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0Vaccine xxx (2017) xxx-xxx
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Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity and safety of an AS03-adjuvanted H7N1 vaccine in healthy adults: A phase I/II, observer-blind, randomized, controlled trial
Anuradha Madan a'*, Murdo Ferguson b, Eric Sheldonc, Nathan Segalld, Laurence Chue, Azhar Tomaf, Paul Rheaultg, Damien Frielh, Jyoti Soni', Ping Li-1,1, Bruce L. Innisk, Anne Schuindj
a GSK, 1250 South Collegeville Road, Collegeville, PA 19426, USA b Colchester Research Group, 68 Robie Street, Truro, Nova Scotia B2N 1L2, Canada c Miami Research Associates, 6141 Sunset Drive Suite 501, Miami 33143, USA d Clinical Research Atlanta, 175 Country Club Dr. Ste A, Stockbridge 30281, USA eBenchmark Research, 1015 East 32nd Street, Suite 309, Austin, TX 78705, USA fManna Research, 2291 Kipling Avenue Suite 117B, Toronto, Ontario M9W4L6, Canada g Medicor Research Inc, 202-1280 Lasalle Blvd, Sudbury, Ontario P3E 1H5, Canada h GSK, 20 Avenue Fleming, 1300 Wavre, Belgium i GSK Pharmaceuticals Ltd., 5 Embassy Links, SRT Road, Bangalore, India j GSK, 2301 Renaissance Blvd, King of Prussia, PA 19406-2772, USA k GSK, 14200 Shady Grove Road, Rockville, MD 20850, USA
ABSTRACT
Background: H7 influenza strains have pandemic potential. AS03-adjuvanted H7N1 A/mallard/ Netherlands/12/2000 split-virion vaccine formulations were evaluated as model H7-subtype vaccine and tested after H7N9 emerged in China, and caused severe human disease with high mortality. Methods: In this phase I/II, observer-blind, randomized trial in US and Canada, 420 healthy adults (2164 years) were randomized to receive 1 of 4 H7N1 vaccine formulations (3.75 or 7.5 ig hemagglutinin adjuvanted with either AS03a or AS03B), 15 ig unadjuvanted H7N1 hemagglutinin, or saline placebo, given as 2-dose series. Immunogenicity was assessed using hemagglutination-inhibition (HI) and microneutralization (MN) assays, at day 42 (21 days post-dose 2), month 6, and month 12 (HI only) for the per-protocol cohorts (398, 379 and 368 participants, respectively). Safety is reported up to month 12. Results: Beneficial AS03 adjuvant effect was demonstrated. Committee for Medical Products for Human Use, and Center for Biologics Evaluation and Research (CBER) criteria were met for all adjuvanted formulations at day 42 (H7N1 HI assay); seroprotection (SPR) and seroconversion rates (SCR) were 88.5-94.8%, mean geometric increase (MGI) 19.2-34.9, and geometric mean titers (GMT) 98.3-180.7. Unadjuvanted H7N1 vaccine did not meet CBER criteria. In adjuvanted groups, antibody titers decreased over time; month 12 SPRs and GMTs were low (2.0-18.8% and 8.1-12.2). MN antibodies showed similar kinetics, with titers persisting at higher range than HI at month 6. All adjuvanted groups showed cross-reactivity against H7N9, with HI responses similar to H7N1.
The most frequent solicited symptom in adjuvanted groups was injection site pain (71.2-86.7%); grade 3 solicited symptoms were infrequent. Nine participants reported 17 serious adverse events; none were considered causally related to vaccination.
Conclusions: Adjuvanted H7N1 vaccine formulations had an acceptable safety profile and induced an antibody response after 2 doses with cross-reactivity to H7N9. ClinicalTrials.gov: NCT01934127
© 2017 Published by Elsevier Ltd.
ARTICLE INFO
Article history:
Received 19 October 2016
Received in revised form 19 January 2017
Accepted 20 January 2017
Available online xxxx
Keywords:
Pandemic flu H7 influenza vaccine AS03 adjuvant
Abbreviations: AE, adverse event; CBER, Center for Biologics Evaluation and Research; CHMP, Committee for Medical Products for Human Use; CI, confidence interval; GMT, geometric mean titers; HA, hemagglutinin; HI, hemagglutination-inhibition; MGI, mean geometric increase; MN, microneutralization; pIMD, potential immunemediated disease; PP, per-protocol; RBC, red blood cells; SAE, serious adverse event; SAS, statistical analysis system; SBIR, internet-based randomization system; SCR, seroconversion rate; SPR, seroprotection rate; TVC, total vaccinated cohort; VRR, vaccine response rate.
* Corresponding author at: 1250 South Collegeville Road, Collegeville, PA 19426, USA.
E-mail addresses: Anu.2.Madan@gsk.com (A. Madan), azhar.toma@mannaresearch.com (A. Toma), ping.li4@pfizer.com (P. Li). 1 Present address: Pfizer, 500 Arcola Rd, Collegeville, PA 19426, USA.
http://dx.doi.org/10.1016/j.vaccine.2017.01.054 0264-410X/© 2017 Published by Elsevier Ltd.
1. Introduction
Most H7 influenza viruses are considered marginally pathogenic avian influenza viruses causing only mild disease in birds. However, some highly pathogenic viruses evolved from low-pathogenic precursors and caused important avian influenza outbreaks in poultry in Italy (1999-2001) [1], the Netherlands (2003) [2] or Canada (2004) [3]. In 2003, 89 human infections were reported in an H7N7 outbreak in the Netherlands, mainly with mild symptoms except for one fatal case [2,4]. However, in 2013, a novel avian influenza virus (H7N9) emerged, causing human infections in China [5] with high morbidity and mortality (322 deaths among 800 laboratory-confirmed cases, reported up to November 2016) [6]. Although there has been no evidence of sustained human-to-human transmission, the genetic changes observed in the H7N9 virus suggest adaptation to mammals, indicating its pandemic potential [7].
The World Health Organization recognizes development of vaccines as an important aspect of the global strategy for pandemic preparedness. Following the emergence of H7N9 human infections and before availability of H7N9 strains for vaccine development, this study was initiated with an AS03-adjuvanted H7N1 strain as a model for H7-subtype influenza vaccines. Several H7-subtype inactivated influenza vaccines and H7 live-attenuated influenza vaccines have been tested in clinical trials [8-11]: some have been reported to induce strong immune memory [11,12], and modest immunogenicity was observed for an H7N9 live attenuated influenza vaccine candidate [13]. On the other hand, adjuvantation has shown to increase immunogenicity [14-18].
This trial assessed immunogenicity and safety of different vaccine formulations comprising H7N1 split-virus hemagglutinin (HA) antigen (A/mallard/Netherlands/12/2000) with AS03 in adults aged 21-64 years.
2. Methodology
2.1. Study design and participants
This study was a phase I/II, observer-blind, randomized, controlled, multi-country study with a staggered dosing scheme (Supplementary methods), conducted in the US and Canada between August 2013 and October 2014. Persons eligible for enrolment were healthy men and women 21-64 years of age at the time of first study vaccination and satisfying preset inclusion criteria (exclusion criteria in Supplementary methods). Participants who attended the screening visit (Supplementary methods) had to have results of all safety laboratory tests within reference ranges.
Two doses of vaccine or placebo were administered at day 0 and 21. The studies comprised 7 study visits (day 0, 7, 21, 28, 42, and month 6 and 12), and 2 telephone contacts (month 3 and 9).
The study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation Good Clinical Practice guidelines. The protocol was approved by the independent ethics committee or institutional review board of each study center. Written informed consent was obtained from each participant before any study procedure.
The study is registered at www.ClinicalTrials.gov (NCT01934127) and available at http://www.gsk-clinicalstudyregister.com (study ID: 115415).
2.2. Randomization
Participants were randomized (1:1:1:1:1:2) to receive 1 of 5 H7N1 vaccine formulations, or saline placebo. The vaccine formu-
lations contained 3.75 ig HA adjuvanted with AS03a (H7N1-3.75A) or AS03B (H7N1-3.75B), or 7.5 ig HA adjuvanted with AS03a (H7N1-7.5A) or AS03B (H7N1-7.5B), or 15 ig plain antigen (H7N1-15).
Allocation to groups was performed using an internet-based randomization system (SBIR). The randomization algorithm used a minimization procedure accounting for center, age (21-40 years/4164 years), and receipt of seasonal influenza vaccine in the past 3 years.
2.3. Blinding
The trial was conducted in an observer-blind manner; participants, those responsible for evaluation of any study endpoint, and the sponsor were unaware of the administered product. The laboratory was also blinded to the treatment, and individual data were not available during the course of the study to the sponsor, any investigator, or any person involved in data collection.
2.4. Study vaccine
The monovalent H7N1 split-virus vaccine was manufactured by GSK, Quebec, Canada. Vaccine formulations contained 3.75 or 7.5 ig HA of the A/mallard/Netherlands/12/2000 N1BRG-63 strain, adjuvanted with AS03a or AS03b. AS03 is an Adjuvant System containing a-tocopherol and squalene in an oil-in-water emulsion (11.86 mg tocopherol for AS03a and 5.93 mg for AS03b), manufactured by GSK Biologicals. The placebo control was phosphate-buffered saline. The vaccines or placebo (0.5 mL per dose) were administered intramuscularly in the deltoid region of the arm.
2.5. Study objectives
Co-primary objectives were to evaluate whether vaccination with adjuvanted vaccine formulations resulted in an immune response that met or exceeded the Center for Biologics Evaluation and Research (CBER) [19] and Committee for Medical Products for Human Use (CHMP) [20] immunogenicity acceptance criteria, 21 days post-dose 2; and to evaluate safety and reactogenicity through the day 42 visit.
The first secondary objective was to demonstrate adjuvant effect of AS03 for adjuvanted formulations that met CBER/CHMP criteria 21 days post-dose 2. Another secondary objective assessed whether unadjuvanted vaccine resulted in an immune response that met or exceeded CBER/CHMP immunogenicity acceptance criteria, 21 days post-dose 2. Hemagglutination-inhibition (H1) immune response to the vaccine strain and vaccine-heterologous H7N9 strain up to month 12, and of vaccine-strain and vaccine-heterologous H7N9 microneutralization (MN) antibodies up to month 6 was described. Safety of the different vaccine formulation between day 42 and month 12 was also evaluated.
2.6. Immunogenicity assessments
Blood samples were taken at day 0, 21, 42 and month 6 and 12. H1 antibody titers were measured in serially-diluted serum samples. Horse red blood cells (RBC) were used because a significantly improved sensitivity to detect H1 antibodies was observed for H7 strains [21]. Serum pre-treatment procedure was adapted (adding a receptor-destroying enzyme treatment step after horse RBC hemadsorption) to remove nonspecific agglutinins of horse RBC and nonspecific virus inhibitors introduced by the hemadsorption step. Assay specificity was assessed. The assay cut-off was 10 (reciprocal of the initial serum dilution). Antibody geometric mean
A. Madan et al./Vaccine xxx (2017) xxx-xxx
titers (GMTs), seropositivity rates, mean geometric increase (MG1), seroconversion rate (SCR) and seroprotection rate (SPR) were evaluated with 98.75% confidence intervals (CIs) regarding the primary objective and 95% CIs for the secondary objectives. The assay was performed with A/mallard/Netherlands/12/2000 (vaccine-homologous H7N1 strain; all participants) and A/Shanghai/2/2013 (vaccine-heterologous H7N9 strain; all vaccine recipients and a subset of placebo recipients).
Virus neutralization by serum antibodies was determined by a MN assay on heat-treated sera (56 °C for 30 min); sera were assessed in single tests (no duplicates). The assay was performed for a subset of participants in each study group, with A/mallard/ Netherlands/12/2000 as vaccine-homologous strain and with A/Anhui/1/2013 as vaccine-heterologous H7N9 strain. A standardized amount of virus was mixed with serial serum dilutions and incubated to allow antibody binding to the virus. A cell suspension, containing a defined amount of Madin-Darby Canine Kidney cells was then added to the serum/virus mixture and incubated at 37 °C. After incubation, virus replication was visualized by hemagglutination of RBC added to culture fluids. The neutralizing titer corresponded to the highest serum dilution able to neutralize 50% of the virus; in practice, this titer corresponds to the GMT between the last dilution without haemagglutination (100% neutralization) and the next dilution. For the MN assay, titers <28 were considered to be below the cut-off. MN antibody GMTs, seropositivity rates, and vaccine response rates (VRRs) were evaluated with 95% CIs.
2.7. Safety and reactogenicity assessments
Blood samples for clinical safety laboratory (hematology/bio-chemistry; Supplementary methods) evaluations were drawn at day 0, 7, 21, 28, and 42; an additional blood sample was drawn at the screening visit for participants of step 1. Diary cards were used to capture solicited symptoms during the 7-day post-vaccination period and spontaneously reported adverse events (AEs) up to 21 days post-vaccination. Potential immune-mediated diseases (pIMDs) and serious adverse events (SAEs) were recorded throughout the study. SAEs were defined as any medical occurrence that resulted in death, was life-threatening, resulted in disability or incapacity, or required hospitalization or prolonged existing hospitalization.
2.8. Statistical analyses
The target sample size was 420 participants (60 in each vaccine group and 120 in the placebo group). Assuming an enrolment of 60 participants per vaccine group and a non-evaluable rate of 5%, the overall study power to assess the primary immunogenicity endpoint was 88.80%. The type 1 error was split equally so that if any of the adjuvanted H7N1 vaccines met CBER/CHMP criteria, the co-primary study objective of immunogenicity was considered met.
According to CBER immunogenicity acceptance criteria, the primary objective was met if the lower limit of the 98.75% CI for SCR met or exceeded 40%, and the lower limit of the 98.75% CI for the incidence rate of HI reciprocal titers p40 (potential SPR) met or exceeded 70%. CHMP criteria were: point estimate for SCR exceeds 40%, point estimate for SPR exceeds 70%, and MG1 exceeds 2.5.
GMT calculations were performed by taking the anti-log of the mean of the log10 transformed inverse titers. A seronegative participant was defined as having an antibody titer below the cut-off value; antibody titers below the cut-off were given an arbitrary value of half the cut-off for GMT calculation.
SCR was defined as percentage of participants with either pre-vaccination titer <1:10 and post-vaccination titer p1:40, or pre-vaccination titer p1:10 and p 4-fold increase in post-vaccination titer. SPR was defined as percentage of participants with H1 titer p 1:40. VRR for MN titers was defined as proportion of participants
with either pre-vaccination (Day 0) reciprocal titer <28 and postvaccination reciprocal titer p56, or pre-vaccination reciprocal HI titer p28 and p 4-fold increase in post-vaccination reciprocal titer against the tested virus.
H1 antibody response was also calculated by age stratum (2140 and 41-64 years).
The secondary objective concerning adjuvant effect was met when the lower limit of the 98.75% CI for HI GMT ratio (adjuvanted to non-adjuvanted group) exceeded 1.5 and the SCR difference (adjuvanted minus non-adjuvanted group) exceeded 10%.
Safety analyses were performed on the total vaccinated cohort (TVC), including all participants who received at least 1 study dose. 1mmunogenicity analyses were based on the per-protocol (PP) cohorts for immunogenicity (at day 42, month 6, and month 12). This included the participants who complied with protocol procedures, had received 2 study doses, and for whom the assay results for H7N1 antibodies were available. An adapted PP cohort was defined, which includes day 0, 21 and 42 data obtained from the PP day 42 cohort, month 6 data obtained from the PP month 6 cohort, and month 12 data obtained from the PP month 12 cohort.
Statistical analyses were performed using Statistical Analysis System (SAS) version 9.22 on Windows.
3. Results
3.1. Study population
Overall, 420 participants received at least 1 study dose and constituted the TVC; 398 of them were included in the day 42 PP cohort for immunogenicity (Fig. 1). 413 (98.3%) participants completed the day 42 visit, while 386 (91.9%) participants returned to the month 12 visit.
Baseline characteristics were balanced in the vaccine and placebo groups (Supplementary Table 1).
3.2. Immune response to the H7N1 vaccine strain
1n adults 21-64 years of age, the CBER and CHMP criteria as described in the co-primary objective were met for all adjuvanted formulations at day 42 (21 days post-dose 2) (Fig. 2, Table 1).
The adjuvant effect of AS03 was demonstrated for all adju-vanted vaccine groups, 21 days post-dose 2 (Table 2). Administration of unadjuvanted vaccine did not meet CBER or CHMP SPR criteria at day 42; only the CHMP criteria for SCR and MG1 were met (Table 1). MG1 point estimates tended to be higher for AS03a-adjuvanted formulations than for AS03b, but the 95% C1s were overlapping.
At day 21, GMTs were low across all treatment groups (Fig. 2). Following a peak 21 days post-dose 2 (day 42), H1 titers declined at month 6 and further at month 12, but GMTs remained above baseline levels in the adjuvanted groups (month 6: 11.8-21.2, month 12: 8.1-12.2). SPR, SCR and MG1 were low at 6 and 12 months (Table 1). No clinically meaningful differences in immune response were observed between the 3.75 and 7.5 ig HA formulations.
Analyses of H1 antibody response by age stratum showed a trend toward higher H1 GMT titers in the younger (21-40 years) than older age stratum (41-64 years) (Supplementary Table 2).
3.3. Immune response to the cross-reactive H7N9 strain
Cross-reactivity against vaccine-heterologous H7N9 virus was observed by H1 assay 21 days post-dose 2 (day 42) and at month 6 in all groups who received an adjuvanted formulation, with H1 GMTs ranging from 187.2-421.0 at day 42 and 10.0-19.7 at month
A. Madan et al./Vaccine xxx (2017) xxx-xxx
Fig. 1. Participant flow diagram. N, number of participants per group; n, number of participants eliminated from per-protocol cohort due to the specified reason.
6 (Fig. 2, Table 1). By month 12, H7N9 HI GMTs had fallen back to baseline levels.
3.4. Vaccine-homologous (H7N1) and heterologous (H7N9) MN antibody profiles
Vaccine-homologous (H7N1) and heterologous (H7N9) MN antibody parameters (seropositivity rates, GMT, and VRR) showed an increase from day 0 to day 42, and then a decrease until month 6 with values remaining above baseline for groups H7N1-3.75B, H7N1-3.75A, H7N1-7.5B, and H7N1-7.5A(Fig. 3). In the adjuvanted groups, VRR values for the homologous H7N1 MN antibody titers
were p86.7% 21 days post-dose 2 and p50% at month 6, while VRR values for H7N9 MN antibody titers were p83.3% and 51.9%, respectively, at the same timepoints (Fig. 3).
3.5. Safety and reactogenicity
The most common solicited injection site symptom was pain, which occurred at a higher frequency in the vaccine groups; incidences of injection site pain were also noticeably higher in the adjuvanted vaccine groups than in the H7N1-15 group (Fig. 4A). The incidence of grade 3 injection site pain was low (63 [5.0%] participants per group). Incidences of injection site redness and
Fig. 2. Hemagglutination inhibition geometric mean titers (Adapted PP cohort for immunogenicity). Groups received the following H7N1 vaccine formulations: 3.75 ig HA adjuvanted with AS03a (H7N1-3.75A) or AS03b (H7N1-3.75B), 7.5 ig HA adjuvanted with AS03a (H7N1-7.5A) or AS03b (H7N1-7.5B), or 15 ig unadjuvanted HA (H7N1-15). HI, hemagglutination-inhibition; GMT, geometric mean titer; Pre-vac, pre-vaccination (day 0); PP, per protocol. Error bars indicate 95% confidence intervals. In the placebo group, H7N9 HI GMTs were assessed for a subset of participants.
swelling were also low; no participants reported grade 3 redness, and grade 3 swelling was reported for 1 participant (1.7%) in the H7N1-3.75A group.
Muscle aches, headache, and fatigue were the most commonly reported solicited general symptoms (Fig. 4B). Incidences of muscle ache and fatigue tended to be higher in the groups who received an adjuvanted formulation than in those who received the unadjuvanted vaccine or placebo, except for fatigue in the H7N1-7.5B group. Incidences of joint pain tended to be higher in all vaccinated groups compared to placebo. The incidence of grade 3 solicited general symptoms was low; each grade 3 solicited general symptom was reported by 64 (6.7%) participants per group.
At least 1 unsolicited AE was reported by 617 (27.9%) participants per group. A total of 16 unsolicited AEs considered by the investigator to be causally related to vaccination were reported by 11 participants (1 [1.7%] in the H7N1-3.75B, H7N1-7.5B and H7N1-15 groups, 2 [3.3%] in the H7N1-3.75A and H7N1-7.5A groups, and 4 [3.3%] in the placebo group) (Supplemental Table 3). No clinically relevant changes in the laboratory parameters of the participants were observed up to day 42. Up to the month 12 visit, no pIMDs were reported, and none of the participants experienced an AE leading to withdrawal from the study.
Nine participants (2 [3.3%] in the H7N1-3.75B group, 1 [1.7%] in the H7N1-3.75A, H7N1-7.5B and H7N1-15 groups, 1 [1.6%] in the H7N1-7.5A group and 3 [2.5%] in the placebo group) reported 17
Table 1
Immune response for vaccine-homologous H7N1 and vaccine-heterologous H7N9 HI antibodies (adapted PP cohort for immunogenicity). H7N1 HI antibodies
H7N1-3.75B H7N1-3.75A H7N1-7.5B H7N1-7.5A H7N1-15 Placebo
N Value or % N Value or % N Value or % N Value or % N Value or % N Value or %
(95% CI)a (95% CI)a (95% CI)a (95% CI)a (95% CI)a (95% CI)a
Seroprotection rate
PRE 58 0.0% (0.0; 6.2) 52 0.0% (0.0; 6.8) 57 0.0% (0.0; 6.3) 58 0.0% (0.0; 6.2) 58 0.0% (0.0; 6.2) 115 0.0% (0.0; 3.2)
D21 58 20.7% (11.2; 33.4) 52 30.8% (18.7; 45.1) 56 26.8% (15.8;40.3) 58 34.5% (22.5; 48.1) 58 13.8% (6.1; 25.4) 115 0.0% (0.0; 3.2)
D42 58 89.7% (75.7; 97.1) 52 88.5% (73.1; 96.8) 57 91.2% (77.6; 98.0) 58 94.8% (82.8; 99.4) 58 41.4% (28.6; 55.1) 115 0.0% (0.0; 3.2)
M6 52 15.4% (6.9; 28.1) 51 29.4% (17.5; 43.8) 55 14.5% (6.5; 26.7) 54 31.5% (19.5; 45.6) 56 5.4% (1.1; 14.9) 111 0.0% (0.0; 3.3)
M12 50 2.0% (0.1; 10.6) 48 18.8% (8.9; 32.6) 53 9.4% (3.1; 20.7) 56 16.1% (7.6; 28.3) 53 0.0% (0.0; 6.7) 108 0.0% (0.0; 3.4)
Seroconversion rate
D21 58 19.0% (9.9; 31.4) 52 30.8% (18.7; 45.1) 56 26.8% (15.8; 40.3) 58 32.8% (21.0; 46.3) 58 13.8% (6.1; 25.4) 115 0.0% (0.0; 3.2)
D42 58 89.7% (75.7; 97.1) 52 88.5% (73.1; 96.8) 57 91.2% (77.6; 98.0) 58 94.8% (82.8; 99.4) 58 41.4% (28.6; 55.1) 115 0.0% (0.0; 3.2)
M6 52 13.5% (5.6; 25.8) 51 29.4% (17.5; 43.8) 55 14.5% (6.5; 26.7) 54 31.5% (19.5; 45.6) 56 5.4% (1.1; 14.9) 110 0.0% (0.0; 3.3)
M12 50 2.0% (0.1; 10.6) 48 18.8% (8.9; 32.6) 53 9.4% (3.1; 20.7) 56 16.1% (7.6; 28.3) 53 0.0% (0.0; 6.7) 107 0.0% (0.0; 3.4)
Mean geometric increase
D21 58 2.3 (1.8; 3.0) 52 2.4 (1.7; 3.3) 56 2.9 (2.3; 3.8) 58 3.8 (2.9; 5.0) 58 1.7 (1.4; 2.2) 115 1.0(1.0; 1.0)
D42 58 19.2 (14.7; 25.1) 52 29.8 (20.8; 42.8) 57 22.0 (17.3; 27.8) 58 34.9 (27.0; 45.1) 58 4.3 (3.1; 5.9) 115 1.0(1.0; 1.0)
M6 52 2.5 (1.9; 3.1) 51 3.7 (2.8; 5.0) 55 2.3 (1.8; 3.0) 54 4.1 (3.2; 5.3) 56 1.4 (1.2; 1.7) 110 1.0(1.0; 1.0)
M12 50 1.6(1.3; 1.9) 48 2.4 (1.9; 3.2) 53 1.6 (1.3; 2.1) 56 2.2 (1.7; 2.8) 53 1.2 (1.0; 1.3) 107 1.0(1.0; 1.0)
Seroprotection rate
PRE 57 1.8% (0.0; 9.4) 52 0.0% (0.0; 6.8) 57 0.0% (0.0; 6.3) 58 0.0% (0.0; 6.2) 58 1.7% (0.0; 9.2) 57 0.0% (0.0; 6.3)
D21 57 28.1% (17.0; 41.5) 52 42.3% (28.7; 56.8) 56 41.1% (28.1; 55.0) 58 55.2% (41.5; 68.3) 58 29.3% (18.1; 42.7) 57 0.0% (0.0; 6.3)
D42 58 94.8% (85.6; 98.9) 52 96.2% (86.8; 99.5) 57 98.2% (90.6; 100) 58 100% (93.8; 100) 58 62.1% (48.4; 74.5) 57 0.0% (0.0; 6.3)
M6 52 11.5% (4.4; 23.4) 51 31.4% (19.1; 45.9) 55 16.4% (7.8; 28.8) 54 31.5% (19.5; 45.6) 56 5.4% (1.1; 14.9) 53 0.0% (0.0; 6.7)
M12 50 14.0% (5.8; 26.7) 48 31.3% (18.7; 46.3) 53 18.9% (9.4; 32.0) 55 36.4% (23.8; 50.4) 53 1.9% (0.0; 10.1) 53 0.0% (0.0; 6.7)
Seroconversion rate
D21 57 26.3% (15.5; 39.7) 52 42.3% (28.7; 56.8) 56 41.1% (28.1; 55.0) 58 55.2% (41.5; 68.3) 58 27.6% (16.7; 40.9) 57 0.0% (0.0; 6.3)
D42 57 93.0% (83.0; 98.1) 52 96.2% (86.8; 99.5) 57 98.2% (90.6; 100) 58 100% (93.8; 100) 58 56.9% (43.2; 69.8) 57 0.0% (0.0; 6.3)
M6 51 11.8% (4.4; 23.9) 51 31.4% (19.1; 45.9) 55 16.4% (7.8; 28.8) 54 31.5% (19.5; 45.6) 56 5.4% (1.1; 14.9) 53 0.0% (0.0; 6.7)
M12 49 14.3% (5.9; 27.2) 48 31.3% (18.7; 46.3) 53 18.9% (9.4; 32.0) 55 34.5% (22.2; 48.6) 53 1.9% (0.0; 10.1) 53 0.0% (0.0; 6.7)
Mean geometric increase
D21 57 3.6 (2.7; 4.8) 52 5.4 (3.7; 7.8) 56 4.5 (3.3; 6.0) 58 6.3 (4.5; 8.8) 58 2.8 (2.0; 3.7) 57 1.0(1.0; 1.0)
D42 57 35.3 (26.2; 47.6) 52 63.5 (43.4; 93.0) 57 40.8 (32.1; 51.9) 58 76.2 (57.8; 100.4) 58 6.9 (4.9; 9.9) 57 1.0(1.0; 1.0)
M6 51 2.4(1.9; 3.1) 51 4.1 (3.1; 5.4) 55 2.3 (1.8; 2.9) 54 4.1 (3.1; 5.5) 56 1.3 (1.1; 1.6) 53 1.0 (0.9; 1.1)
M12 49 1.9 (1.4; 2.4) 48 3.6 (2.7; 4.9) 53 2.1 (1.6; 2.8) 55 3.5 (2.6; 4.8) 53 1.2 (1.0; 1.4) 53 1.0 (0.9; 1.0)
H1, hemagglutination-inhibition; PP, per-protocol; N, number of participants with results available (for seroprotection rate) or number of participants with both pre and post results available (for seroconversion rate and mean geometric increase); 95% C1, 95% confidence interval; PRE, before vaccination; D, day; M, month. 1mmunogenicity acceptance criteria at 21 days post-dose 2: Center for Biologics Evaluation and Research (CBER) = the lower limit of the 98.75% confidence interval for seroconversion rate meets or exceeds 40%, and the lower limit of the 98.75% C1 for the incidence rate of hemagglutination inhibition reciprocal titers p40 (potential seroprotection rate) meets or exceeds 70%; Committee for Medical Products for Human Use (CHMP) = the point estimate for seroconversion exceeds 40%; the point estimate for potential seroprotection rate exceeds 70% and the mean geometric increase exceeds 2.5. Day 42 criteria met are shown in bold.
a 98.75% C1 for SPR and SCR at day 42 for all adjuvanted groups. SCR was defined as percentage of participants with either a pre-vaccination titer <1:10 and a postvaccination titer p1:40, or a pre-vaccination titer p1:10 and a p4-fold increase in post-vaccination titer. SPR was defined as percentage of participants with an H1 titer p1:40.
Table 2
Criteria for adjuvant effect (Day 42 PP cohort for immunogenicity).
Group Adjusted GMT ratio (vs H7N1-15) SCR difference (minus H7N1-15) Criteria met
98.75% CI (LL; UL) 98.75% CI (LL; UL)
H7N1-3.75B 4.42 (2.69; 7.27) 48.28% (27.54; 65.19) Yes
H7N1-3.75A 6.70 (4.02; 11.17) 47.08% (25.43; 64.41) Yes
H7N1-7.5B 4.84 (2.93; 7.99) 49.85% (29.41; 66.45) Yes
H7N1-7.5A 8.20 (4.98; 13.51) 53. 45% (34.22; 69.25) Yes
Bold = criteria for adjuvant effect met: LL of the 98.75% C1 for GMT ratio >1.5 and SCR difference >10%. GMT, geometric mean titer; SCR, seroconversion rate; C1, confidence interval; LL, lower limit; UL, upper limit; PP, per protocol.
SAEs during the study (Supplemental Table 4). None of these SAEs were considered related to vaccination; 13 SAEs resolved by the end of the study. The 4 non-resolved SAEs were reported by 2 participants: 1 reported anal cancer, while another participant reported ovarian cyst, salpingitis and uterine leiomyoma.
4. Discussion
We report here the first assessment of an AS03-adjuvanted H7N1 vaccine, developed initially as model H7 vaccine in light of
the H7N9 influenza outbreak in China which started in 2013. All adjuvanted formulations were immunogenic, with an acceptable safety profile.
Adjuvantation allows antigen sparing, which is key for pandemic vaccines. Moreover, previously evaluated non-adjuvanted H7 vaccine candidates were found to have poor immunogenicity in humans [8,9]. After vaccination with two doses of split H7N1 virus vaccine containing 12 or 24 ig HA, only 21% and 23% of vaccinees had detectable antibodies. While adjuvantation with aluminum hydroxide enhanced the number of responders (50% and
A. Madan et al./Vaccine xxx (2017) xxx-xxx
Fig. 3. Vaccine-homologous (H7N1) and heterologous (H7N9) microneutralization antibody profiles (Adapted PP cohort for immunogenicity). Vaccination groups received the following H7N1 vaccine formulations: 3.75 ig HA adjuvanted with AS03a (H7N1-3.75A) or AS03b (H7N1-3.75B), 7.5 ig HA adjuvanted with AS03a (H7N1-7.5A) or AS03b (H7N1-7.5B), or 15 ig unadjuvanted HA (H7N1-15). GMT, geometric mean titer; MN, microneutralization; VRR, vaccine response rate; Pre-vac, pre-vaccination (day 0); PP, per protocol. Error bars indicate 95% confidence intervals. MN GMTs were assessed for a subset of participants in each study group.
62%, respectively), none of the formulations met CHMP licensing criteria [9]. Similarly, after 2-dose administration of inactivated subunit H7N7 vaccine formulations containing up to 90 ig HA, only 1 vaccinee out of 25 developed a p 4-fold HI antibody response and final titer of p 1:40; only 5 vaccinees had an MN titer p 1:40 [8]. In our study, adjuvant effect of AS03 was demonstrated in all groups; the unadjuvanted formulation containing 15 ig plain H7N1 antigen did not meet CBER criteria or CHMP SPR criteria. Similarly, AS03 adjuvant was shown to enhance the immune response against H7 antigens for antigen-sparing doses of an AS03-adjuvanted H7N9 vaccine candidate [14].
There was no clinically meaningful difference between the adjuvant effects of AS03b and AS03a. There was a trend towards higher MGI for AS03A-adjuvanted formulations compared to AS03b, but the 95% CIs were overlapping. The study did not show a marked impact of HA content (3.75 vs 7.5 ig) on immune
response. This finding is in agreement with observations of other H7 vaccine candidates; while adjuvanted low-antigen-content formulations elicit higher immune responses than unadjuvanted high-antigen-content formulations, minimal to no effect of antigen doses is observed within formulations using the same adjuvant [14,16-18]. Similarly, a study assessing various formulations of adjuvanted and non-adjuvanted H5N1 vaccinations (containing 3.75, 7.5, 15 or 30 ig HA) in adults 18-60 years of age observed no antigen-dose effect on the HI antibody response for adjuvanted formulations after a 2-dose vaccination course [22].
CBER and CHMP immunogenicity acceptance criteria were met at 21 days post-dose 2 for all adjuvanted formulations. Results were in similar ranges as those reported for an AS03A-adjuvanted H5N1 vaccine, where SPR and SCR in adults 18-64 years of age were 90.8% (95% CI: 89.3%; 92.2%) [23]. As observed with other vaccines, immunogenicity decreased with age. Nevertheless, all
A. Madan et al./Vaccine xxx (2017) xxx-xxx
Fig. 4. Solicited local and general symptoms (Total vaccinated cohort). Groups received the following H7N1 vaccine formulations: 3.75 ig HA adjuvanted with AS03a (H7N1-3.75A) or AS03b (H7N1-3.75B), or 7.5 ig HA adjuvanted with AS03a (H7N1-7.5A) or AS03b (H7N1-7.5B), or 15 ig plain antigen (H7N1-15). GI, gastrointestinal. Error bars represent 95% confidence intervals. Symptom intensity was graded on a scale of 1 (mild) to 3 (severe). Grade 3 symptoms were defined as follows: for redness or swelling, a diameter >100 mm; for fever, body temperature P39.0-40.0 "C; and for all other events, preventing normal activities.
adjuvanted formulations were immunogenic in both age strata (21-40 and 41-64 years).
Immune responses after the first dose were low, indicating the need for a 2-dose vaccination schedule to achieve robust immuno-genicity. Likewise, the adjuvanted H5N1 vaccine formulation containing 3.8 ig HA needed 2 doses to meet CBER and CHMP immunogenicity acceptance criteria [22], and also AS03-adjuvanted H7N9 vaccine formulations require 2 doses to induce an adequate immune response [14].
Overall, all formulations were well tolerated. Although injection site pain was reported in 71.2-86.7% of the participants, incidences of grade 3 pain did not exceed 5% of study participants, and other grade 3 solicited symptoms were also uncommon. Compliance with receipt of two vaccine doses was high (98.3%). Incidences of solicited injection site symptoms were higher in the adjuvanted vaccine groups than in the unadjuvanted and placebo groups, especially for pain, which was the most commonly reported injection site symptom. Incidences of fatigue and muscle ache also tended to be higher in the adjuvanted groups generally, and incidences of joint pain tended to be higher in all vaccine groups compared to placebo. For the other solicited general symptoms, no distinct differences between the vaccinated groups and the placebo group could be observed.
Until month 12, no participants discontinued participation due to an SAE. Unsolicited AEs were reported at similar rates in the various vaccine groups and placebo group. SAEs were uncommon
(reported by 9 participants), and none were considered by the investigators to be causally related to vaccination. Reactogenicity and safety profiles did not vary notably between AS03b and AS03a formulations, or between formulations containing 3.75 vs 7.5 ig HA.
Although it has been suggested that the evaluation of cellmediated immune responses might bring additional information, our analysis focused on endpoints used for CBER and CHMP criteria, which may be a potential limitation of this trial. Nevertheless, there was a notable increase in immune response from dose 1 to dose 2 for groups receiving adjuvanted formulations, which indicates the induction of a T-cell response.
In conclusion, in the current study in adults 21-64 years of age, 2-dose vaccination with AS03-adjuvanted H7N1 vaccine formulations induced a strong humoral immune response with a clinically acceptable safety profile. High cross-reactivity against vaccine-heterologous H7N9 virus was observed by HI assay 21 days postdose 2, in all adjuvanted groups. Our results show that AS03 adju-vantation elicits robust immunogenicity at antigen-sparing doses and thus represents an avenue for protection against various H7 subtype infections.
Funding
The trial was funded by the Biomedical Advanced Research and Development Authority of the United States Department of Health and Human Services (HHS/BARDA; contract number
A. Madan et al./Vaccine xxx (2017) xxx-xxx
HHS0100200700029C) and GlaxoSmithKline Biologicals SA. GlaxoSmithKline Biologicals SA was involved in all stages of the study conduct and analysis. GlaxoSmithKline Biologicals SA paid for all costs associated with the development and the publishing of the present manuscript.
Conflict of interest
AM, DF, JS, PL, BLI and AS are employees of the GSK group of companies. AM, PL, BLI, AS and PR own stock/stock options/ restricted shares in GSK. NS declares receiving funding from GSK for multiple influenza clinical trials. ES conducted several clinical trials with GSK. MF declares payment from Colchester Research Group (CRG) for work as an investigator outside the submitted work. MF's wife is CEO/owner of CRG and they have conducted numerous clinical trials with multiple sponsors over the last ten years. The CEO/owner of CRG received payment as per guidelines to present papers at conferences. LC has nothing to disclose. AT conducted several clinical trials with GSK as an investigator at Manna Research.
Authors contribution
All authors participated in the design, implementation or analysis, or the interpretation of the study, and the development of this manuscript. All authors had full access to the data and gave final approval before submission. The corresponding author was responsible for submission of the publication.
Acknowledgements
We are grateful to the principal investigators Benjamin Lasko and Matthew G. Davis. The authors also thank Janine Linden (GSK) for writing the study protocol, Stephanie Sharp for writing the study report, Manon André and Sridevi Pallem (Keyrus Bio-pharma C/0 GSK), and Thierry Ollinger (GSK) for contributing to immunological data generation, Joke Vandewalle and Iudit-Hajnal Filip (XPE Pharma & Science C/0 GSK) for drafting the manuscript, Shirin Khalili and Julie Todoroff (XPE Pharma & Science C/0 GSK), and Vincent Laporte (Business & Decision Life Science C/0 GSK) for manuscript coordination and editing.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.01. 054.
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