Key words: Adjuvant, Influenza, Randomised control trials, Vaccine. Introduction ... exposure to influenza occurs late in the season [22, 23]. In the effort to ...
European Journal of Epidemiology 17: 135±140, 2001. Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Increased immunogenicity of the MF59-adjuvanted in¯uenza vaccine compared to a conventional subunit vaccine in elderly subjects R. Gasparini1, T. pozzi1, E. Montomoli1, E. Fragapane1, F. Senatore2, M. Minutello2 & A. Podda2 1
Institute of Hygiene, University of Siena; 2Chiron Vaccines Clinical Research, Chiron SpA, Siena, Italy
Accepted in revised form 11 April 2001
Abstract. Three-hundred and eight outpatient elderly subjects (P65 years) were randomly assigned to receive the MF59-adjuvanted in¯uenza vaccine (FLUAD; n 204) or a conventional subunit in¯uenza vaccine (AGRIPPAL S1; n 104) in order to compare the safety and immunogenicity of the two vaccines. Although mild pain at the injection site was reported more frequently by subjects immunised with the adjuvanted vaccine, both vaccines were shown to be safe and well tolerated. The adjuvanted vaccine was more immunogenic as indicated by higher post-
immunisation geometric mean titres (GMTs) and by higher proportions of subjects with post-immunisation Pfour fold increases of antibody titres or subjects with P1/160 post-immunisation HI titres. These dierences, statistically signi®cant for all three strains after immunisation, indicated that, by addition of the MF59 adjuvant emulsion, conventional subunit in¯uenza antigens acquire an enhanced immunogenicity without any clinically signi®cant increase of their reactogenicity.
Key words: Adjuvant, In¯uenza, Randomised control trials, Vaccine
Introduction In¯uenza is still a very important epidemic viral infection in humans. Worldwide there is almost one local outbreak every year [1±4]. On average in the US, there are every year 17±50 million in¯uenza illnesses, 165±233 million in¯uenza sick days, 43±70 million in¯uenza bed and activity restriction days and 20,000 deaths [5]. Mortality associated with in¯uenza infection is in part a result of pulmonary or cardiovascular complications. Elderly people and subjects with underlying chronic diseases are at increased risk from death due to in¯uenza [6± 11]. In¯uenza vaccination prevents hospitalisation due to in¯uenza, pneumonia, emphysema and other cardiopulmonary conditions, such as congestive heart failure, with an ecacy rate ranging from 30 to 50% and due to all respiratory conditions with an ecacy rate ranging from 15 to 34% [12±14]. Additionally, vaccination is also eective in reducing mortality from all causes among elderly subjects, with an ecacy rate ranging from 27 to 75% [12, 14, 15]. Current in¯uenza vaccines are only modestly protective among the elderly population, with eectiveness rates ranging from 23 to 37% [16±19]. Vaccine ecacy may be reduced or compromised in years
when the vaccine viruses and circulating strains do not match [20, 21]. Additionally, the relatively short duration of antibody persistence after immunisation can further decrease the eectiveness of in¯uenza vaccines if exposure to in¯uenza occurs late in the season [22, 23]. In the eort to improve the immunogenicity of in¯uenza vaccine, many potential adjuvants have been evaluated. These include Cholera toxin B subunit, Escherichia coli enterotoxin heat labile fraction and various types of liposomes [24±27]. Additionally, trials have been conducted administering immunostimulants such as interleukin-2, acetylsalicylic acid, tymopentin (TP-5) and dehydroepiandrosterone sulphate (DHEAS), simultaneously with immunisation [27±32]. In addition to this context, an oil in water adjuvant emulsion containing squalene (MF59) was developed and was shown to enhance the immune response to a variety of viral antigens [33±36]. Among other MF59adjuvanted vaccine formulations, the in¯uenza vaccine FLUAD was evaluated in clinical trials and showed increased immunogenicity compared to conventional in¯uenza vaccines [37±40]. In this paper, we report the results from a phase II, double-blinded comparison of FLUAD and the conventional subunit vaccine AGRIPPAL S1 in out patient elderly subjects.
136 Materials and methods Study design In this prospective, double-blind, randomised, parallel-group, multi-centre study, FLUAD or AGRIPPAL S1 in¯uenza vaccines were administered prior to the beginning of the local in¯uenza season. The study population consisted of noninstitutionalised, mentally competent, healthy elderly people (P65 years of age) recruited in ®ve clinics of the Local Health Unit (USL) in Siena, Italy. Vaccinees were observed in the clinic for 30 min after immunisation. Follow-up medical visits were made at 28 and approximately 180 days post-immunisation. During the 7 days post-immunisation interval (study days 0±6), local reactions and selected systemic reactions to immunisation were recorded, as were all other adverse events on a diary card by the study nurse. In addition, during the 28 days following immunisation, any adverse event necessitating a physician visit or consultation was noted. During the study days 28±180, only hospitalisations and deaths were collected and recorded as serious adverse events. Blood samples were drawn at day-0, day-28 and day-180 post-immunisation to measure the hemagglutination inhibition (HI) antibodies against the vaccine antigens. Vaccines FLUAD (subunit in¯uenza vaccine combined with MF59 adjuvant emulsion, Chiron SpA, Siena, Italy) and the comparator vaccine AGRIPPAL S1 (subunit in¯uenza vaccine, Chiron SpA, Siena, Italy) were provided in pre®lled, single-dose syringes. For each immunisation, the antigenic content of FLUAD was identical to that of the AGRIPPAL S1 vaccine and was consistent with the World Health Organisation (WHO) requirements of at least 15 lg of hemagglutinin for each virus subtype [41]. The antigenic contents of the vaccines were: A/Shangdong/9/93 (H3N2 strain), A/Texas/36/91 (H1N1 strain) and B/Panama/ 45/90 (B strain). Only H3N2 strain in the studied vaccines diered from the strain included in the previous ¯u season vaccine (A/Beijing/32/92). Vaccines were given as single IM injections in the deltoid muscle prior to the onset of the in¯uenza season according to the recommended in¯uenza vaccine schedule. Immunogenicity The HI assays were conducted using the method of Dowdle modi®ed to use 0.4% guinea pig RBCs instead of 0.5% chicken RBCs [42]. Antibody titration was made in duplicate; pre- and post-vaccination sera were titrated simultaneously. The titre assigned to
each sample was the geometric mean of two independent determinations. The immunogenicity measures used to evaluate the results were the day-28 HI geometric mean titre (GMT), the day-28/day-0 geometric mean ratio (GMR), the FLUAD: Comparator day-28 GMT ratio, the percentage of subjects with a day-28 fourfold increase of antibody titres and the percentages of subjects with day-28 titres P1/40, thought to be associated with 50% protection from in¯uenza, and P1/160 associated with more complete protection [23]. Additional immunogenicity variables included the day-180 GMT, the day-180/day-0 GMR, and the percentage of subjects with day-180 titre P40 and P160. Statistical methods All statistical analyses were performed using SAS version 6.08 or higher (SAS Institute, Cary, NC). All reported p-values are two-sided. Signi®cance was assessed at 0.05. The analyses were carried out utilising analysis of covariance (ANCOVA), analysis of variance (ANOVA) and Pearson's v2 test, or Fisher's exact test where appropriate. Ethics The study was conducted in accordance with the Declaration of Helsinki, as amended, and the Italian laws. The study protocol and consent form were approved by the Italian Ministry of Health and by the Local Ethical Committee. Results Vaccinees A total of 308 subjects were recruited. Two-hundred and four elderly subjects received FLUAD and 104 received AGRIPPAL S1 vaccine during the 1994/ 1995 season. As shown in Table 1, the demographic characteristics of subjects immunised with FLUAD Table 1. Demographic characteristics of subjects enrolled in the FLUAD and AGRIPPAL S1 vaccine groups Demographic variable
FLUAD N = 204
AGRIPPAL S1 N = 104
Males (%) Females (%) Mean age (years) Mean weight (Kg) Mean height (cm)
91 (45) 113 (55) 75.9 65.5 160.8
46 (44) 58 (56) 75.4 65.2 161.3
No statistical dierences were observed among both vaccine groups.
137 Table 2. HI titre results according to the type of vaccine HI variable
Antigen
FLUAD N = 192
95% CI
Day-0 GMT
B A/H1N1 A/H3N2
39 100 31
35±42 90±110 27±35
36 111 25
31±42 94±132 20±32
Day-28 GMT
B A/H1N1 A/H3N2
102* 191 103*
92±114 173±211 89±119
70 167 55
59 83 141±197 43±70
Day-28/Day-0 GMR
B A/H1N1 A/H3N2
Subjects with P fourfold increase (%)
B
35***
28.3±41.7
27
18.3±35.7
A/H1N1 A/H3N2
20** 52*
14.3±25.6 44.9±59.1
11 29
4.8±17.2 20.1±37.9
Subjects with titre P1/160 (%)
B A/H1N1 A/H3N2
54* 88 51*
46.9±61.0 83.4±92.6 43.9±58.1
35 85 34
25.6±44.4 78.0±92.0 24.7±43.3
2.6** 1.9*** 3.3*
2.4±2.9 1.7±2.1 2.9±3.8
AGRIPPAL S1 95% CI N = 99
2.0 1.5 2.1
1.7±2.4 1.3±1.8 1.7±2.6
* p O 0:001; ** p O 0:01; *** p O 0:05.
or AGRIPPAL S1 vaccine were not signi®cantly dierent according to age, weight and height. Immunogenicity Two-hundred and ninety-one of the 308 subjects receiving the ®rst immunisation provided day-0 and day-28 blood samples: 192 were enrolled in the FLUAD group and 99 in the control group. The day180 blood sample was obtained from 237 subjects, of which 158 were in the FLUAD group and 79 were enrolled in the control group. Both FLUAD and AGRIPPAL S1 vaccine induced a signi®cant increase of HI antibodies, against all three in¯uenza antigens at day-28 post-immunisation compared to baseline. As shown in Table 2, at day-28 after immunisation, the HI GMT in the FLUAD group was significantly higher than that in the AGRIPPAL S1 group for the B and A/H3N2 antigens (p O 0:001). The day-28/day-0 GMR and the percentage of subjects with a fourfold increase in HI titres at day-28 were signi®cantly higher in the FLUAD group for all three in¯uenza antigens (virus B: p O 0:01; virus A/ H1N1: p O 0:05; virus A/H3N2: p O 0:001 and virus B: p O 0:05; virus A/H1N1: p O 0:01; virus A/H3N2: p O 0:001 respectively). Additionally, for the B and A/H3N2 antigens, the percentage of subjects achieving titres P1/160 at day-28 were also signi®cantly higher in FLUAD vaccinees (Table 2). The FLUAD: AGRIPPAL S1 day-28 GMT ratio, which is a measure of the relative immunogenicity of the two vaccines, showed that, for the B and H3N2 strains, not only the point estimates, but also the entire con®dence intervals were above 1.0 (Figure 1).
At 180 days after immunisation, no signi®cant dierences in the GMT of the two vaccines were shown for any of the three antigens. However, the FLUAD vaccine group had higher percentage of subjects with antibody titres P1/160 for B and H3N2 antigens, while the opposite was true for the H1N1 antigen (data not shown). Furthermore, the percentage of subjects with antibody levels P1/160 was signi®cantly higher for the FLUAD recipients for the B strain (p < 0:01) and the A/H1N1 (p < 0:05). Safety No serious adverse events attributable to the administration of the study vaccines were observed during the follow-up period. As shown in Table 3,
Figure 1. Fluad: Comparator day-28 GMT Ratios for B, A/H3N2 and A/H1N1 strains.
138 Table 3. Rates of local and systemic post-immunisation reactions (days 0±7) according to the type of vaccine
Post-immunisation reactions
FLUAD N = 204
AGRIPPAL S1 N = 104
%
95% CI
%
95% CI
Local reactions Pain Warmth Erythema Induration
19 13 7 5
13.6±24.4 11 8.4±17.6 9 3.5±10.5 5 2.0±7.9 3
4.9±17.0 3.5±14.5 0.8±9.2 0±6.3
Systemic reactions Chills Nausea Malaise Myalgia Rash Arthalgia Headache Axillary temp. P38 °C
4 2 6 5
