The A/H5N1 monovalent split-virion recombinant influenza prepandemic candidate vaccine was manufactured by GlaxoSmithKline (GSK) Biologicals in Quebec, Canada. The vaccine contained 3.75 μg HA of the A/Indonesia/5/2005-like IBCDC-RG2; Clade 2.1 (H5N1) strain (Centers for Disease Control and Prevention [CDC], Atlanta, USA) adjuvanted with AS03A (an O/W emulsion-based Adjuvant System containing 11.86 mg of α-tocopherol).
This open-label, single-group study (NCT00742885) was conducted at two centres in Japan. The study set out to evaluate the humoral immune response generated by two doses of the adjuvanted prepandemic A/Indonesia/5/2005 H5N1 vaccine in terms of H5N1 haemagglutination inhibition (HI) antibody titres against the vaccine strain (Clade 2.1) 21 days after the second dose. Secondary and exploratory endpoints included the assessment of the humoral response in terms of neutralising antibody titres, the response against additional H5N1 strains (Clade 1 and Clade 2.2), as well as the evaluation of safety and reactogenicity. The latter were assessed in terms of the occurrence of solicited local and general adverse events (AEs), unsolicited AEs, serious AEs (SAEs) and by the evaluation of medically attended visits and selected laboratory parameters.
Healthy Japanese men and women aged between 20 and 64 years at the time of first vaccination were eligible for inclusion if they were in good general health and provided written informed consent before enrolment. Subjects were stratified by age (20-40 years and 41-64 years) in a 1:1 ratio. Subjects were excluded from the study if they had an axillary temperature ≥37.5°C, or acute symptoms of more than mild severity on the scheduled date of first vaccination; any confirmed or suspected immunosuppressive or immunodeficient condition including history of human immunodeficiency virus (HIV) infection; administration of any registered vaccine within 30 days before study vaccination or planned administration within the first vaccination period up to blood sampling at Day 42; use of any investigational or non-registered product (drug or vaccine) within 30 days prior to study enrolment or planned use during the study period, and history of previous H5N1 vaccination; or history of H5N1 influenza infection.
Subjects received two doses of the prepandemic (H5N1) influenza candidate vaccine. The vaccine was administered intramuscularly in the deltoid region of the non-dominant arm on Day 0 and the second dose was given 21 days later in the non-dominant arm. Blood samples were collected for serological testing on Day 0, 7, 21, 42 and 182, and telephone contact was made on Day 84 to record any unsolicited AEs (Figure 1). Solicited AEs were assessed up to 7 days after each vaccination and, additionally, unsolicited AEs, including SAEs, were recorded throughout the duration of the study.
The protocol and study documents were approved by the Institutional Review Boards of the respective study centres - National Hospital Organization Tokyo National Hospital and Haradoi Hospital. The study was conducted in accordance with Good Clinical Practice (GCP) and the Declaration of Helsinki. GSK Biologicals (Wavre, Belgium) sponsored the study and was involved in all stages of the study conduct, including analysis of data. All authors had full access to the data and were involved in the analysis of data and preparation of the manuscript.
Assessment of immunogenicity
The humoral immune response against the vaccine strain (A/Indonesia/5/2005; Clade 2.1), as well as against the heterologous strains (A/turkey/Turkey/1/2005; Clade 2.2 and A/Vietnam/1194/2004; Clade 1) was measured in terms of the standard HI antibody response according to the guidelines of the Committee for Medicinal Products for Human Use (CHMP) . In addition, neutralising antibodies against the vaccine (A/Indonesia/5/2005) and one heterologous strain (A/Vietnam/1194/2004) were measured by the microneutralisation (MN) assay and are further referred to as H5N1 neutralising antibodies. Studies have shown that neutralisation assays may be more sensitive than the HI test in detecting both greater increases in antibody levels and in detecting infected individuals who are seronegative according to the HI assay .
Specific HI antibody titres were determined at GSK Biologicals' laboratories using methods described elsewhere . Antibody titre measurements were conducted on thawed frozen serum samples with a standardised and validated micromethod using four haemagglutination-inhibiting units (HIU) of the appropriate antigens and a 0.5% horse erythrocyte suspension. Non-specific serum inhibitors were removed by subjecting the sera to heat treatment (56°C) and receptor-destroying enzyme. The sera obtained were evaluated for HI antibody levels. Starting with an initial dilution of 1:10, a dilution series (by a factor of 2) was prepared up to an end dilution of 1:20,480. The titration endpoint was taken as the highest dilution step that showed complete inhibition (100%) of haemagglutination. All assays were performed in duplicate.
The titre of H5N1 virus neutralising antibody contained in the serum was determined by an MN assay on thawed frozen serum samples. Each sample was tested in triplicate. Non-specific serum inhibitors were removed by subjecting the sera to heat treatment (56°C). A standardised amount of virus (100 infectious Unit [TCID50] in 0.05 mL) was mixed with serial dilutions of sera and incubated to allow binding of the antibodies to the virus. A cell suspension, containing a defined number of Madin-Darby canine kidney (MDCK) cells was then added to the mixture of virus and antiserum and incubated at 33°C for 7 days. After the incubation period, virus replication was visualised by haemagglutination of chicken red blood cells. The 50% neutralisation titre of a serum was calculated by the method of Reed and Muench .
Assessment of safety
Adverse events were classified according to the Medical Dictionary for Regulatory Activities (MedDRA). The occurrence of AEs was recorded by the subjects themselves using diary cards. In addition, investigators solicited information on specific local AEs (swelling/induration, redness and pain at injection site) and general AEs (fever, headache, fatigue, muscle aches, sweating, joint pain and shivering) occurring within 7 days of each vaccination. Symptom intensity was assessed on a 3-point scale where grade 3 represents the most intense. For both unsolicited AEs and solicited general AEs, the investigators determined the likely relationship of vaccination to symptoms. Intensity and relationship to vaccination of unsolicited local and general AEs were recorded during a 21-day follow-up period from each vaccine administration, as well as overall (Day 0 through to Day 84). All solicited local (injection site) reactions were considered causally related to vaccination. The occurrence of SAEs was recorded during the entire study (up to Day 182).
Haematological and biochemical parameter testing was performed by SRL Medisearch Inc, Japan. The number and percentage of subjects with normal or abnormal haematological and biochemical values, and with normal or abnormal urine values at Day 0, 7 and 42, were calculated. An assessment of these haematological, biochemical and urine parameters was performed at Day 7 and 21 - all parameters were reviewed at Day 7 by the investigators before administering the second vaccine dose at Day 21. Blood parameters assessed were complete blood count, blood urea nitrogen (BUN), creatinine, alanine amino transferase (ALT) and aspartate aminotransferase (AST). Urine parameters measured were protein, glucose, blood and urobilinogen.
The statistical methods for all immunogenicity analyses were performed using the per protocol group. The primary objective of this study was to evaluate the humoral immune response induced by two doses of the H5N1 influenza candidate vaccine in terms of H5N1 HI antibody titres against the vaccine strain. The immunogenicity assessments were based on the surrogate HI endpoints as required by regulatory authorities (CBER and CHMP). In order to meet or exceed these immunogenicity guidance criteria, a target sample size of 100 subjects was required in order to ensure 90 evaluable subjects. Taking into account a 10% drop-out rate and considering a true seroconversion rate (SCR for HI antibodies was defined as the percentage of subjects with either a pre-vaccination titre <1:10 and a post-vaccination titre ≥1:40 or a pre-vaccination titre ≥1:10 and at least a 4-fold increase in post-vaccination titre) of 83.7% and a true seroprotection rate (SPR; defined as the percentage of subjects with a serum H5N1 HI antibody titre ≥1:40) of 84.3%, the proposed sample size allowed for an overall probability of above 85% of meeting the lower limits of 95% confidence intervals (CIs) for SCRs and SPRs of 40% and 70%, respectively. Ninety five percent CIs were calculated for geometric mean titres (GMTs) by exponential transformation of the 95% CI for the mean of log-transformed titres, assuming normal distribution of log-transformed titres.
The immunogenicity analysis was performed for each age stratum and overall. The humoral immune response endpoints in terms of H5N1 HI antibodies were measured using the GMTs of H5N1 HI antibody titres at Day 0, 21, 42 and 182, SCR at Day 21, 42 and Day 182 and seroconversion factors (SCF; defined as the fold increase in serum H5N1 HI antibody GMTs post-vaccination compared with Day 0) at Day 21, 42 and Day 182. The CHMP cut off for SCF is defined as a ratio of >2.5. SPRs were also calculated at Day 0, 21, 42 and 182. Seropositivity was defined as an HI titre >1:10.
For neutralizing antibodies, the endpoints (with 95% CIs) were seropositivity, GMTs and SCRs (SCR for MN antibodies was defined as the percentage of subjects with at least four-fold increase in post-vaccination neutralising antibody titres). The GMTs of neutralising antibody titres were calculated at Day 0, 42 and 182, and the SCRs in terms of neutralising antibody titres were calculated at Day 42 and 182.
The safety analysis was performed on all subjects receiving at least one vaccination (the total vaccinated cohort [TVC]). The percentage of subjects with at least one local AE (solicited and unsolicited), at least one general AE (solicited and unsolicited) and any AE during the solicited follow-up period was tabulated, with exact 95% CI after each vaccination and overall per subject considering both post-immunisation periods.
Solicited symptoms and any pain relief and/or antipyretics taken by the subject to correct the symptoms of local and/or general solicited symptoms were recorded during the 7-day follow-up period after each H5N1 vaccination.