Evaluation of non-inferiority of intradermal versus adjuvanted seasonal influenza vaccine using two serological techniques: a randomised comparative study

The study was approved by ethics committees in participating countries (Comité de Protection des Personnes, France; Ethics Committee of Universitair Ziekenhuis Antwerpen, Belgium), and also by the French Health Products Safety Agency (Agence Française de Sécurité Sanitaire des Produits de Santé, AFSSaPS) in France and by the Federal Agency for Medicines and Health Products (Federaal Agentschap voor Geneesmiddelen en Gezondheidsproducten, FAGG) in Belgium. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Participants gave written informed consent before enrolment. The study was registered on ClinicalTrials.gov; identifier: NCT00554333.

This phase-III, multicentre, randomised, controlled, open-label, parallel-group study was carried out in adults aged 65 years or older who were recruited at three centres in Belgium and seven centres in France between October and December 2007. Participants attended the study centre on two occasions. At visit 1, participants who were eligible for inclusion were randomised to receive intradermal vaccine or an adjuvanted intramuscular influenza vaccine. Investigators were provided with a masked randomised list generated using a 1:1 ratio based on balanced permuted blocks stratified by centre. A pre-vaccination blood sample was taken and a single dose of allocated vaccine given. At visit 2, which was scheduled 21 ± 3 days post-vaccination, a second blood sample was taken for assessment of immunogenicity and collection of vaccine safety data.

Individuals were invited to participate by the investigators via a phone call, an invitation letter, or during a consultation. Participants aged 65 years or older were eligible for enrolment in the study provided they met none of the exclusion criteria: acute febrile illness (oral temperature ≥ 37.5°C); systemic hypersensitivity to egg or chicken proteins or any of the vaccine constituents; thrombocytopenia or a bleeding disorder contraindicating intramuscular vaccination; and unstable chronic illness (defined as illness requiring hospitalisation or a clinically significant change in medication in the previous 12 weeks). Individuals were also excluded if they had: congenital or acquired immunodeficiency; received treatment with immunosuppressive therapy within the previous 6 months or long-term treatment with systemic corticosteroids; received blood or blood-derived products in the previous 3 months. Additional exclusion criteria were: current abuse of alcohol or drug addiction; any vaccination within the previous 4 weeks or planned vaccination within the 4 weeks after the first vaccination; seasonal influenza vaccination in the previous 6 months; or previous seasonal influenza vaccination by the intradermal route.

Both vaccines were formulated according to the WHO recommendations for the 2007-2008 Northern Hemisphere influenza season: A/Solomon Islands/3/2006 (H1N1)-like strain; A/Wisconsin/67/2005 (H3N2)-like strain; and B/Malaysia/2506/2004-like strain.

The intradermal vaccine (Intanza^® 15 μg; Sanofi Pasteur, Lyon, France) is an inactivated, split-virion influenza vaccine containing 15 μg HA/strain per 0.1-ml dose, all propagated in fertilised hen's eggs. The manufacturing process is based on that used for the intramuscular seasonal vaccine, with an additional concentration step to obtain a lower volume for intradermal use. The vaccine was administered in the deltoid region using a pre-filled intradermal micro-needle injection system (Becton Dickinson; Franklin Lakes, NJ, USA) [21].

The adjuvanted vaccine (Addigrip^®/Fluad^®, Novartis Vaccines and Diagnostics S.r.l., Origgio, Italy) is a subunit (HA and neuraminidase) influenza vaccine containing 15 μg HA/strain per 0.5-ml dose, all propagated in fertilised hen's eggs and adjuvanted with the squalene-based adjuvant MF59C.1. The vaccine was administered by injection into the deltoid muscle.

Consistent with previous studies of this intradermal vaccine, the primary immunogenicity endpoints were anti-HA antibody titres (geometric mean titre) for the three strains on day 21 post-vaccination [18, 19]. Antibody titres were determined for each strain before and 21 days after vaccination by haemagglutination inhibition (HI) assay [22]. This was carried out in accordance with standard procedures at sanofi pasteur in which the antibody titre is the highest reciprocal dilution that induced complete inhibition of haemagglutination (1/dilution [1/dil]). The secondary endpoints were anti-HA antibody titres for the three strains assessed using the single radial haemolysis (SRH) method, in which anti-HA antibody titres are measured in square millimetres (mm²) [23]. SRH assays were performed by the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, UK). HI and SRH assays are recognised by the European Medicines Agency (EMA) for the assessment of the immunogenicity of influenza vaccines [24, 25].

The other secondary endpoints were anti-HA individual titre ratios (geometric mean titre ratio [GMTR], day 21/day 0) determined using the HI and SRH methods, the post-vaccination seroprotection rate (defined as the percentage of patients with anti-HA titre ≥ 40 [1/dil] or ≥ 25 mm² for the HI and SRH methods, respectively), and seroconversion or significant increase rate on day 21 according to HI and SRH methods. For the HI method, seroconversion was defined as post-vaccination anti-HA titre ≥ 40 (1/dil) for subjects with a pre-vaccination titre < 10 (1/dil) and a significant increase was defined as ≥ fourfold increase in anti-HA titre for subjects with a pre-vaccination titre ≥ 10 (1/dil). For the SRH method, seroconversion was defined as a post-vaccination anti-HA titre ≥ 25 mm² for subjects with a pre-vaccination titre ≤ 4 mm², and a significant increase was defined as ≥ 1.5-fold increase from pre-to post-vaccination for subjects with a pre-vaccination titre > 4 mm² [24].

Compliance with EMA criteria (as defined for annual re-licensure trials) for the immunogenicity of influenza vaccines in elderly people for both the HI and SRH methods was also assessed: a GMTR >2, a seroprotection rate ≥ 60%, and a rate of seroconversion or significant titre increase ≥ 30% using either test [24].

Participants used diary cards to record details of solicited injection-site reactions (pain, erythema, swelling, induration, ecchymosis, pruritus) and solicited systemic reactions (fever [rectal equivalent temperature ≥ 38.0°C], headache, malaise, myalgia, shivering) occurring within 7 days of vaccination. Participants also recorded unsolicited adverse events and serious adverse events occurring within 21 days of vaccination. If the investigators felt that adverse events were possibly, probably or definitely related to the study vaccine, they were considered to be 'adverse reactions'. Occurrence of solicited adverse reactions according to EMA criteria was also assessed in the three days after vaccination: injection-site induration >5 cm observed for > 3 consecutive days; injection-site ecchymosis; pyrexia (rectal equivalent temperature > 38.0°C) for ≥ 24 hours; malaise; and shivering [24].

Three sets of participants were defined for the assessment of immunogenicity, and one set for the safety analysis (Table 1).

The primary hypothesis was that the immunogenicity of the intradermal vaccine would be non-inferior to that of the adjuvanted vaccine for each virus strain in terms of antibody titres using the HI method. In the study, non-inferiority was tested with HI and SRH methods, and was defined as the upper bound of the 95% confidence intervals (CIs) around the post-vaccination ratios of geometric mean titres (GMT) (adjuvanted vaccine/intradermal vaccine) being < 1.5 for all three strains in the per protocol set [26].

If non-inferiority was achieved for the three strains, the superiority of the intradermal vaccine in the full analysis set was also investigated. Superiority was defined as the upper bound of the 95% CI being < 1 for at least two of the three strains. The primary analysis model did not include adjustments. Baseline antibody titres between the two vaccine groups were different so, to improve the accuracy of the estimates, a post-hoc analysis of covariance (ANCOVA model with group and pre-vaccination titre; no interaction) was performed to compare post-vaccination results adjusted according to pre-vaccination levels [26].

To demonstrate non-inferiority of immune responses induced by the intradermal vaccine compared with the adjuvanted vaccine with a power of >90% and to allow for 10% of participants not being evaluable, enrolment of 395 participants was planned for each group to provide 355 evaluable participants per group in the per protocol analysis. The analysis of the secondary endpoints included calculation of differences or ratios (and 95% CI) between groups, and safety endpoints were analysed using descriptive statistics.

A total of 795 participants were enrolled in the study (intradermal vaccine, n = 398; adjuvanted vaccine, n = 397; Table 1). Two participants withdrew from the study. One participant in the intradermal vaccine group had a cardiac arrest that led to death on day 20 (this event was considered to be unrelated to the vaccine; this 82-year old male had a history of coronary insufficiency and chronic obstructive pulmonary obstruction and several risk factors for myocardial infarction, including hypertension and past smoking). One participant in the adjuvanted vaccine group withdrew consent. After the second study visit, blood samples for analysis of immunogenicity were available for 395 participants in each group. All sera were tested by the HI method and most of them (with adequate samples) were evaluated by the SRH method (Table 1).

The demographic characteristics of the participants randomised in the two groups were comparable (Table 2). The study involved 370 men (46.5%) and 425 women (53.5%), with a mean age of 74.3 ± 6.4 years (range, 60.3-94.2 years). One individual aged 60.3 years and who was therefore below the age for inclusion was randomised by error and received the intradermal vaccine. This individual was excluded from the per protocol analyses but included in all other analyses. Most participants in both groups (89.9%) had past and/or current significant medical conditions, but only 52.7% of participants had underlying diseases identified as risk factors for influenza-related morbidity (mainly heart diseases). Overall, 72.5% of participants had been vaccinated against influenza in the previous season (2006-2007).

The geometric mean antibody titres induced by the intradermal and adjuvanted vaccines for all three virus strains by both the HI and SRH methods are summarised in Table 3. For the primary objective of non-inferiority of the intradermal vaccine immunogenicity compared with that for the adjuvanted vaccine assessed by the HI method in the per protocol population, non-inferiority was not demonstrated for the A/H3N2 strain (upper bound of the 95% CI = 1.53) (Table 3). Non-inferiority was demonstrated for all three strains by the SRH method (Table 3). Similar results for the three strains were observed in the full analysis set with both the HI and the SRH methods (ratios [adjuvanted vaccine/intradermal vaccine] of post-vaccination GMT: A/H1N1 1.12, A/H2N3 1.31, B 1.07; and A/H1N1 1.16, A/H2N3 1.18, B 1.05, respectively). Non-inferiority of the intradermal vaccine was not demonstrated for all three strains by the HI method, so the superiority of the vaccine was not tested. Superiority of the intradermal vaccine by the SRH method was tested on the full analysis set, but superior immunogenicity was not demonstrated for any of the virus strains.

For the A/H1N1 strain (HI method), baseline and post-vaccination antibody titres were correlated (Pearson's correlation coefficient ρ = 0.5 in per protocol set) and baseline antibody titres were slightly different in the two groups (pre-vaccination GMTR of 1.15 in per protocol set). Therefore a post-hoc ANCOVA adjusting for baseline values was carried out. Non-inferiority of the intradermal vaccine compared with the adjuvanted vaccine for all three virus strains by HI and SRH methods was observed (Table 4).

In the immunogenicity assessment by the HI method, the results for the A/H1N1 and A/H3N2 strains with both vaccines satisfied all three EMA criteria, and the results for the B strain with both vaccines satisfied the EMA criterion for GMTR (Figure 1). When immunogenicity was assessed by the SRH method, the results for all three strains with both vaccines satisfied all EMA immunogenicity criteria (Figure 1). There were no significant differences between the two vaccine groups in GMTRs, seroprotection rates, and seroconversion/significant increase rates for the three virus strains by either assay, with the exception of the seroprotection rate for the A/H1N1 strain (Figure 1). Although the seroprotection rates for the A/H1N1 strain were high in both the intradermal and adjuvanted groups (HI method: 81.3% and 87.1%, respectively; SRH method: 84.3% and 90.1%, respectively), they were higher in the adjuvanted vaccine group (difference between groups: HI method: 5.8% [95% CI: 0.7-10.9]; SRH method: 5.8% [95% CI: 1.1-10.5]).

The incidence of reactions within three days after the first vaccination listed in the EMA guideline (most commonly shivering and malaise) was similar in the intradermal and adjuvanted vaccine groups (Table 5). Induration of >5 cm that lasted for >3 days was not observed in either group. More participants in the intradermal vaccine group reported at least one solicited injection-site reaction (70.1%) compared with the adjuvanted vaccine group (33.8%). Erythema was reported more frequently in the intradermal vaccine group (63.1% vs. 13.4%). Swelling (34.2% vs. 8.6%), induration (32.9% vs. 10.6%) and pruritus (28.1% vs 6.5%) were also more frequently reported but at a lower incidence than for erythema. The proportion of participants who reported pain (19.8% vs. 20.9%) or ecchymosis (4.8% vs. 3.0%) was comparable between the two vaccine groups (Figure 2a). Most of the solicited injection-site reactions were of mild intensity or < 2.5 cm, and most occurred between day 0 and day 3 post-vaccination and lasted ≤ 3 days without treatment. Injection-site erythema lasted ≥ 8 days in 22 participants in the intradermal vaccine group compared with 4 participants in the adjuvanted vaccine group.

Overall, 99 participants (24.9%) in the intradermal vaccine group and 96 participants (24.2%) in the adjuvanted vaccine group reported at least one solicited systemic reaction in the first 7 days after vaccination. The incidence of each solicited systemic reaction was comparable for the two vaccine groups (Figure 2b). Most reactions were of mild intensity. Only 4.0% of the participants in the intradermal vaccine group and 5.8% of those in the adjuvanted vaccine group had a fever. In both groups pyrexia was, in general, ≤ 38.5°C (3.3% and 4.0% of subjects, respectively) and none were > 39.6°C, During the 21 days post-vaccination, there were no differences between the intradermal and adjuvanted vaccine groups in the proportion of participants reporting systemic adverse events (31.9% vs 32.5%; severe 3.3% vs 2.5%) or systemic adverse reactions (26.4% vs 25.9%; severe 2.0% vs 1.8%). No unsolicited adverse events occurred within 30 minutes of vaccination in either group. Six participants reported serious adverse events, two of which were considered by the investigator to be possibly related to vaccination: a participant in the intradermal vaccine group presented with pneumonia one day after vaccination, and a participant in the adjuvanted vaccine group presented with facial herpes zoster three days after vaccination.