants' serostatus were not available at the time of randomization. Participants seropositive by Western blot or anti-gD in-house ELISA before vaccination were excluded from the according-to-protocol (ATP) cohort.
Anti-gD and anti-HSV neutralizing antibody titers were measured in serum samples for all subjects at months 0, 2, 7 and 12 in a central laboratory. The anti-gD in-house ELISA was performed
as follows: 96-well plates were coated with the gD antigen, previously diluted in phosphate buffered saline to give a concentration of 2.5 ig/ml. Subsequently participant's sera were added to the plate and allowed to incubate for 2 h at 18-22 °C. After washing five times with NaCl Tween (Merck) 0.05% buffer, the presence of anti-gD antibodies was revealed by addition of an anti-human conjugate. Human control sera with known titers were included in each assay for validation of the results. Anti-gD antibody titers were expressed in ELISA units (EU)/ml, with reference to a standard serum, using the 4-parameter method [21]. The assay cutoff titer was based on the evaluation of 110 sera samples from healthy subjects enrolled in another HSV study that tested seronegative for IgM and IgG antibodies against HSV using two kits from Behring. The cut-off had to be higher than the range of test results for these sera and the lowest limit of quantitation. The lowest limit of quantitation was established by multiplying the lowest dilution of the test by the concentration corresponding to 10% optical density and calculating the values from 100 different standard curves. The results obtained for the control sera ranged from 0 to 31 EU/ml and the lowest limit of quantitation was 30 EU/ml (standard deviation: 7 EU/ml). Based on these data, the cut-off for the in-house ELISA used to determine the anti-gD antibody titers was set at 40 EU/ml. Participants with titers p40 EU/ml were considered seropositive [16]. Anti-HSV neutralizing antibody titers were assessed using an in-house water-soluble tetrazolium salt (WST-1)-based colorimetric assay [22,23]. Serially diluted sera from participants were incubated with HSV-2 virus particles and complement for 2 h at 37 °C in 96-well plates before adding a Vero cell suspension. After a 6-day incubation period at 37 °C in the presence of 5% CO2 in air, the culture medium was removed and cell viability was determined by measuring the optical density at k = 450 nm. The assay cut-off was set at 4 arbitrary units based on the estimated dose at which 90% of the virus is neutralized (ED90). Seropositivity rates and GMTs, with 95% CI, were calculated for anti-gD antibodies and anti-HSV neutralising antibodies by group at all time points for which blood samples were taken.
2.5. Safety evaluation
Safety was assessed through self-reporting of solicited AEs (on the day of vaccination and 6 days following each dose), unsolicited AEs (occurring within 30 days after each dose, reported by investigator). Serious adverse events (SAEs), new onset chronic diseases (NOCD), other medically significant conditions and conditions prompting emergency room (ER) visits or physician visits that were not related to common diseases were recorded in all subjects throughout the entire study period, regardless of causal relationship to vaccination or intensity.
2.6. Statistical analysis
The primary endpoint was anti-gD antibody titer assessed at month 7 in the ATP cohort. The ATP cohort included subjects vaccinated with three doses as per protocol, who were confirmed to be HSV seronegative pre-vaccination by both Western blot and anti-gD in-house ELISA assay and who had immunogenicity results available post dose 3. Consistency analysis was based on the two-sided 90% CI for the GMT ratio between each pair of gD2-AS04 vaccine lots and was demonstrated if the lower and upper limits of the 90% CI were within the [0.67;1.5] consistency interval. The GMT group ratios were derived from group comparison in a one way ANOVA model on log-transformed titer. To be consistent with the industry post-hoc standards for consistency, the 95% CIs for the month 7 anti-gD GMT ratios were also computed between each pair of gD2-AS04 vaccine lots using an ANOVA model that included vaccine group (each group), age (615 years, >15 years at
dose 1) and Western blot status at pre-vaccination (HSV 1-/2-: yes/no) as co-variables. The study was not statistically powered for this analysis because it was not planned per protocol.
Secondary endpoints were: anti-gD antibody titer at months 2 and 12; anti-HSV neutralizing antibody titers at months 2, 7 and 12; occurrence of solicited (day 0-6) and unsolicited symptoms (day 0-29); occurrence of NOCD, other medically significant conditions or SAEs throughout the entire study period. All local injection site symptoms (solicited or unsolicited) were considered related to vaccination.
With respect to the descriptive analysis of immunogenicity, the percentages of subjects with anti-gD and anti-HSV neutralizing antibody titers at or above the pre-defined cut-off values were tabulated with exact 95% CIs [24] and GMTs were tabulated with 95% CIs for anti-gD and anti-HSV neutralizing antibody titers, respectively. With respect to the descriptive analysis of safety, the percentage of subjects reporting safety endpoints was provided with exact 95% CI. These analyses were also performed by age (1015 years old and 16-17 years old) and by weight (655 kg and >55 kg), for the three gD2-AS04 lots pooled.
The sample size of 146 subjects per lot in the ATP cohort was targeted to reach at least 90% power for meeting simultaneously the consistency criteria for the three pairwise lot comparisons. The sample size was based on three equivalence tests with 5% alpha under the alternative hypothesis that the three lots were identical with a population standard error for a log10 transformed titer of 0.384. The study was not powered to be analyzed with 95% CI, i.e. 2.5% alpha. Assuming a rate of non-evaluable participants of 20%, 522 participants were planned to be enrolled in this study, with 184 participants in each study group.
3. Results
3.1. Study population
A total of 554 participants were enrolled and randomized. All participants received at least one dose of study vaccine and were included in the total vaccinated cohort (TVC) (Fig. 1). Twelve participants withdrew from the study prematurely: 4 were lost to follow-up, 3 withdrew consent and 5 moved from the study area. 542 participants (97.8% of the TVC) completed the study.
The ATP cohort for immunogenicity included 312 participants who were negative for both HSV-1 and HSV-2 antibodies by Western blot and anti-gD in-house ELISA. 242 participants were eliminated from the ATP cohort for immunogenicity due to noncompliance with study eligibility criteria (initially seropositive or unknown serostatus), noncompliance with vaccination or blood sampling schedule, or missing serological data (Fig. 1). No participants prematurely discontinued from the study due to AEs.
The mean age in the three treatment groups ranged from 14.2 to 14.4 years; 87.5% of the study participants were White/Caucasian (Table 1). At the time of the first dose, the majority of participants in each group were negative for both HSV-1 and HSV-2 antibodies by Western blot (ranging from 79.9% to 83.8% across the three groups). Approximately 10% of participants were positive for HSV-1 and negative for HSV-2 by Western blot (ranging from 9.2% to 12.4% across the three groups).
3.1. ¡mmunogenicity
The protocol-defined criteria for lot-to-lot consistency at month 7 (1 month after the third dose) were met (primary objective) (Table 2). The two-sided 90% CI of the anti-gD GMT ratio for each pair of groups was within the pre-defined range [0.67; 1.5], thus demonstrating lot-to-lot consistency.
ATP cohort for immunogenicity
(N = 104)
81 participants excluded (6 due to administration of vaccines forbidden; 29 due to noncompliance with study eligibility criteria; 31 initially seropositive or unknown antibody status*; 10 due to noncompliance with the vaccination schedule; 4 due to noncompliance with the blood sampling schedule; 1 with essential serological data missing)
ATP cohort for immunogenicity
(N = 101)
83 participants excluded (3 due to administration of vaccines forbidden; 36 due to noncompliance with study eligibility criteria; 23 initially seropositive or unknown antibody status*; 18 due to noncompliance with the vaccination schedule; 1 due to noncompliance with the blood sampling schedule; 1 with essential serological data missing; 1 other reason)
ATP cohort for immunogenicity
(N = 107)
78 participants excluded (2 due to administration of vaccines forbidden; 37 due to noncompliance with study eligibility criteria; 21 initially seropositive or unknown antibody status*; 15 due to noncompliance with the vaccination schedule; 1 due to noncompliance with the blood sampling schedule; 1 with essential serological data missing; 1 other reason)
Fig. 1. Study flow chart. Group A = gD2-AS04 vaccine lot AHS1020A2; Group B = gD2-AS04 vaccine lot AHS1021A2; Group C = gD2-AS04 vaccine lot AHS1022A2; N = number of subjects; TVC = total vaccinated cohort; ATP = according to protocol; N1N2 = negative for HSV-1 and negative for HSV-2 by Western blot; P1N1 = positive for HSV-1 and negative for HSV-2 by Western blot; »Not confirmed seronegative by both Western blot and anti-gD in-house ELISA.
When using post-hoc consistency criteria based on today's standard, namely two-sided 95% CIs, all but one pair of lots led to CI within the [0.67; 1.5] interval; for the comparison of group B with group C, the 95% CI of the age-adjusted ratio (B over C) was [0.66; 0.96]. As explained, this post-hoc consistency criterion was not planned and therefore the study was not powered for this analysis. When the analysis was repeated on a larger sample also including 82 participants, who were seronegative by ELISA but whose pre-vaccination HSV serostatus was not confirmed by Western blot, the two-sided 95% CIs for each comparison were within the [0.67; 1.5] interval.
3.1.1. HSV glycoprotein D specific antibodies (Fig. 2)
All subjects in the three vaccine lot groups, irrespective of age, weight or menarcheal status, were seropositive for anti-gD after the second dose and remained so through month 12 (approximately 6 months after the third dose). Between month 2 and month 7, anti-gD GMTs levels increased 1.7-2.1-fold in all vaccine lot groups. Levels then decreased 3.6-3.9-fold by month 12.
Descriptive analysis of the anti-gD response over the course of the study showed higher antibody responses in the younger age stratum than the older age stratum and in the 655 kg stratum than
the >55 kg stratum at each post-vaccination time point. GMT ratios of the 10-15 year age group over the 16-17 year age group were: 1.4 at month 2, 1.2 at month 7 and 1.0 at month 12. When GMTs in the 655 kg and >55 kg groups were compared, the ratios were as follows: 1.5 at month 2, 1.2 at month 7 and 1.2 at month 12.
3.1.2. HSV neutralizing antibodies (Fig. 2)
At month 2 (1 month after the second vaccine dose), anti-HSV seropositivity rates for neutralizing antibodies ranged from 93% (group A) to 96.2% (group C) and remained >90% throughout the study duration (ranging from 93.7% to 96.1% across the three vaccine lot groups at month 12).
GMTs for anti-HSV neutralizing antibodies displayed similar kinetic profiles in all the vaccine lot groups through the course of the study. From pre-vaccination to 1 month post dose 2, anti-HSV GMTs increased in all groups, with values ranging from 16.6 ED90 in group A to 21.7 ED90 in group C. GMTs reached peak levels at month 7, 1 month after the third vaccine dose. Between month 2 and month 7, anti-HSV GMTs increased 3.9-fold in group A (GMT = 16.6 ED90 and 65.1 ED90 at month 2 and month 7, respectively), 3.2-fold in group B (GMT = 19.4 ED90 at month 2 and 62.6 ED90 at month 7) and 3.5-fold in group C (GMT = 21.7 ED90
10000 -
pre-vacc Month 2 Month 7 Month 12
pre-vacc Month 2 Month 7 Month 12
Fig. 2. Geometric mean titers for gD specific antibodies (panel A) and HSV neutralizing antibodies (panel B). Group A = gD2-AS04 vaccine lot AHS1020A2; Group B = gD2-AS04 vaccine lot AHS1021A2; Group C = gD2-AS04 vaccine lot AHS1022A2; GMT = geometric mean antibody titer.
at month 2 and 76.2 ED90 at month 7). From month 7 to month 12, GMT values decreased between 3.6- and 3.8-fold in all three vaccine lot groups, but remained higher than the baseline levels.
Data were also analyzed by age groups and weight for the three lots pooled. One month after the second dose, seropositivity rates were 98% in the 655 kg group and 92% in the >55 kg group, increasing to 99.4% and 98.7% after the third dose. At all time points, anti-HSV GMTs were higher in the 655 kg group than in the >55 kg group. GMT ratios for the two weight groups (655 kg and >55 kg) were as follows: 1.3 at month 2, 1.0 at month 7 and 1.1 at month 12. By age stratum, seropositivity rates after the second dose were 93.6% in 16-17 year olds and 95.9% in 10-15 year olds, increasing to 98.5% and 100% after the third dose. Seropositiv-ity rates remained >90% at month 12 irrespective of age.
3.2. Safety and reactogenicity
Overall, the percentage of subjects reporting at least one symptom after all doses (solicited or unsolicited, local or general) ranged from 95.7% to 96.7%. At least one AE (solicited or unsolicited) was reported for 95.7% to 96.7% of subjects. Overall, the incidence of
grade 3 AEs (solicited or unsolicited) ranged from 17.9% to 22.2% (Supplementary Table). The incidence of grade 3 AEs considered by the investigator to be related to study vaccine was 15.7% in group A, 15.8% in group B and 17.3% in group C.
3.2.1. Solicited adverse events (Table 3)
Injection site pain was the most frequently reported solicited local symptom after each vaccine dose in all three vaccine lot groups (reported by 91.4%, 91.8% and 89.2% of participants in groups A, B and C, respectively). At least 7.6% of participants in each group reported grade 3 injection site pain after vaccine administration.
The most frequently reported solicited general symptoms were fatigue (reported by 35.7%, 47.3% and 49.7% of participants in groups A, B and C, respectively) and headache (reported by 41.1%, 48.4% and 53.5% of participant in groups A, B and C, respectively). The most commonly reported grade 3 solicited general symptoms per group were fatigue in group A (2.2% of subjects), fatigue and headache in group B (both reported in 3.3% of subjects) and malaise in group C (7.0% of subjects). Fever (defined as oral
Table 1
Demographic characteristics of vaccinated participants (total vaccinated cohort).
Characteristics
Total N =554
Group A N =185
Group B N =184
Group C N =185
Age (years)
Mean ± SD 14.3 ±2.1
Median 15.0
Range (min-max) 10-17
BMI (kg/m2)
Mean ± SD 21.7 ±4.2
Median 20.9
Race, n (%)
White/Caucasian 485 (87.5)
Black 25(4.5)
East/South East Asian 9 (1.6)
South Asian 5 (0.9)
American Hispanic 15 (2.7)
Other 15 (2.7)
Menarcheal status at baseline, n (%)
Pre-menarcheal 112 (20.2)
Post-menarcheal 442 (79.8)
Western blot at baseline, n (%)
N1N2 450 (81.2)
P1N2 58 (10.5)
other 46 (8.3)
14.2 ±2.2
15.0 10-17
22.1 ±4.5 20.9
160 (86.5) 10 (5.4) 3(1.6) 3(1.6) 6 (3.2) 3(1.6)
39 (21.1) 146 (78.9)
155 (83.8) 17 (9.2) 13 (7.0)
14.3 ± 2.1 14.0 10-17
21.4 ±4.4 20.7
157 (85.3) 7 (3.8) 6 (3.3) 2(1.1) 3 (1.6) 9 (4.9)
32 (17.4) 152 (82.6)
147 (79.9)
18 (9.8)
19 (10.3)
14.4 ± 2.1 15.0 10-17
21.5 ±3.8 20.9
168 (90.8) 8 (4.3) 0 (0.0) 0 (0.0) 6 (3.2) 3 (1.6)
41 (22.2) 144 (77.8)
148 (80.0) 23 (12.4) 14 (7.6)
Group A = subjects receiving gD2-AS04 vaccine lot AHS1020A2. Group B = subjects receiving gD2-AS04 vaccine lot AHS1021A2. Group C = subjects receiving gD2-AS04 vaccine lot AHS1022A2.
N = number of subjects; n/% = number/percentage of subjects in a given category; SD = standard deviation; N1N2 = negative for HSV-1 and negative for HSV-2; P1N2 = positive for HSV-1 and negative for HSV-2.
Table 2
Ratios of post-vaccination anti-gD GMT at month 7 between the three gD2-AS04 vaccine lots (according to protocol cohort for immunogenicity).
ATP cohort for immunogenicity
GMT ratio
Ratio order
Value (90% CI)
Group A Group A Group B
104 104
9012.6 9012.6 8233.6
Group B Group C Group C
99 106 106
8233.6
10,417.6
10,417.6
ATP cohort for immunogenicity
Group A/Group B Group A/Group C Group B/Group C
Adjusted GMT ratio*
1.09 (0.94-1.28) 0.87 (0.74-1.01) 0.79 (0.68-0.92)
Group description
Adjusted GMT
Group description
Adjusted GMT*
Ratio order
Value (95% CI)
Group A Group A Group B
104 104
8833.4 8833.4 8104.4
Group B Group C Group C
99 106 106
8104.4
10,142.8
10,142.8
ATP cohort for immunogenicity and subjects excluded because they were not confirmed N1N2 at baseline
Group A/Group B 1.09 (0.90-1.31)
Group A/Group C 0.87 (0.72-1.05)
Group B/Group C 0.80 (0.66-0.96)
Adjusted GMT ratio§
Group description
Adjusted GMT
Group description
Adjusted GMT§
Ratio order
Value (95% CI)
Group A Group A Group B
125 125 128
8133.4 8133.4 7689.7
Group B Group C Group C
128 138 138
7689.7
9586.8 9586.8
Group A/Group B Group A/Group C Group B/Group C
1.06 (0.90-1.25) 0.85 (0.72-1.00) 0.80 (0.68-0.94)
Group A = gD2-AS04 vaccine lot AHS1020A2; Group B = gD2-AS04 vaccine lot AHS1021A2; Group C = gD2-AS04 vaccine lot AHS1022A2.
N = number of subjects with both pre- and post-vaccination results available; GMT = unadjusted geometric mean antibody titer; GMT* = geometric mean antibody titer adjusted for age strata; GMT§ = geometric mean antibody titer adjusted for age strata and Western blot status (N1N2 Yes/No); N1N2 = negative for HSV-1 and negative for HSV-2; 95% CI = 95% confidence interval for the adjusted GMT ratio (ANOVA model)
temperature P37.5 °C) occurred with a frequency of 11.4% in groups B and C and 12.4% in group A.
3.2.2. Unsolicited adverse events (Table 4)
During the 30 days after vaccination, 60.5% participants in group A, 60.9% in group B and 61.1% in group C reported at least one unsolicited adverse event. Upper respiratory tract infections were the most frequently reported unsolicited AE in all treatment groups, with an incidence of 8.1% in groups A and C and 14.7% in group B. At least one grade 3 unsolicited AE was reported for 6.3% of participants. The most commonly reported grade 3 unsolicited AE was upper respiratory tract infection, occurring in 5
participants overall (0.9%). Unsolicited AEs prompting medical attention were reported with an incidence ranging from 21.6% in group A to 24.9% in group C.
3.2.3. Serious adverse events and new onset chronic diseases
During the study period, 13 non-fatal SAEs were reported in 13 participants: 2 in group A (1.1%), 5 in group B (2.7%) and 6 in group C (3.2%). None were considered by the investigator as being related to vaccination and all resolved.
At least one NOCD was identified by the investigator in 8.1%, 7.6% and 4.9% of participants in groups A, B and C, respectively. The most commonly reported NOCD was asthma: 2 subjects in
Table 3
Incidence of solicited local and general symptoms within the 7-day post-vaccination period (total vaccinated cohort)
Lot A N =185 Lot B N = 184 Lot C N =185 Total N = 554
n % (95% CI) n % (95% CI) n % (95% CI) n % (95% CI)
Pain 169 91.4 (86.3-95.0) 169 91.8 (86.9-95.4) 165 89.2 (83.8-93.3) 503 90.8 (88.1-93.1)
Grade 3 14 7.6 (4.2-12.4) 19 10.3 (6.3-15.7) 22 11.9 (7.6-17.4) 55 9.9 (7.6-12.7)
Redness 66 35.7 (28.8-43.0) 55 29.9 (23.4-37.1) 69 37.3 (30.3-44.7) 190 34.3 (30.3-38.4)
Grade 3a 1 0.5 (0.0-3.0) 1 0.5 (0.0-3.0) 1 0.5 (0.0-3.0) 3 0.5 (0.1-1.6)
Swelling 50 27.0 (20.8-34.0) 49 26.6 (20.4-33.6) 48 25.9 (19.8-32.9) 147 26.5 (22.9-30.4)
Grade 3a 8 4.3 (1.9-8.3) 3 1.6 (0.3-4.7) 3 1.6 (0.3-4.7) 14 2.5 (1.4-4.2)
General
Arthralgia 41 22.2 (16.4-28.8) 42 22.8 (17.0-29.6) 36 19.5 (14.0-25.9) 119 21.5 (18.1-25.1)
Grade 3 3 1.6 (0.3-4.7) 2 1.1 (0.1-3.9) 3 1.6 (0.3-4.7) 8 1.4 (0.6-2.8)
Fatigue 66 35.7 (28.8-43.0) 87 47.3 (39.9-54.8) 92 49.7 (42.3-57.2) 245 44.2 (40.0-48.5)
Grade 3 4 2.2 (0.6-5.4) 6 3.3 (1.2-7.0) 8 4.3 (1.9-8.3) 18 3.2 (1.9-5.1)
Headache 76 41.1 (33.9-48.5) 89 48.4 (41.0-55.8) 99 53.5 (46.0-60.9) 264 47.7 (43.4-51.9)
Grade 3 2 1.1 (0.1-3.9) 6 3.3 (1.2-7.0) 7 3.8 (1.5-7.6) 15 2.7 (1.5-4.4)
Malaise 56 30.3 (23.7-37.4) 57 31.0 (24.4-38.2) 70 37.8 (30.8-45.2) 183 33.0 (29.1-37.1)
Grade 3 3 1.6 (0.3-4.7) 5 2.7 (0.9-6.2) 13 7.0 (3.8-11.7) 21 3.8 (2.4-5.7)
Rash 5 2.7 (0.9-6.2) 13 7.1 (3.8-11.8) 8 4.3 (1.9-8.3) 26 4.7 (3.1-6.8)
Grade 3 0 0.0 (0.0-2.0) 0 0.0 (0.0-2.0) 0 0.0 (0.0-2.0) 0 0.0 (0.0-0.7)
Fever 23 12.4 (8.0-18.1) 21 11.4(7.2-16.9) 21 11.4 (7.2-16.8) 65 11.7 (9.2-14.7)
Grade 3b 1 0.5 (0.0-3.0) 1 0.5 (0.0-3.0) 1 0.5 (0.0-3.0) 3 0.5 (0.1-1.6)
Urticaria 2 1.1 (0.1-3.9) 2 1.1 (0.1-3.9) 5 2.7 (0.9-6.2) 9 1.6 (0.7-3.1)
Grade 3c 0 0.0 (0.0-2.0) 0 0.0 (0.0-2.0) 0 0.0 (0.0-2.0) 0 0.0 (0.0-0.7)
Group A = gD2-AS04 vaccine lot AHS1020A2; Group B = gD2-AS04 vaccine lot AHS1021A2; Group C = gD2-AS04 vaccine lot AHS1022A2; Total = data from the three treatment groups pooled; N = number of subjects with available results; n/% = number/percentage of subjects reporting the symptom at least once; 95% CI = 95% confidence interval.
a Grade 3 local injection site redness and swelling was defined as any injection site redness and swelling >30 mm and persisting for more than 24 h, i.e., for two consecutive days.
b Grade 3 fever was defined as oral temperature >39.0 "C.
c Grade 3 urticaria was defined as urticaria distributed on at least four body areas. All other symptoms were recorded as grade 3 if they prevented normal, everyday activities (in adults/adolescents, such an AE would, for example, prevent attendance at work/school and would necessitate the administration of corrective therapy).
Table 4
Percentage (%) of participants with unsolicited local and general AEs ( p5 cases) within the 30-day post-vaccination period (total vaccinated cohort).
Primary system organ class Group A N =185 Group B N =184 Group C N =185 Total N =554
n % (95% CI) n % (95% CI) n % (95% CI) n % (95% CI)
At least one symptom 112 60.5 (53.1-67.6) 112 60.9 (53.4-68.0) 113 61.1 (53.7-68.1) 337 60.8 (56.6-64.9)
Abdominal pain 5 2.7 (0.9-6.2) 6 3.3 (1.2-7.0) 6 3.2 (1.2-6.9) 17 3.1 (1.8-4.9)
Diarrhea 3 1.6 (0.3-4.7) 5 2.7 (0.9-6.2) 4 2.2 (0.6-5.4) 12 2.2 (1.1-3.8)
Nausea 9 4.9 (2.2-9.0) 6 3.3 (1.2-7.0) 6 3.2 (1.2-6.9) 21 3.8 (2.4-5.7)
Vomiting 8 4.3 (1.9-8.3) 8 4.3 (1.9-8.4) 9 4.9 (2.2-9.0) 25 4.5 (2.9-6.6)
Influenza-like illness 5 2.7 (0.9-6.2) 3 1.6 (0.3-4.7) 2 1.1 (0.1-3.9) 10 1.8 (0.9-3.3)
Injection site reaction 1 0.5 (0.0-3.0) 3 1.6 (0.3-4.7) 5 2.7 (0.9-6.2) 9 1.6 (0.7-3.1)
Nasopharyngitis 14 7.6 (4.2-12.4) 11 6.0 (3.0-10.4) 15 8.1 (4.6-13.0) 40 7.2 (5.2-9.7)
Upper respiratory tract infection 15 8.1 (4.6-13.0) 27 14.7 (9.9-20.6) 15 8.1 (4.6-13.0) 57 10.3 (7.9-13.1)
Headache 12 6.5 (3.4-11.1) 18 9.8 (5.9-15.0) 13 7.0 (3.8-11.7) 43 7.8 (5.7-10.3)
Dysmenorrhea 5 2.7 (0.9-6.2) 3 1.6 (0.3-4.7) 4 2.2 (0.6-5.4) 12 2.2 (1.1-3.8)
Cough 1 0.5 (0.0-3.0) 6 3.3 (1.2-7.0) 8 4.3 (1.9-8.3) 15 2.7 (1.5-4.4)
Oropharyngeal pain 12 6.5 (3.4-11.1) 9 4.9 (2.3-9.1) 11 5.9 (3.0-10.4) 32 5.8 (4.0-8.1)
Sinus congestion 5 2.7 (0.9-6.2) 4 2.2 (0.6-5.5) 2 1.1 (0.1-3.9) 11 2.0 (1.0-3.5)
Group A = gD2-AS04 vaccine lot AHS1020A2; Group B = gD2-AS04 vaccine lot AHS1021A2; Group C = gD2-AS04 vaccine lot AHS1022A2. Total = data from the three treatment groups pooled. N = number of subjects
n/% = number/percentage of subjects reporting the symptom at least once 95% CI = 95% confidence interval.
group A, 2 subjects in group B and 1 subject in group C. No death or pregnancy occurred during the study.
4. Discussion
The present study was designed to assess the consistency of the immune response elicited by three different commercial scale vaccine production lots in participants representing the target population of the vaccine. Demonstration of lot-to-lot consistency is
critical in the development of a vaccine [17,18]. The main objective of this trial was achieved: the two-sided 90% CIs for each GMT ratio comparing groups receiving the different vaccine lots were within the [0.67; 1.5] interval defining lot-to-lot consistency.
All vaccine lots induced a strong immune response that persisted up to the end of the trial. All subjects in the three gD2-AS04 vaccine lot groups were seropositive for anti-gD antibodies after the second dose and remained so through the end of the study. Analysis of HSV-2 neutralizing antibodies also indicated seropositivity rates >90% at each post-vaccination time point (after
the second and the third vaccine dose and at month 12). These results in adolescent girls aged 10-17 years are consistent with those previously observed in adult women [16,19]. Furthermore, the administration of the third vaccine dose was followed by a robust increase in GMT levels followed by a decrease in antibody levels over the next 6 months.
The observed profile of the GMT levels, both for the anti-gD antibodies and neutralizing antibodies in all study groups, was consistent with previous findings in adults [16,19]. The magnitude of the immune response after the third vaccine dose was higher compared to that previously reported in HSV-1 and HSV-2 seronegative 18-30-year-old women included in the HERPEVAC trial [19]. In that trial (in which 8323 women were randomized to receive the same HSV-2 vaccine at the same dose as in the study presented here or the control vaccine Havrix™), anti-gD GMTs at month 7 were 6809 EL.U/ml and HSV neutralizing antibody levels were 29 ED90. The difference in these levels could be explained by differences in the age of participants: adolescents in the present study and adults in the HERPEVAC trial. Other clinical trials have shown higher immunogenicity of vaccines in younger age groups. For example, administration of three doses of HPV-16/18 AS04-adjuvanted vaccine (Cervarix®) in 10-14 years old girls induced 2-fold higher GMTs for antibodies to HPV-16 and -18 VLPs compared with 15-25 years old young women [25]. Another explanation for the difference in the magnitude of the immune responses could be methodological differences in the assays used to determine anti-gD and anti-HSV neutralizing antibody titers.
One of the secondary objectives of this trial was to evaluate the safety of the gD2-AS04 candidate vaccine in all treatment groups. The safety data suggest that the three commercial scale production lots of gD2-AS04 vaccine have similar and acceptable overall reac-togenicity profiles. Solicited symptoms after each dose and overall were most commonly local (injection site).
Our study has several limitations. The pre-specified lot-to-lot consistency criteria for the present study did not reach current standards based on 95% CI; however, the study sample size was not planned for reaching consistency with 95% CI. According to a post-hoc analysis based on 95% CI, failure was borderline, with a lower limit of the 95% CI of 0.66 (below the pre-defined 0.67 margin). This study was conducted in accordance with industry standards that were in place at time of study initiation, which supported the analysis of lot-to-lot consistency to be based on 90% CIs. Subsequent to this study, consistency analyses based on 95% CIs became more widely used. GlaxoSmithKline, therefore, proactively conducted a post-hoc analysis using 95% CIs in order to support the primary analysis. However, the study sample size was not sufficient to meet the lot-to-lot equivalence criteria with 95% CIs. Another limitation of this study was the high rate of exclusions from the ATP cohort for immunogenicity. A total of 242 (43.7%) subjects were not eligible for inclusion in this cohort. Since this was more than 5% of enrolled subjects, and as stated in the protocol, a supplemental immunogenicity analysis on the TVC was performed (data not shown). The results of the TVC analysis were consistent with those obtained for participants eligible for inclusion in the ATP cohort for immunoge-nicity; thus it is unlikely that the results of this study were affected by the number of exclusions. A further limitation of the study was the absence of a control group for comparison of immunogenicity and safety data.
In conclusion, this study demonstrated the lot-to-lot consistency of three different commercial scale production lots of the herpes simplex candidate vaccine. The vaccine was immunogenic and was shown to have a clinically acceptable safety profile when administered in HSV-1 and HSV-2 seronegative girls 1017 years of age. The development of this gD2-AS04 candidate
vaccine was stopped due to lack of efficacy [19]. However, newer candidate vaccines based at least partially on immune responses to HSV gD are currently under development and questions regarding the mechanism underlying the failure of previous gD-based candidate vaccines remain. Data such as those presented here may have important implications for future vaccine development activities.
5. Trademarks
Cervarix® and Havrix™ are registered trademarks of the Glaxo-SmithKline group of companies.
Enzygnost® is a registered trademark of Behring. HerpeSelect-2™ is a trademark of Focus Technologies.
6. Financial support
GlaxoSmithKline Biologicals SA sponsored this study and was involved in all stages of its conduct and analysis. GlaxoSmithKline Biologicals SA took responsibility for all costs associated with the development and publishing of the present manuscript.
7. Conflict of interest statement
All participating institutions received compensation for study involvement. Dr G. Leroux-Roels & I. Leroux-Roels report payments from Baxter, GlaxoSmithKline group of companies, Novartis, Immune Targeting Systems and UK for consultancy. Drs G. Dubin, T. Heineman and B. Cheuvart are employees of GlaxoSmithKline group of companies. Drs G. Dubin, T. Heineman report ownership of stock options. Dr G. Dubin reports royalties payments from Wyeth Vaccines. Dr D. Bernstein reports royalties payments for another GlaxoSmithKline Vaccines product.
Acknowledgements
The authors thank all investigators who participated in the trials, especially Dr R. Rupp and Dr M. Sperling. The authors also thank the study participants, staff members at the study site and GlaxoSmithKline Vaccines staff members involved in the study.
Medical writing assistance was provided by Adriana Rusu (XPE Pharma & Science). We thank Linda Gibbs (BD Life Sciences, Belgium) for editorial support and Jeremie Dedessus le Moutier (BD Life Sciences, on behalf of GlaxoSmithKline Vaccines) for manuscript coordination and editorial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.trivac.2013. 03.001.
References
[1] K.J. Looker, G.P. Garnett, G.P. Schmid, An estimate of the global prevalence and incidence of herpes simplex virus type 2 infection, Bull. World Health Organ. 86 (2008) 805-812. A.
[2] J.S. Smith, N.J. Robinson, Age-specific prevalence of infection with herpes simplex virus types 2 and 1: a global review, J. Infect. Dis. 186 (Suppl. 1) (2002) S3-S28.
[3] R.G. Pebody, N. Andrews, D. Brown, R. Gopal, H. De Melker, G. Francois, et al., The seroepidemiology of herpes simplex virus type 1 and 2 in Europe, Sex. Transm. Infect. 80 (2004) 185-191.
[4] A.M. Eis-Hubinger, E. Nyankiye, D.M. Bitoungui, J. Ndjomou, Prevalence of herpes simplex virus type 2 antibody in Cameroon, Sex. Transm. Dis. 29 (2002) 637-642.
[5] D.T. Fleming, G.M. McQuillan, R.E. Johnson, A.J. Nahmias, S.O. Aral, F.K. Lee, et al., Herpes simplex virus type 2 in the United States, 1976 to 1994, N. Engl. J. Med. 337 (1997) 1105-1111.
[6] F. Xu, M.R. Sternberg, B.J. Kottiri, G.M. McQuillan, F.K. Lee, A.J. Nahmias, et al., Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States, JAMA 296 (2006) 964-973.
[7] Centers for Disease Control and Prevention (CDC), Seroprevalence of herpes simplex virus type 2 among persons aged 14-49 years-United States, 20052008, MMWR Morb Mortal Wkly Rep 59 (2010) 456-459.
[8] L. Corey, Synergistic copathogens - HIV-1 and HSV-2, N. Engl. J. Med. 356 (2007) 854-856.
[9] C. Johnston, D.M. Koelle, A. Wald, HSV-2: in pursuit of a vaccine, J. Clin. Invest. 121 (2011)4600-4609.
[10] E.E. Freeman, H.A. Weiss, J.R. Glynn, P.L. Cross, J.A. Whitworth, R.J. Hayes, Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies, AIDS 20 (2006) 73-83.
[11] E. Anzivino, D. Fioriti, M. Mischitelli, A. Bellizzi, V. Barucca, F. Chiarini, et al., Herpes simplex virus infection in pregnancy and in neonate: status of art of epidemiology, diagnosis, therapy and prevention, Virol. J. 6 (2009) 40.
[12] A. Chatterjee, S.A. Chartrand, C.J. Harrison, A. Felty-Duckworth, C. Bewtra, Severe intrauterine herpes simplex disease with placentitis in a newborn of a mother with recurrent genital infection at delivery, J. Perinatol. 21 (2001) 559564.
[13] Z.A. Brown, J. Benedetti, R. Ashley, S. Burchett, S. Selke, S. Berry, et al., Neonatal herpes simplex virus infection in relation to asymptomatic maternal infection at the time of labor, N. Engl. J. Med. 324 (1991) 1247-1252.
[14] G.L. Armstrong, J. Schillinger, L. Markowitz, A.J. Nahmias, R.E. Johnson, G.M. McQuillan, et al., Incidence of herpes simplex virus type 2 infection in the United States, Am. J. Epidemiol. 153 (2001) 912-920.
[15] L.R. Stanberry, S.L. Spruance, A.L. Cunningham, D.I. Bernstein, A. Mindel, S. Sacks, et al., Glycoprotein-D-adjuvant vaccine to prevent genital herpes, N. Engl. J. Med. 347 (2002) 1652-1661.
[16] D.I. Bernstein, F.Y. Aoki, S.K. Tyring, L.R. Stanberry, C. St-Pierre, S.D. Shafran, et al., Safety and immunogenicity of glycoprotein D-adjuvant genital herpes vaccine, Clin. Infect. Dis. 40 (2005) 1271-1281.
[17] European Medicines Agency, Note for guidance on the clinical evaluation of vaccines. Available from: <http://www.ema.europa.eu/docs/en_GB/document_ library/Scientific_guideline/2009/09/WC500003875.pdf> (accessed June 2012).
[18] World Health Organization, Clinical considerations for evaluation of vaccines for prequalification. Points to consider for manufacturers of human vaccines. Available from: <http://www.who.int/immunization_standards/vaccine_ quality/clinical_considerations_oct10.pdf> (accessed June 2012).
[19] R.B. Belshe, P.A. Leone, D.I. Bernstein, A. Wald, M.J. Levin, J.T. Stapleton, et al., Efficacy results of a trial of a herpes simplex vaccine, N. Engl. J. Med. 366 (2012) 34-43.
[20] R. Ashley, A. Cent, V. Maggs, A. Nahmias, L. Corey, Inability of enzyme immunoassays to discriminate between infections with herpes simplex virus types 1 and 2, Ann. Intern. Med. 115 (1991) 520-526.
[21] K.F. Karpinski, S. Hayward, H. Tryphonas, Statistical considerations in the quantitation of serum immunoglobulin levels using the enzyme-linked immunosorbent assay (ELISA), J. Immunol. Methods 103 (1987) 189194.
[22] A.G. Langenberg, R.L. Burke, S.F. Adair, R. Sekulovich, M. Tigges, C.L. Dekker, et al., A recombinant glycoprotein vaccine for herpes simplex virus type 2: safety and immunogenicity, Ann. Intern. Med. 122 (1995) 889-898. Erratum, Ann. Intern. Med. 123 (1995) 395.
[23] S.M. Garland, T.N. Lee, R.L. Ashley, L. Corey, S.L. Sacks, Automated microneutralization: method and comparison with Western blot for type-specific detection of herpes simplex antibodies in two pregnant populations, J. Virol. Methods 55 (1995) 285-294.
[24] C.J. Clopper, E.S. Pearson, The use of confidence or fiducial limits illustrated in the case of binomial, Biometrika 26 (1934) 404-413.
[25] C. Pedersen, T. Petaja, G. Strauss, H.C. Rumke, A. Poder, J.H. Richardus, et al., Immunization of early adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant, J. Adolesc. Health 40 (2007) 564-571.
