Conceived and designed the experiments: AB RG ST AP. Analyzed the data: AB RG FLP TS PD EM PC PdG LS CG AH VB ST AP. Wrote the paper: AB RG FLP TS PD EM PC PdG LS CG AH VB ST AP.
Dr Angelika Banzhoff, Dr Anke Hilbert, Dr Volker Brauer, Sandrine Tilman and Dr Audino Podda are employees of Novartis Vaccines, the sponsor of the study. Dr Franco Laghi-Pasini has received a travel grant from Novartis Vaccines. No other potential conflicts of interest were declared by the authors.
Pathogenic avian influenza virus (H5N1) has the potential to cause a major global pandemic in humans. Safe and effective vaccines that induce immunologic memory and broad heterotypic response are needed.
Healthy adults aged 18–60 and >60 years (n = 313 and n = 173, respectively) were randomized (1∶1) to receive two primary and one booster injection of 7.5 μg or 15 μg doses of a subunit MF59-adjuvanted H5N1 (A/Vietnam/1194/2004) (clade 1) vaccine. Safety was monitored until 6 months after booster. Immunogenicity was assessed by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization assays (MN). Mild injection-site pain was the most common adverse reaction. No serious adverse events relating to the vaccine were reported. The humoral immune responses to 7.5 μg and 15 μg doses were comparable. The rates for seroprotection (HI>40; SRH>25mm2; MN ≥40) after the primary vaccination ranged 72–87%. Six months after primary vaccination with the 7.5 μg dose, 18% and 21% of non-elderly and elderly adults were seroprotected; rates increased to 90% and 84%, respectively, after the booster vaccination. In the 15 μg group, seroprotection rates among non-elderly and elderly adults increased from 25% and 62% after primary vaccination to 92% and 88% after booster vaccination, respectively. A heterologous immune response to the H5N1/turkey/Turkey/05 strain was elicited after second and booster vaccinations.
Both formulations of MF59-adjuvanted influenza H5N1 vaccine were well tolerated. The European Union requirement for licensure for pre-pandemic vaccines was met by the lower dose tested. The presence of cross-reactive antibodies to a clade 2 heterologous strain demonstrates that this vaccine may be appropriate for pre-pandemic programs.
ClinicalTrials.gov
The highly pathogenic avian influenza H5N1 virus, first reported in China in 1996, is responsible for severe avian influenza outbreaks
Since future pandemic virus strains cannot be clearly anticipated, vaccines using strains with pandemic potential, such as H5N1, that induce immunologic memory and cross-reactivity, could form the first line of defense
Immunogenicity data on conventional non-adjuvanted H5N1 vaccines are not encouraging. A previous study showed that two vaccinations with 90 μg hemagglutinin (HA) of a non-adjuvanted vaccine induced an antibody response at protective levels in only half of an immunologically naïve population
The use of adjuvants in vaccines is an established method for increasing the immune response and cross-reactivity and reducing the antigen content
With the exception of the virus strain and the amount of antigen, the MF59-adjuvanted H5N1 vaccine used in this trial and the licensed seasonal influenza vaccine, Fluad®, are identical. Previous clinical trials using other potential pandemic influenza strains such as H5N3
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
Healthy adults 18 years of age and above were eligible to participate in the study. Principal criteria for exclusion from the study were history of anaphylactic shock; allergy to eggs or any vaccine component; immunodeficiency or immunosuppressive therapy; participation in another clinical trial; previous receipt of an H5N1, H3 or H9 vaccine; current acute febrile disease; current use of antibiotics or antivirals; planned surgery during the study period; and, for women, pregnancy or refusal to use reliable contraception. The study was approved by the Ethics Review Committee of each participating center and was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. The study is registered in the National Institutes of Health ClinicalTrials.gov register (NCT00311480). Written informed consent was obtained from each eligible volunteer before enrollment.
Eligible participants were randomized at a 1∶1 ratio, stratified by age group, to receive two vaccinations of either 7.5 μg or 15 μg HA H5N1 (A/Vietnam/1194/2004; NIBRG-14) inactivated subunit influenza virus vaccine adjuvanted with MF59. A subgroup of the first 50% of participants enrolled at each site also received a booster vaccination with the same dose as that used for primary vaccination. Both formulations were supplied in pre-filled syringes (0.5 mL) and administered into the deltoid muscle by an un-blinded individual who had sole access to the randomization code at each site. All other site personnel and participants were blinded to the vaccine group assignment. The first two vaccinations were administered 21 days apart, and the third (booster) vaccination was administered 6 months later.
Each participant was observed for 30 minutes post-injection for anaphylactic reactions, and was instructed to complete a diary card on the day of vaccination and each of the 6 subsequent days to report solicited local reactions at the site of injection (i.e., ecchymosis, redness, induration, swelling and pain) and systemic reactions (i.e., chills, malaise, myalgia, arthralgia, nausea, headache, sweating, fatigue and fever). Severe solicited reactions were defined as injection-site reactions >50 mm diameter, reactions preventing the performance of normal daily activities or body temperature ≥40°C. All unsolicited adverse events were recorded for 3 weeks after each vaccination and assessed by the investigator for severity, seriousness and relationship to the study vaccine. In addition, all adverse events necessitating a physician's visit or leading to premature study discontinuation, and all serious adverse events were recorded during the 6-month follow-up periods after the second and booster vaccinations.
Blood samples were collected from study participants at baseline before the first vaccination, before the second vaccination, and at 3 weeks and 6 months after the second vaccination (pre-booster). In the booster subset, blood samples were also collected 3 weeks and 6 months post-booster. The modified hemagglutinin-inhibition (HI) test using horse erythrocytes was performed in the laboratory of Clinical Serology, Novartis Vaccines, Marburg, Germany and was based on the method of Stephenson and colleagues
The planned sample size of 460 individuals (i.e., 230 subjects per study arm) was sufficient to demonstrate that the immune response to primary vaccination with 7.5 μg HA was non-inferior to the 15 μg HA formulation. For each vaccine group within each age cohort and at each blood sampling time point, the proportion of subjects seroprotected, defined as an SRH area ≥25 mm2 or MN titer ≥40, were calculated and two-sided 95% confidence intervals (CIs) were determined using the Clopper-Pearson method. MN titers below the detection limit (1∶20) were arbitrarily assigned to half that limit for the purpose of the analysis. Undetectable SRH areas were assigned an area of 4 mm2. The immune response elicited by two vaccinations formulated with 7.5 μg HA was considered non-inferior to that containing 15 μg HA if the lower limit of the two-sided 95% CI around the ratio of geometric mean titer (GMT) at 3 weeks after the second injection (GMT7.5/GMT15) exceeded 0.5. Participants were excluded from the immunogenicity analysis if they did not receive all designated vaccinations or if the planned blood collections were missed or performed outside the specified time window. Safety was analyzed in participants who received at least one vaccination and reported solicited or unsolicited adverse events.
The target was to enrol and randomize 520 subjects to treatment, but due to slow recruitment a total of 486 individuals were enrolled in the study, of whom 313 were 18–60 years of age and 173 were >60 years of age (
A total of 486 healthy adults were enrolled in the study and randomized in a 1∶1 ratio, stratified by age group (non-elderly adults, aged 18–60 years, and elderly adults, aged >60 years) to receive two vaccinations of either 7.5 μg or 15 μg HA H5N1 (A/Vietnam/1194/2004; NIBRG-14) inactivated subunit influenza virus vaccine adjuvanted with MF59. The first two vaccinations were administered 21 days apart, and a subset of the first participants also received a third (booster) vaccination 6 months later.
18–60 years | >60 years | |||
7.5 μg HA | 15 μg HA | 7.5 μg HA | 15 μg HA | |
157 | 156 | 87 | 86 | |
43.4 (18–60) | 42.3 (18–60) | 71 (62–88) | 70.1 (61–90) | |
99 | 97 | 100 | 100 | |
45∶55 | 45∶55 | 60∶40 | 56∶44 | |
52 | 56 | 89 | 85 |
HA, hemagglutinin
Both the 7.5 μg and 15 μg formulations were well tolerated, and there were no reports of serious adverse events related to the study vaccine during the study period or during follow-up (mean 13 months) (
Classified as mild/moderate (white bars) or severe (grey bars) after the first, second and third vaccination in non-elderly adults (18–60 years; part a) and in elderly adults (>60 years; part b).
The most commonly reported solicited systemic adverse reactions in both age cohorts were myalgia and headache, with the highest rates occurring after the first vaccination (in the 7.5 μg group: myalgia in 28% of non-elderly and 8% of elderly and headache in 15% of non-elderly and 9% of elderly; in the 15 μg group: myalgia in 26% non-elderly and 13% elderly and headache in 19% non-elderly and 9% elderly). Severe solicited reactions were reported by less than 2% of non-elderly adults and 1% of the elderly participants (
After primary vaccination, an axillary temperature of 38°C or greater was reported by 1% and 3% of non-elderly adults in the 7.5 μg and 15 μg groups, respectively, while no elderly participants reported fever. No participants reported severe fever (≥40°C) and no participant of any age reported fever after the booster vaccination. Fewer than 3% of individuals in either dose group reported mild to moderate non-solicited adverse events that were possibly related to the vaccine.
During the 3-week to 6-month follow-up period after the second vaccination, fewer than 3% of participants reported adverse events. One participant (18–60 years) in the 7.5 μg group reported a non-solicited adverse event possibly related to vaccination (generalized myalgia and arthralgia of moderate severity 30 days after the second vaccination). One pregnancy occurred before the second vaccination and the participant withdrew from the study and subsequently delivered a healthy full-term infant.
After the third vaccination, one non-elderly adult (1%) and one elderly (3%) participant in the 7.5 μg group and two (2%) non-elderly adults and one (4%) elderly participant in the 15 μg group reported non-solicited adverse events, all judged as unrelated to the study vaccine, during the 3-week to 6-month follow-up period. No vaccine-related serious adverse events were reported during the entire study period.
A total of 466 of the 486 enrolled individuals were included in the immunogenicity analyses.
At baseline in both dose groups, HI titers, SRH areas and MN titers, respectively, were detectable in 0–3%, 5–9% and 3% of non-elderly adults and 11–12%, 11–24% and 15–18% of elderly participants (
18–60 years | >60 years | |||
7.5 μg HA | 15 μg HA | 7.5 μg HA | 15 μg HA | |
n = 151 | n = 147 | n = 81 | n = 74 | |
GMT | 5.1 (4.8–5.4) | 5.5 (5.2–5.9) | 8.1 (6.3–11) | 8.2 (6.3–11) |
Seroprotection rate, % | 0 (0–2) | 3 (1–7) | 12 (6–22) | 11(5–20) |
GMT | 17 (13–23) | 21 (16–28) | 31 (20–49) | 40 (25–63) |
GMR | 3.4 (2.6–4.5) | 3.8 (2.8–5.0) | 3.9 (2.6–5.7) | 4.9 (3.3–7.3) |
Seroprotection rate, % | 34 (26–42) | 39 (31–47) | 51 (39–62) | 53 (41–64) |
Seroconversion rate, % | 34 (26–42) | 35 (28–44) | 38 (28–50) | 46 (34–58) |
GMT | 82 (62–110) | 85 (64–110) | 77 (53–114) | 82 (55–123) |
GMR | 16 (12–21) | 15 (12–21) | 9.52 (6.6–1.4) | 10 (6.8–1.5) |
Seroprotection rate, % | 73 (65–80) | 72 (64–79) | 75 (64–84) | 76(64–85) |
Seroconversion rate, % | 73 (65–80) | 69 (61–77) | 67 (55–77) | 70 (59–80) |
GMT | 12 (9.0–17) | 16 (12–21) | 26 (13–49) | 42 (21–85) |
Seroprotection rate, % | 27 (17–39) | 34 (24–45) | 54 (37–71) | 62 (41–80) |
GMT | 138 (93–205) | 104 (72–151) | 129 (83–201) | 212 (130–345) |
GMR to pre-booster | 11 (7.6–16) | 6.54 (4.6–9.3) | 5.02 (2.8–9.0) | 5.07 (237–9.6) |
Seroprotection rate, % | 83 (72–91) | 76 (65–84) | 92 (78–98) | 96 (80–100) |
Seroconversion rate, % | 73 (61–83) | 62 (51–73) | 51 (34–68) | 54 (33–73) |
GMT | 27 (18–41) | 29 (20–42) | 36 (20–68) | 71 (34–145) |
Seroprotection rate, % | 52 (40–64) | 46 (35–58) | 57 (39–74) | 77 (55–92) |
Numbers in parenthesis are two-sided 95% confidence intervals; Seroprotection = HI titer ≥40; Seroconversion = negative pre-vaccination serum (i.e., HI titer <10) and post-vaccination HI titer ≥40 or significant increase (at least a fourfold increase in HI titer in subjects who were positive pre-vaccination, i.e., HI titer ≥10); HA, hemagglutinin; GMT, geometric mean titer; GMR, the geometric mean of the ratio over baseline.
18–60 years | >60 years | |||
7.5 μg HA n = 149 | 15 μg HA n = 149 | 7.5 μg HA n = 84 | 15 μg HA n = 80 | |
GMA | 4.8 (4.3−5.3) | 5.2 (4.7−5.8) | 6.0 (4.9−7.4) | 7.6 (6.2−9.5) |
Seroprotection rate, % | 5 (2−10) | 9 (5−14) | 11 (5−19) | 24 (15−35) |
GMA | 11 (9.5−14) | 15 (12−18) | 17 (13−22) | 19 (14−25) |
GMR | 2.4 (2.0−2.9) | 2.8 (2.4−3.4) | 2.9 (2.2−3.7) | 2.5 (1.9−3.2) |
Seroprotection rate, % | 40 (32−49) | 51 (43−59) | 52 (41−63) | 58 (46−68) |
Seroconversion rate, % | 38 (30–47) | 42 (34–51) | 44 (33–55) | 43 (32–54) |
GMA | 37 (32−43) | 36 (31−41) | 30 (24−37) | 31 (25−39) |
GMR | 7.7 (6.6−9.1) | 6.9 (5.9−8.0) | 5.0 (3.9−6.4) | 4.1 (3.2−5.3) |
Seroprotection rate, % | 85 (79−91) | 85 (79−91) | 80 (70−88) | 81 (71−89) |
Seroconversion rate, % | 85 (78–90) | 80 (73–86) | 70 (59–80) | 69 (57–79) |
GMA | 6.8 (5.4−8.6) | 8.0 (6.5−9.9) | 6.3 (4.3−9.1) | 16 (10−24) |
Seroprotection rate, % | 18 (10−29) | 25 (16−36) | 21 (10−37) | 62 (41−80) |
GMA | 41 (34−48) | 42 (36−49) | 32 (23−46) | 42 (29−62) |
GMR to pre-booster | 6.0 (4.7−7.5) | 5.2 (4.2−6.5) | 5.2 (3.5−7.7) | 2.7 (1.7−4.2) |
Seroprotection rate, % | 89 (79−95) | 92 (83−97) | 84 (69−94) | 88 (70−98) |
Seroconversion rate, % | 83 (72–91) | 81 (71–89) | 63 (46–78) | 65 (44–83) |
GMA | 17 (13−21) | 17 (14−21) | 13 (8.4−19) | 25 (16−39) |
Seroprotection rate, % | 55 (43−67) | 55 (43−67) | 43 (26−61) | 77 (55−92) |
Numbers in parenthesis are two-sided 95% confidence intervals; Seroprotection = SRH ≥25mm2; Seroconversion = negative pre-vaccination serum (i.e., SRH titer ≤ 4mm2) and post-vaccination SRH area ≥25 mm2 or significant increase (at least a 50% increase in SRH area in subjects who were positive pre-vaccination, i.e., SRH area >4mm2); HA, hemagglutinin; GMA, geometric mean area; GMR, the geometric mean of the ratio over baseline
18–60 years | >60 years | |||
7.5 μg HA n = 151 | 15 μg HA n = 151 | 7.5 μg HA n = 84 | 15 μg HA n = 80 | |
GMT | 11 (10−12) | 11 (10−12) | 18 (14−22) | 16 (13−20) |
MN ≥1∶40, % | 3 (1−7) | 3 (1−7) | 18 (10−28) | 15 (8−25) |
GMT | 26 (22−32) | 35 (29−42) | 42 (31−59) | 51 (37–71) |
MN ≥1∶40, % | 33 (26−41) | 44 (36−52) | 49 (38−60) | 51 (40−63) |
GMT | 117 (98−140) | 102 (85−122) | 80 (61−105) | 90 (68−119) |
MN ≥1∶40, % | 85 (78−90) | 81 (74−87) | 79 (68−87) | 76 (65−85) |
GMT | 33 (26−42) | 43 (34−54) | 31 (20−47) | 63 (39−101) |
MN ≥1∶40, % | 41 (29−53) | 53 (42−64) | 42 (26−59) | 69 (48−86) |
GMT | 210 (168−262) | 214 (174−263) | 154 (105−227) | 225 (146−345) |
MN ≥1∶40, % | 94 (86−98) | 96 (90−99) | 97 (86−100) | 96 (80−100) |
GMT | 34 (26−44) | 35 (27−44) | 25 (17−39) | 46 (28−75) |
MN ≥1∶40, % | 41 (29−53) | 46 (35−58) | 37 (21−55) | 64 (41−83) |
Numbers in parenthesis are two-sided 95% confidence intervals; HA, hemagglutinin; GMT, geometric mean titer
Following two vaccinations with either formulation, 81–86% of non-elderly adults and 76–79% of elderly adults had neutralizing antibody titers of at least 40 (
Measured 3 weeks after a second injection and 3 weeks after a booster dose in non-elderly adults (18–60 years; parts a and c, respectively) and in elderly adults (>60 years; parts b and d, respectively).
After primary vaccination (two-dose regimen) both the 7.5 μg and the 15 μg MF59-adjuvanted vaccine formulations met all the Committee for Medicinal Products for Human Use criteria for evaluation of influenza vaccines (HI and SRH assays) in both non-elderly and elderly participants
Seroprotection rates assessed by HI assay increased from 27% (18–60 years) and 54% (>60 years) immediately before the 7.5 μg booster dose to 83% and 92%, respectively, 3 weeks following the booster dose. Similar increases in seroprotection rates were observed in the 15 μg group, rising from 34% (18–60 years) and 62% (>60 years) before the booster dose to 76% and 96%, respectively, after the booster dose (
An antibody response against a heterologous clade 2 strain (H5N1/turkey/Turkey/1/05) was detectable by HI assay, with cross-immunogenicity to the clade 2 strain increasing from 28–36% after the second dose to 59–70% after the third dose, across dosage groups and age cohorts. Seroprotection rates as measured by SRH were 57–77% and 78–88% after the second and third doses, respectively. The MN GMTs ranged 12–19 after the second vaccination and 44–93 after the booster for the clade 2 strain (
Assessment Parameter | 7.5 μg HA | 15 μg HA | |||
Post-second injection (day 43) | GMT | 18 (13–24) (n = 69) | 14 (10–19) (n = 78) | ||
Seroprotection rate | % | 36 (25–49) | 28 (19–40) | ||
Post-booster injection (day 223) | GMT | 58 (39–86) | 46 (31–67) | ||
Seroprotection rate | % | 70 (58–80) | 59 (48–70) | ||
Post-second injection (day 43) | GMT | 19 (15–24) | 19 (15–24) | ||
Titer ≥40 | % | 27 (17–39) | 21 (13–31) | ||
Post-booster injection (day 223) | GMT | 77 (60–100) | 93 (74–118) | ||
Titer ≥40 | % | 73 (61–83) | 79 (69–87) | ||
Post-second injection (day 43) | GMA | 23 (19–28) | 24 (20–28) | ||
Seroprotection rate | % | 70 (58–80) | 70 (59–80) | ||
Post-booster injection (day 223) | GMA | 30 (26–36) | 34 (29–39) | ||
Seroprotection rate | % | 83 (72–91) | 88 (78–94) | ||
Post-second injection (day 43) | GMT | 14 (8.4–22) | 13 (7.74–23) (N = 23) | ||
Seroprotection rate | % | 36 (21–54) | 35 (16–57) | ||
Post-booster injection (day 223) | GMT | 36 (19–68) | 49 (25–97) | ||
Seroprotection rate | % | 67 (49–81) | 65 (44–83) | ||
Post-second injection (day 43) | GMT | 12 (9.4–16) | 17 (13–23) | ||
Titer ≥40 | % | 11 (3–25) | 31 (14–52) | ||
Post-booster injection (day 223) | GMT | 44 (29–67) | 61 (39–95) | ||
Titer ≥40 | % | 62 (45–78) | 65 (44–83) | ||
Post-second injection (day 43) | GMA | 15 (9.91–22) | 24 (16–36) | ||
Seroprotection rate | % | 57 (39–73) | 77 (56–91) | ||
Post-booster injection (day 223) | GMA | 24 (17–34) | 33 (23–48) | ||
Seroprotection rate | % | 78 (62–90) | 88 (70–98) |
Numbers in parenthesis are two-sided 95% confidence intervals; GMT/GMA, geometric mean titer/geometric mean area ;Seroprotection rate = SRH ≥25mm2 or MN ≥40.
The H5N1 virus is the most likely candidate for an emerging pandemic strain. Owing to the anticipated rapid spread of the pandemic virus, together with the time required to produce a vaccine that matches the circulating strain, the first wave of the pandemic may have passed in many countries before a significant amount of pandemic vaccine is available. The success of national pandemic preparedness strategies may depend on providing appropriate H5N1 pre-pandemic vaccines (i) in a proactive controlled manner (ii) that induce immunologic memory and demonstrate cross-reactivity in the whole population, including the elderly, and (iii) with a proven safety profile. Evidence for the safety of the MF59 adjuvant is provided not only by the present trial, but also from a review of the database (>14,000 clinical trial participants and >30 million distributed doses post-licensure) for the interpandemic influenza vaccine Fluad®, which differs from the H5N1 vaccine only in its HA content and viral strain composition (2×7.5 μg or 2×15 μg H5N1 versus 1×45 μg HA seasonal viral strains). It has been well established that repeated yearly vaccination with Fluad® does not lead to increased reactogenicity or other side effects
As was the case in previous H5N1 vaccine trials
Regardless of antigen content, the good immune response observed after two primary injections of adjuvanted H5N1 vaccine exceeded all requirements of the European Regulatory Authority
As expected, seroprotection rates fell significantly 6 months after primary vaccination but increased after the booster dose to levels higher than those achieved 3 weeks after the second vaccination, indicating a memory response in both elderly and non-elderly adults. As the timing of the next pandemic influenza outbreak is unknown, the memory response may be more important than persistence, as subsequent exposure to the pandemic vaccine or wild-type virus could trigger a full immunologic response.
A heterologous immune response against H5N1/turkey/Turkey/1/05 (NIBRG-23) was detectable after the primary and booster vaccinations, indicating that there is cross-reactivity between the clade 1 and clade 2 strains. Increased heterologous immune responsiveness against antigenically drifted strains presents a significant public health advantage in the event of a pandemic outbreak. We have shown elsewhere that, among primed subjects, protective cross-reactive antibody titers to diverse H5N1 virus variants can be achieved within 7 days after administration of a single dose of M59-adjuvanted vaccine [Stephenson, manuscript submitted]. In addition, the availability of a 7.5 μg formulation of MF59-adjuvanted influenza vaccine would allow a threefold increase in the number of subjects who could be vaccinated compared with seasonal trivalent influenza vaccines, enabling production of higher quantities of vaccine, in pre-pandemic and pandemic situations, as encouraged by the World Health Organization
In conclusion, H5N1/Vietnam/1194/2004 influenza vaccine adjuvanted with MF59 can be safely used as a pre-pandemic vaccine. Primary vaccination of non-elderly and elderly adults induces a sufficient immune response and cross-reactivity against the clade 2 H5N1/turkey/Turkey/05 strain, and booster vaccination leads to a strong and sustained immunologic response. A low-dose antigen formulation (7.5 μg) resulted in a comparable seroprotection benefit when compared with a higher dose (15 μg). These results, in conjunction with the extensive safety data on the MF59 adjuvant, suggest that this vaccine would be a suitable choice for proactive priming in advance of pandemic influenza.
Trial protocol
(0.25 MB PDF)
CONSORT checklist
(0.06 MB DOC)
At the final stages, Dr John Clarke from Health Interactions Limited, helped to revise the manuscript in conjunction with the authors.