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Safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons
PERGAMON
Vaccine 17 (1999) 99±104
Safety and immunogenicity of an inactivated subunit in¯uenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive in¯uenza seasons M. Minutello a, F. Senatore a, G. Cecchinelli b, M. Bianchi b, T. Andreani b, A. Podda a, *, P. Crovari c a
Chiron Vaccine Clinical Research, Siena, Italy Centro Medicina di Gruppo, Ortonovo (SP), Italy c Istituto di Igiene, University of Genoa, Genoa, Italy b
Abstract 1
A clinical trial to evaluate the safety and tolerability of a new in¯uenza adjuvanted vaccine (FLUAD , Chiron Vaccines), compared with a conventional non adjuvanted in¯uenza vaccine, was conducted in elderly ambulatory patients. Subjects were vaccinated with one dose of either vaccine each year for three consecutive years; 92 subjects received the ®rst immunization, 74 subjects received the second and 67 subjects received the third. The primary objective of this study was to evaluate the safety of repetitive injections of the adjuvanted vaccine in elderly subjects. There were no reports of any vaccine-related serious adverse event or of safety concerns related to study vaccines after the ®rst, second or third immunization. The adjuvanted vaccine induced more local reactions than the conventional vaccine; however, the reactions were normally mild and limited to the ®rst 2±3 days after immunization. No statistically signi®cant dierence between groups in systemic postimmunization reactions was reported except for a mild, transient malaise after the ®rst immunization. Compared with the ®rst immunization, no increase in postimmunization reactions was seen after the second and third immunizations. Despite the small sample size of the trial, which was not powered to test immunogenicity dierences, the antibody response was tested and resulted higher in the adjuvanted vaccine recipients, not only against the current season's vaccine strains, but also against heterologous vaccine strains. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: In¯uenza; Vaccine; Adjuvant
1. Introduction In industrialized countries, in¯uenza is a most serious epidemic viral infection in humans. Elderly persons are at increased risk from in¯uenza-related complications [1±4]. Current in¯uenza vaccines are less eective in elderly persons than in young persons [5, 6]. Additionally, vaccine ecacy may be compromised whenever the epidemic strain does not match the vaccine strain [7]. Furthermore, if exposure to in¯uenza occurs late in the season, the eectiveness of current in¯uenza vaccines may be compromised by the rela* Corresponding author. Chiron Vaccine Clinical Research, Via Fiorentina 1 Siena, Italy. Tel: +39(0)577 243496; fax: +39(0)577 243551. 0264-410X/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 8 ) 0 0 1 8 5 - 6
tively short duration of antibody persistence after immunization [8]. The development of adjuvanted in¯uenza vaccines is one approach to enhance the eectiveness of vaccination in the elderly population. Micro¯uidized Emulsion 59 (MF59) is a milky white liquid with a particle size of 155 nm and a density of 0.996 g ml ÿ 1; the viscosity is close to that of water. The adjuvant emulsion contains 0.5% Polysorbate 80 (Tween 80), 0.5% Sorbitan Trioleate and 4.3% Squalene [9]. MF59 has enhanced the response to a variety of viral antigens, including in¯uenza, in several dierent animal models [9, 10] and in humans [11]; therefore, it represents an ideal candidate for improving the immunogenicity of a conventional ¯u vaccine. This study was the ®rst phase II trial in elderly subjects to evaluate the reactogenicity of an in¯uenza vac-
100
M. Minutello et al. / Vaccine 17 (1999) 99±104
Table 1 Antigenic content of in¯uenza vaccines by season Season strain
1992/93
1993/94
1994/95
B A/H3N2 A/H1N1
B/Yamagata/16/88 A/Beijing/353/89 A/Taiwan/1/86
B/Panama/45/90 A/Beijing/32/92 A/Texas/36/91
B/Panama/45/90 A/Shangdong/9/93 A/Texas/36/92
1
cine adjuvanted with MF59 (FLUAD ) compared with a conventional inactivated ¯u vaccine. Recipients of both vaccines received a dose of the same vaccine in three consecutive in¯uenza seasons, allowing us to address the issue that multiple immunizations with an adjuvanted vaccine might be theoretically associated with an increase in reactogenicity. This is particularly important since in¯uenza vaccine is indicated for annual use among the elderly. 2. Materials and methods 2.1. Vaccine The in¯uenza vaccine for the ®rst immunization in this trial was provided in one vial as a 2 concentrate sterile ¯uid for i.m. injection. MF59 adjuvant emulsion was provided in a second vial as 2 concentrate sterile ¯uid for i.m. injection. A volume of 0.35 ml of Chiron subunit in¯uenza vaccine was diluted with either 0.35 ml of MF59 or 0.35 ml of 2 buer solution just 1 prior to vaccination to yield 0.7 ml of FLUAD or 1 AGRIPPAL S1 , respectively. A single 0.5-ml i.m. injection was then administered to the subjects randomly assigned to receive one of the two vaccines (1:1 ratio). For the second and third immunization, 1 FLUAD and the conventional subunit in¯uenza vaccine were both provided in pre®lled, single-dose syr1 inges. For the ®rst immunization, FLUAD was hypotonic (due to the absence of buer in the MF59 vial), whereas the non-adjuvanted control vaccine was isotonic. Study vaccines used for the second and third immunizations were both isotonic. For each immuniz1 ation, the antigenic content of FLUAD was identical to that of the conventional in¯uenza vaccine used as a control, and was consistent with the World Health Organization [12±14] recommendations of at least 15 mg of hemagglutinin for each vaccine strain (Table 1). Vaccines were given as single i.m. injections prior to the start of the in¯uenza season according to the recommended in¯uenza vaccine schedule. 2.2. Clinical study The trial was organized as a phase II, prospective, observer-blind, parallel-group, randomized, single-cen1 ter study comparing FLUAD and the conventional
in¯uenza vaccine. The clinical trial, approved by the Italian Ministry of Health and by an Ethical Committee, was conducted following the Good Clinical Practices (GCP) European Guidelines [15]. A written informed consent was obtained from each subject prior to the enrollment for each immunization. The study population in this trial consisted of healthy, ambulatory, elderly (r65 years of age) subjects. All subjects were recruited from the community by the local `Centro di Medicina di Gruppo' of Ortonovo, Italy, in collaboration with the University of Genoa, Italy. Ninety-two subjects were enrolled for the ®rst immunization, 74 of the same cohort were enrolled for the second immunization, and 67 were enrolled for the third. Immunizations were administered prior to the beginning of the 1992, 1993 and 1994 in¯uenza seasons. Subjects were observed in the clinic for 30 min after immunization and were then asked to return to the clinic at 1, 2 and 28 days for follow-up visits. During these visits, postimmunization reactions were collected directly by the study sta and, together with medical problems and concomitant medications, reported on the study Case Report Form (CRF). Additionally, subjects were given a diary card on which to note any observed reaction occurring in the ®rst week after immunization (days 0±6). For the measurement of body temperature, calibrated thermometers, obtained by the same supplier, were used. Hematology, urinalysis, and serum chemistry samples were obtained at day 0 and day 28 (®rst immunization only), as were blood samples for determination of immunogenicity. Assignment to vaccine groups for the second and third immunizations was identical to that for the ®rst immunization. For the second and third immunizations, the protocol speci®ed that an additional clinic visit would be made 6 months after the immunization. The purpose of this visit was to assess the subject's health status and to obtain a blood sample so that persistence of the antibody could be assessed at the end of the in¯uenza season following the second and third immunizations. 2.3. Laboratory tests and statistical analysis Immunogenicity was evaluated by hemagglutination inhibition (HI) using the modi®ed method of Dowdle et al. [16] (0.4% guinea-pig RBCs were used rather than 0.5% chicken RBCs). The HI assays were
M. Minutello et al. / Vaccine 17 (1999) 99±104
101
Table 2 Number of subjects (%) with local and systemic reactions within 6 days of ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1) First immunization Postimmunization reaction Local reactions Soreness Erythema Induration Injection site warmth Systemic reactions Fever (r388C) Chills Malaise Myalgia Arthralgia Nausea Headache
Second immunization
Third immunization
Fluad (n = 46)
AGR S1 (n = 46)
Fluad (n = 39)
AGR S1 (n = 35)
Fluad (n = 35)
AGR S1 (n = 32)
19 14 6 6
(41%)** (30%) (13%) (13%)
3 7 6 5
(6.5%) (15%) (13%) (11%)
7 15 12 2
(18%) (38.5%)* (31%) (5%)
5 5 6 2
(14%) (14%) (17%) (6%)
8 10 3 4
(23%) (29%) (9%) (11%)
7 4 3 6
(22%) (12.5%) (9%) (19%)
0 3 7 4 0 1 2
(0%) (7%) (15%)* (9%) (0%) (2%) (4%)
0 1 0 0 0 0 1
(0%) (2%) (0%) (0%) (0%) (0%) (2%)
(0%) (0%) (0%) (3%) (0%) (0%) (5%)
0 0 1 3 3 2 2
(0%) (0%) (3%) (9%) (9%) (6%) (6%)
0 1 2 2 1 1 2
(0%) (3%) (6%) (6%) (3%) (3%) (3%)
0 0 1 2 2 2 3
(0%) (0%) (3%) (6%) (6%) (6%) (9%)
0 0 0 1 0 0 2
* p = 0.05; **p = 0.01.
conducted by Viral and Rickettsial Disease Laboratories (VRDL; Berkeley, CA), using the respective season's vaccine strains (Table 1). Additionally, for a subset of subjects, HI assays were also conducted using heterologous vaccine strains (next season vaccine strains) in order to evaluate the heterovariant immunity induced by the adjuvanted vaccine with respect to the conventional one. The primary criterion for immunogenicity was the HI geometric mean titer (GMT) at 28 days after immunization for each antigen. A 50% increase in the 1 28-day GMT in the FLUAD vaccine group relative to the conventional subunit in¯uenza vaccine group was considered clinically important. In the evaluation of persistence of antibody titers at the end of the in¯uenza season, subjects with a day 180 titer rfourfold than that at day 28 were excluded from the analysis because of a possible infection with the epidemic virus. Data were summarized by study vaccine groups. All reported p-values are two-sided. Signi®cance was assessed with a set at 0.05. No formal sample size calculation or power analysis was performed for this study. Since this was the ®rst time that the adjuvanted in¯uenza vaccine had been administered to elderly people, the number of subjects to be enrolled was conservatively limited to approximately 40±50 subjects per vaccine group, a number considered sucient to address the primary study objective (i.e. to obtain a preliminary evaluation of the safety and tolerability of FLUAD compared with the conventional vaccine in a target population for in¯uenza immunization). The actual enrollment was 46 subjects per treatment group.
3. Results Of the 92 subjects enrolled for the ®rst immunization, 46 received Fluad and 46 received Agrippal S1. All enrolled subjects were white. Their mean age was 71.5 years (range: 65±81) for the Fluad group and 73.4 years (range, 65±90) for Agrippal S1. Males and females were 43.5 and 56.5% in the Fluad group and 39.1 and 60.9% in the Agrippal S1 group. Sixty-three per cent of Fluad recipients and 72% of Agrippal S1 recipients had received in¯uenza vaccination in the previous year. No signi®cant dierence in demographic characteristics between the two groups emerged during the second and third immunization trials. 3.1. Safety All subjects receiving at least one vaccine dose were included in the safety analyses. The percentages of subjects who experienced speci®c postimmunization reactions during days 0±6 following the three immunizations are shown by vaccine group in Table 2. The single i.m. administration of both in¯uenza vaccines was well tolerated by the elderly subjects. Overall, the postimmunization reactions were generally mild and transient and were reported mostly in the ®rst 48 h after vaccination. After the ®rst immunization, the percentages of subjects reporting injection-site tenderness and malaise 1 were higher in the FLUAD vaccine group. The same was true for injection-site erythema after the second immunization. No statistically signi®cant dierence
64 44 83 71 51 103 70 63 92 91 91 141* 39 36 63 43 37 80 37 79 65 51 100 85 111 202 147 142 306 230* 48 43 68 54 51 102 33 55 14 38 57 20 74 149 31 115* 189 45 * pR0.05; **pR0.01.
18 29 9 15 24 9 B A/H3N2 A/H1N1
AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 (n=46) (n=46) (n=46) (n=39) (n=35) (n=39) (n=35) (n=39) (n=35) (n=36) (n=32) (n=35) (n=32) (n=35) (n=32) (n=35) (n=29) FLUAD (n=46)
Day 28
between vaccine groups was seen for any postimmunization reaction obtained after the third immunization. The above mentioned signi®cant dierences between the two vaccines were reported for subjective (soreness) and objective (erythema) reactions at the local level and for subjective reactions only (malaise) at the systemic level. There were no reports of any vaccine-related serious adverse events after the ®rst, second or third immunization.
3.2. Immunogenicity
Antigen
Day 0 Day 0 Day 0
Day 360
Second First
Day 28
Day 180
Third
Day 28
Day 180
M. Minutello et al. / Vaccine 17 (1999) 99±104 Table 3 Serum antibody response against the respective seasonal vaccine strains before and after ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1): geometric mean titers
102
The addition of MF59 adjuvant emulsion to the conventional vaccine resulted in improved immune response to immunization as shown in Tables 3 and 4. After the ®rst immunization of the 92 subjects in the original study cohort, compared with the conventional vaccine, FLUAD had higher day-28 HI GMT for all three antigens (by 55% for B, by 27% for H3N2 and by 45% for H1N1). The day 28:day 0 geometric mean ratio (GMR), the percentage of subjects with fourfold or greater rises in HI titer (for two of three in¯uenza antigens), and the percentage of subjects with HI titers of 128 or more were similarly higher. In the subset of subjects (74 of the original 92) who provided month 12 serum samples after the ®rst immunization, the 1 FLUAD vaccine group had a higher month 12 HI GMT, higher month 12:day 0 GMR, a higher percentage of subjects with a fourfold or greater rise in HI titers month 12 relative to day 0, and a higher percentage of subjects with HI titers of 128 or more, for all three antigens. Following the second immunization of 1 74 subjects, the FLUAD group had, for all three antigens, a higher day 28 HI GMT (by 28% for B, by 52% for A/H3N2 and by 56% for A/H1N1), a higher day 28:day 0 GMR, and a higher percentage of subjects with fourfold or greater increases from baseline. Following the third immunization of 67 subjects, the 1 FLUAD group had, for all three antigens, higher HI GMT for both day 28 and day 180 showing a longlasting higher performance. Day 28 GMT was 30% higher for B, 44% higher for A/H3N2, and 53% higher for A/H1N1. A higher percentage of subjects in 1 the FLUAD group versus the conventional vaccine group showed a fourfold or greater rise in HI titers and HI titers of 128 or more for all three antigens. The HI response to the 1993/94 heterovariant antigens, after immunization with the 1992/93 vaccine, was evaluated in the 74 subjects who participated into both the ®rst and the second immunization trials. As shown in Table 5, the day 28 Fluad:Agrippal S1 GMT ratios against heterologous strains were higher compared with those seen against homologous vaccine strains (by
M. Minutello et al. / Vaccine 17 (1999) 99±104
103
Table 4 Subjects with day 28:day 0rfourfold increase and with day 28 HI titre r128 after the ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1)
Day-28 hi titer variable Percentage of subjects with rfour-fold increase Percentage of subjects with titer r128
Antigen B A/H3N2 A/H1N1 B A/H3N2 A/H1N1
First immunization
Second immunization
Third immunization
Fluad (n = 46)
AGR S1 (n = 46)
FLUAD (n = 39)
AGR S1 (n = 35)
FLUAD (n = 35)
AGR S1 (n = 32)
89
67
36
26
20
9
70 70 63*
72 50 41
74 28 74
60 23 60
37* 20 43
13 9 34
83 22
61 17
92 87
74 77
51 77*
38 47
* pR0.05; **pR0.01.
90% for B, by 75% for A/H3N2 and by 103% for H1N1). 4. Discussion The single i.m. administration of both in¯uenza vaccines was well tolerated by the elderly subjects. No serious adverse events or safety concerns related to study vaccine administration were observed. The addition of MF59 adjuvant emulsion to control vaccine appeared to increase the frequency of reports of some postimmunization reactions, including both local (i.e. injection-site soreness and erythema) and systemic (i.e. malaise) reactions. However, these reactions tended to be of a mild-to-moderate nature, did not aect routine daily activities of the vaccinees, and were transient, lasting only 1±2 days postimmunization. Following the second and third immunizations, local soreness and systemic symptoms were less frequent than after the ®rst immunization. This dierence may re¯ect the hypotonicity of the FLUAD formulation administered as a ®rst immunization compared with that used for the second and third immunizations where the ®nal salt concentration was identical for the two vaccines. Based on the results of this small-scale trial performed throughout three consecutive in¯uenza seasons, we can conclude that the adjuvanted vaccine is very well toler-
ated, despite an increased incidence of local reactions, and that, more importantly, there is no trend for an increased reactogenicity with subsequent immunizations. This latter point is particularly important since in¯uenza immunization is normally administered each year to elderly subjects. From the immunogenicity perspective, the addition of MF59 adjuvant emulsion to the conventional in¯uenza vaccine consistently improved the immune response to in¯uenza vaccination in these elderly subjects. The FLUAD group exhibited higher and longer lasting HI antibody titres against homologous vaccine strains. Additionally and more importantly, the immunogenicity bene®t oered by Fluad was even higher against heterologous vaccine strains, suggesting that the use of the adjuvant can provide a broader immune response than conventional vaccines, which is particularly useful in case of antigenic drifts of epidemic viruses. All of these responses would be important improvements over responses to the currently marketed in¯uenza vaccines. However, because of the small numbers of subjects, statistical signi®cance was not shown for any of the dierences between the 1 FLUAD and the control group in the immunogenicity variables. Overall, these results are encouraging and can support that further clinical studies should be performed
Table 5 Heterovariant activity after immunization with the 1992/93 in¯uenza formulation of either Fluad or Agrippal S1 (AGRS1): geometric mean titres against the 1993/94 vaccine strains Day 0
Day 28
Day 180
Antigen
Fluad (n = 35)
AGRS1 (n = 39)
Fluad (n = 35)
AGRS1 (n = 39)
Fluad (n = 35)
AGRS1 (n = 39)
B A/H3N2 A/H1N1
22 22 51
27 24 53
200 173 270
105 99 133
54 51 102
48 43 69
104
M. Minutello et al. / Vaccine 17 (1999) 99±104
to evaluate better the potential advantages oered by this new in¯uenza vaccine. [9]
References [1] Barker WH. Excess pneumonia and in¯uenza associated hospitalization during in¯uenza epidemics in the United States, 1970± 78. Am. J. Pub. Hlth 1986;76:761±5. [2] Barker WH, Mullooly JP. Impact of epidemic type A in¯uenza in a de®ned adult population. Am. J. Epidemiol. 1980;112:798± 811. [3] Glezen WP, Six HR, Frank AL, Taber LH, Perrotta DM, Decker M. Impact of epidemics upon communities and families. In: Options for the Control of In¯uenza (Eds Kendal AP.and Patriarca PA). Alan R. Liss, New York, 1986, pp. 63±73. [4] Sprenger MJW, Beyer WEP, Kempen BM, Mulder PGH, Masurel N. Risk factors for in¯uenza mortality? In: Options for the Control of In¯uenza II (Eds Hannoun C, Kendal AP, Klenk HD and Ruben FL). Excerpta Medica, New York, 1993, pp. 15±23. [5] Arden NH, Patriarca PA, Kendal AP. Experiences in the use and ecacy of inactivated in¯uenza vaccine in nursing homes. In: Options for the Control of In¯uenza (Eds Kendal AP and Patriarca PA). Alan R. Liss, New York, 1986, pp. 155±168. [6] Barker WH, Mullooly JP. Eectiveness of inactivated in¯uenza vaccine among non-institutionalized elderly persons. In: Options for the Control of In¯uenza (Eds Kendal AP and Patriarca PA). Alan R. Liss, New York, 1986, pp. 169±182. [7] Advisory Committee on Immunization Practices. Prevention and Control of In¯uenza: Part 1, Vaccines Recommendations of the ACIP, 1997, 46 (RR-9), pp. 1±25. [8] Palache AM. Dose-comparative serological studies: critical evaluation of the parameters to assess the antibody response to in¯uenza vaccination. In: In¯uenza Vaccination. The Eect of
[10]
[11]
[12] [13] [14] [15] [16]
Dose and Age on the Antibody Response (Ed. Palache AM). Broos Amsterdam, Amsterdam, The Netherlands, 1991, Chap. 4. pp. 81±86. Van Nest GA, Steimer KS, Haigwood NL, Burke RL, Ott G. Advanced adjuvant formulations for use with recombinant subunit vaccines. In: Vaccines 92. (Eds Brown F, Chanock RH, Ginsberg HS and Lerner RA.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992, pp. 57±62. Ott G, Barchfeld GL, Cherno D, Radhakrishnan R, van Hoogevest P, Van Nest G. MF59: design and evaluation of a safe and potent adjuvant for human vaccines. In: Vaccine Design: The Subunit and Adjuvant Approach (Eds Powell MF and Newman MJ). Plenum Press, New York, 1995, Chap. 10. pp. 227±296. Kahn JO, Sinangil F, Baenziger J et al. Clinical and immunologic responses to Human Immunode®ciency Virus (HIV) Type 1 SF2 gp 120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoil phosphatidylethanolamine in non-HIV-infected human volunteers. J. Infect. Dis. 1994;170:1288±91. World Health Organization. Recommended composition of in¯uenza virus vaccines for use in the 1992±1993 season. Weekly Epidemiol. Rec. 1992;67(77):2. World Health Organization. Recommended composition of in¯uenza virus vaccines for use in the 1993±1994 season. Weekly Epidemiol. Rec. 1993;68(9):57. World Health Organization. Recommended composition of in¯uenza virus vaccines for use in the 1993±1994 season. Weekly Epidemiol. Rec. 1994;69(8):53. CPMP. Good clinical practice for trials on medicinal products in the European Community. III/3976/88-EN Final. Pharmacol. Toxicol. 1990;67:361±72. Dowdle WN, Kendal AP, Noble GR. In¯uenza viruses. In: Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections (Eds Lenette EH and Schmidt NJ), 5th ed. American Public Health Association, Washington, DC, 1979, pp. 603±605.
Vaccine 17 (1999) 99±104
Safety and immunogenicity of an inactivated subunit in¯uenza virus vaccine combined with MF59 adjuvant emulsion in elderly subjects, immunized for three consecutive in¯uenza seasons M. Minutello a, F. Senatore a, G. Cecchinelli b, M. Bianchi b, T. Andreani b, A. Podda a, *, P. Crovari c a
Chiron Vaccine Clinical Research, Siena, Italy Centro Medicina di Gruppo, Ortonovo (SP), Italy c Istituto di Igiene, University of Genoa, Genoa, Italy b
Abstract 1
A clinical trial to evaluate the safety and tolerability of a new in¯uenza adjuvanted vaccine (FLUAD , Chiron Vaccines), compared with a conventional non adjuvanted in¯uenza vaccine, was conducted in elderly ambulatory patients. Subjects were vaccinated with one dose of either vaccine each year for three consecutive years; 92 subjects received the ®rst immunization, 74 subjects received the second and 67 subjects received the third. The primary objective of this study was to evaluate the safety of repetitive injections of the adjuvanted vaccine in elderly subjects. There were no reports of any vaccine-related serious adverse event or of safety concerns related to study vaccines after the ®rst, second or third immunization. The adjuvanted vaccine induced more local reactions than the conventional vaccine; however, the reactions were normally mild and limited to the ®rst 2±3 days after immunization. No statistically signi®cant dierence between groups in systemic postimmunization reactions was reported except for a mild, transient malaise after the ®rst immunization. Compared with the ®rst immunization, no increase in postimmunization reactions was seen after the second and third immunizations. Despite the small sample size of the trial, which was not powered to test immunogenicity dierences, the antibody response was tested and resulted higher in the adjuvanted vaccine recipients, not only against the current season's vaccine strains, but also against heterologous vaccine strains. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: In¯uenza; Vaccine; Adjuvant
1. Introduction In industrialized countries, in¯uenza is a most serious epidemic viral infection in humans. Elderly persons are at increased risk from in¯uenza-related complications [1±4]. Current in¯uenza vaccines are less eective in elderly persons than in young persons [5, 6]. Additionally, vaccine ecacy may be compromised whenever the epidemic strain does not match the vaccine strain [7]. Furthermore, if exposure to in¯uenza occurs late in the season, the eectiveness of current in¯uenza vaccines may be compromised by the rela* Corresponding author. Chiron Vaccine Clinical Research, Via Fiorentina 1 Siena, Italy. Tel: +39(0)577 243496; fax: +39(0)577 243551. 0264-410X/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 8 ) 0 0 1 8 5 - 6
tively short duration of antibody persistence after immunization [8]. The development of adjuvanted in¯uenza vaccines is one approach to enhance the eectiveness of vaccination in the elderly population. Micro¯uidized Emulsion 59 (MF59) is a milky white liquid with a particle size of 155 nm and a density of 0.996 g ml ÿ 1; the viscosity is close to that of water. The adjuvant emulsion contains 0.5% Polysorbate 80 (Tween 80), 0.5% Sorbitan Trioleate and 4.3% Squalene [9]. MF59 has enhanced the response to a variety of viral antigens, including in¯uenza, in several dierent animal models [9, 10] and in humans [11]; therefore, it represents an ideal candidate for improving the immunogenicity of a conventional ¯u vaccine. This study was the ®rst phase II trial in elderly subjects to evaluate the reactogenicity of an in¯uenza vac-
100
M. Minutello et al. / Vaccine 17 (1999) 99±104
Table 1 Antigenic content of in¯uenza vaccines by season Season strain
1992/93
1993/94
1994/95
B A/H3N2 A/H1N1
B/Yamagata/16/88 A/Beijing/353/89 A/Taiwan/1/86
B/Panama/45/90 A/Beijing/32/92 A/Texas/36/91
B/Panama/45/90 A/Shangdong/9/93 A/Texas/36/92
1
cine adjuvanted with MF59 (FLUAD ) compared with a conventional inactivated ¯u vaccine. Recipients of both vaccines received a dose of the same vaccine in three consecutive in¯uenza seasons, allowing us to address the issue that multiple immunizations with an adjuvanted vaccine might be theoretically associated with an increase in reactogenicity. This is particularly important since in¯uenza vaccine is indicated for annual use among the elderly. 2. Materials and methods 2.1. Vaccine The in¯uenza vaccine for the ®rst immunization in this trial was provided in one vial as a 2 concentrate sterile ¯uid for i.m. injection. MF59 adjuvant emulsion was provided in a second vial as 2 concentrate sterile ¯uid for i.m. injection. A volume of 0.35 ml of Chiron subunit in¯uenza vaccine was diluted with either 0.35 ml of MF59 or 0.35 ml of 2 buer solution just 1 prior to vaccination to yield 0.7 ml of FLUAD or 1 AGRIPPAL S1 , respectively. A single 0.5-ml i.m. injection was then administered to the subjects randomly assigned to receive one of the two vaccines (1:1 ratio). For the second and third immunization, 1 FLUAD and the conventional subunit in¯uenza vaccine were both provided in pre®lled, single-dose syr1 inges. For the ®rst immunization, FLUAD was hypotonic (due to the absence of buer in the MF59 vial), whereas the non-adjuvanted control vaccine was isotonic. Study vaccines used for the second and third immunizations were both isotonic. For each immuniz1 ation, the antigenic content of FLUAD was identical to that of the conventional in¯uenza vaccine used as a control, and was consistent with the World Health Organization [12±14] recommendations of at least 15 mg of hemagglutinin for each vaccine strain (Table 1). Vaccines were given as single i.m. injections prior to the start of the in¯uenza season according to the recommended in¯uenza vaccine schedule. 2.2. Clinical study The trial was organized as a phase II, prospective, observer-blind, parallel-group, randomized, single-cen1 ter study comparing FLUAD and the conventional
in¯uenza vaccine. The clinical trial, approved by the Italian Ministry of Health and by an Ethical Committee, was conducted following the Good Clinical Practices (GCP) European Guidelines [15]. A written informed consent was obtained from each subject prior to the enrollment for each immunization. The study population in this trial consisted of healthy, ambulatory, elderly (r65 years of age) subjects. All subjects were recruited from the community by the local `Centro di Medicina di Gruppo' of Ortonovo, Italy, in collaboration with the University of Genoa, Italy. Ninety-two subjects were enrolled for the ®rst immunization, 74 of the same cohort were enrolled for the second immunization, and 67 were enrolled for the third. Immunizations were administered prior to the beginning of the 1992, 1993 and 1994 in¯uenza seasons. Subjects were observed in the clinic for 30 min after immunization and were then asked to return to the clinic at 1, 2 and 28 days for follow-up visits. During these visits, postimmunization reactions were collected directly by the study sta and, together with medical problems and concomitant medications, reported on the study Case Report Form (CRF). Additionally, subjects were given a diary card on which to note any observed reaction occurring in the ®rst week after immunization (days 0±6). For the measurement of body temperature, calibrated thermometers, obtained by the same supplier, were used. Hematology, urinalysis, and serum chemistry samples were obtained at day 0 and day 28 (®rst immunization only), as were blood samples for determination of immunogenicity. Assignment to vaccine groups for the second and third immunizations was identical to that for the ®rst immunization. For the second and third immunizations, the protocol speci®ed that an additional clinic visit would be made 6 months after the immunization. The purpose of this visit was to assess the subject's health status and to obtain a blood sample so that persistence of the antibody could be assessed at the end of the in¯uenza season following the second and third immunizations. 2.3. Laboratory tests and statistical analysis Immunogenicity was evaluated by hemagglutination inhibition (HI) using the modi®ed method of Dowdle et al. [16] (0.4% guinea-pig RBCs were used rather than 0.5% chicken RBCs). The HI assays were
M. Minutello et al. / Vaccine 17 (1999) 99±104
101
Table 2 Number of subjects (%) with local and systemic reactions within 6 days of ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1) First immunization Postimmunization reaction Local reactions Soreness Erythema Induration Injection site warmth Systemic reactions Fever (r388C) Chills Malaise Myalgia Arthralgia Nausea Headache
Second immunization
Third immunization
Fluad (n = 46)
AGR S1 (n = 46)
Fluad (n = 39)
AGR S1 (n = 35)
Fluad (n = 35)
AGR S1 (n = 32)
19 14 6 6
(41%)** (30%) (13%) (13%)
3 7 6 5
(6.5%) (15%) (13%) (11%)
7 15 12 2
(18%) (38.5%)* (31%) (5%)
5 5 6 2
(14%) (14%) (17%) (6%)
8 10 3 4
(23%) (29%) (9%) (11%)
7 4 3 6
(22%) (12.5%) (9%) (19%)
0 3 7 4 0 1 2
(0%) (7%) (15%)* (9%) (0%) (2%) (4%)
0 1 0 0 0 0 1
(0%) (2%) (0%) (0%) (0%) (0%) (2%)
(0%) (0%) (0%) (3%) (0%) (0%) (5%)
0 0 1 3 3 2 2
(0%) (0%) (3%) (9%) (9%) (6%) (6%)
0 1 2 2 1 1 2
(0%) (3%) (6%) (6%) (3%) (3%) (3%)
0 0 1 2 2 2 3
(0%) (0%) (3%) (6%) (6%) (6%) (9%)
0 0 0 1 0 0 2
* p = 0.05; **p = 0.01.
conducted by Viral and Rickettsial Disease Laboratories (VRDL; Berkeley, CA), using the respective season's vaccine strains (Table 1). Additionally, for a subset of subjects, HI assays were also conducted using heterologous vaccine strains (next season vaccine strains) in order to evaluate the heterovariant immunity induced by the adjuvanted vaccine with respect to the conventional one. The primary criterion for immunogenicity was the HI geometric mean titer (GMT) at 28 days after immunization for each antigen. A 50% increase in the 1 28-day GMT in the FLUAD vaccine group relative to the conventional subunit in¯uenza vaccine group was considered clinically important. In the evaluation of persistence of antibody titers at the end of the in¯uenza season, subjects with a day 180 titer rfourfold than that at day 28 were excluded from the analysis because of a possible infection with the epidemic virus. Data were summarized by study vaccine groups. All reported p-values are two-sided. Signi®cance was assessed with a set at 0.05. No formal sample size calculation or power analysis was performed for this study. Since this was the ®rst time that the adjuvanted in¯uenza vaccine had been administered to elderly people, the number of subjects to be enrolled was conservatively limited to approximately 40±50 subjects per vaccine group, a number considered sucient to address the primary study objective (i.e. to obtain a preliminary evaluation of the safety and tolerability of FLUAD compared with the conventional vaccine in a target population for in¯uenza immunization). The actual enrollment was 46 subjects per treatment group.
3. Results Of the 92 subjects enrolled for the ®rst immunization, 46 received Fluad and 46 received Agrippal S1. All enrolled subjects were white. Their mean age was 71.5 years (range: 65±81) for the Fluad group and 73.4 years (range, 65±90) for Agrippal S1. Males and females were 43.5 and 56.5% in the Fluad group and 39.1 and 60.9% in the Agrippal S1 group. Sixty-three per cent of Fluad recipients and 72% of Agrippal S1 recipients had received in¯uenza vaccination in the previous year. No signi®cant dierence in demographic characteristics between the two groups emerged during the second and third immunization trials. 3.1. Safety All subjects receiving at least one vaccine dose were included in the safety analyses. The percentages of subjects who experienced speci®c postimmunization reactions during days 0±6 following the three immunizations are shown by vaccine group in Table 2. The single i.m. administration of both in¯uenza vaccines was well tolerated by the elderly subjects. Overall, the postimmunization reactions were generally mild and transient and were reported mostly in the ®rst 48 h after vaccination. After the ®rst immunization, the percentages of subjects reporting injection-site tenderness and malaise 1 were higher in the FLUAD vaccine group. The same was true for injection-site erythema after the second immunization. No statistically signi®cant dierence
64 44 83 71 51 103 70 63 92 91 91 141* 39 36 63 43 37 80 37 79 65 51 100 85 111 202 147 142 306 230* 48 43 68 54 51 102 33 55 14 38 57 20 74 149 31 115* 189 45 * pR0.05; **pR0.01.
18 29 9 15 24 9 B A/H3N2 A/H1N1
AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 FLUAD AGRS1 (n=46) (n=46) (n=46) (n=39) (n=35) (n=39) (n=35) (n=39) (n=35) (n=36) (n=32) (n=35) (n=32) (n=35) (n=32) (n=35) (n=29) FLUAD (n=46)
Day 28
between vaccine groups was seen for any postimmunization reaction obtained after the third immunization. The above mentioned signi®cant dierences between the two vaccines were reported for subjective (soreness) and objective (erythema) reactions at the local level and for subjective reactions only (malaise) at the systemic level. There were no reports of any vaccine-related serious adverse events after the ®rst, second or third immunization.
3.2. Immunogenicity
Antigen
Day 0 Day 0 Day 0
Day 360
Second First
Day 28
Day 180
Third
Day 28
Day 180
M. Minutello et al. / Vaccine 17 (1999) 99±104 Table 3 Serum antibody response against the respective seasonal vaccine strains before and after ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1): geometric mean titers
102
The addition of MF59 adjuvant emulsion to the conventional vaccine resulted in improved immune response to immunization as shown in Tables 3 and 4. After the ®rst immunization of the 92 subjects in the original study cohort, compared with the conventional vaccine, FLUAD had higher day-28 HI GMT for all three antigens (by 55% for B, by 27% for H3N2 and by 45% for H1N1). The day 28:day 0 geometric mean ratio (GMR), the percentage of subjects with fourfold or greater rises in HI titer (for two of three in¯uenza antigens), and the percentage of subjects with HI titers of 128 or more were similarly higher. In the subset of subjects (74 of the original 92) who provided month 12 serum samples after the ®rst immunization, the 1 FLUAD vaccine group had a higher month 12 HI GMT, higher month 12:day 0 GMR, a higher percentage of subjects with a fourfold or greater rise in HI titers month 12 relative to day 0, and a higher percentage of subjects with HI titers of 128 or more, for all three antigens. Following the second immunization of 1 74 subjects, the FLUAD group had, for all three antigens, a higher day 28 HI GMT (by 28% for B, by 52% for A/H3N2 and by 56% for A/H1N1), a higher day 28:day 0 GMR, and a higher percentage of subjects with fourfold or greater increases from baseline. Following the third immunization of 67 subjects, the 1 FLUAD group had, for all three antigens, higher HI GMT for both day 28 and day 180 showing a longlasting higher performance. Day 28 GMT was 30% higher for B, 44% higher for A/H3N2, and 53% higher for A/H1N1. A higher percentage of subjects in 1 the FLUAD group versus the conventional vaccine group showed a fourfold or greater rise in HI titers and HI titers of 128 or more for all three antigens. The HI response to the 1993/94 heterovariant antigens, after immunization with the 1992/93 vaccine, was evaluated in the 74 subjects who participated into both the ®rst and the second immunization trials. As shown in Table 5, the day 28 Fluad:Agrippal S1 GMT ratios against heterologous strains were higher compared with those seen against homologous vaccine strains (by
M. Minutello et al. / Vaccine 17 (1999) 99±104
103
Table 4 Subjects with day 28:day 0rfourfold increase and with day 28 HI titre r128 after the ®rst, second and third immunization with either Fluad or Agrippal S1 (AGRS1)
Day-28 hi titer variable Percentage of subjects with rfour-fold increase Percentage of subjects with titer r128
Antigen B A/H3N2 A/H1N1 B A/H3N2 A/H1N1
First immunization
Second immunization
Third immunization
Fluad (n = 46)
AGR S1 (n = 46)
FLUAD (n = 39)
AGR S1 (n = 35)
FLUAD (n = 35)
AGR S1 (n = 32)
89
67
36
26
20
9
70 70 63*
72 50 41
74 28 74
60 23 60
37* 20 43
13 9 34
83 22
61 17
92 87
74 77
51 77*
38 47
* pR0.05; **pR0.01.
90% for B, by 75% for A/H3N2 and by 103% for H1N1). 4. Discussion The single i.m. administration of both in¯uenza vaccines was well tolerated by the elderly subjects. No serious adverse events or safety concerns related to study vaccine administration were observed. The addition of MF59 adjuvant emulsion to control vaccine appeared to increase the frequency of reports of some postimmunization reactions, including both local (i.e. injection-site soreness and erythema) and systemic (i.e. malaise) reactions. However, these reactions tended to be of a mild-to-moderate nature, did not aect routine daily activities of the vaccinees, and were transient, lasting only 1±2 days postimmunization. Following the second and third immunizations, local soreness and systemic symptoms were less frequent than after the ®rst immunization. This dierence may re¯ect the hypotonicity of the FLUAD formulation administered as a ®rst immunization compared with that used for the second and third immunizations where the ®nal salt concentration was identical for the two vaccines. Based on the results of this small-scale trial performed throughout three consecutive in¯uenza seasons, we can conclude that the adjuvanted vaccine is very well toler-
ated, despite an increased incidence of local reactions, and that, more importantly, there is no trend for an increased reactogenicity with subsequent immunizations. This latter point is particularly important since in¯uenza immunization is normally administered each year to elderly subjects. From the immunogenicity perspective, the addition of MF59 adjuvant emulsion to the conventional in¯uenza vaccine consistently improved the immune response to in¯uenza vaccination in these elderly subjects. The FLUAD group exhibited higher and longer lasting HI antibody titres against homologous vaccine strains. Additionally and more importantly, the immunogenicity bene®t oered by Fluad was even higher against heterologous vaccine strains, suggesting that the use of the adjuvant can provide a broader immune response than conventional vaccines, which is particularly useful in case of antigenic drifts of epidemic viruses. All of these responses would be important improvements over responses to the currently marketed in¯uenza vaccines. However, because of the small numbers of subjects, statistical signi®cance was not shown for any of the dierences between the 1 FLUAD and the control group in the immunogenicity variables. Overall, these results are encouraging and can support that further clinical studies should be performed
Table 5 Heterovariant activity after immunization with the 1992/93 in¯uenza formulation of either Fluad or Agrippal S1 (AGRS1): geometric mean titres against the 1993/94 vaccine strains Day 0
Day 28
Day 180
Antigen
Fluad (n = 35)
AGRS1 (n = 39)
Fluad (n = 35)
AGRS1 (n = 39)
Fluad (n = 35)
AGRS1 (n = 39)
B A/H3N2 A/H1N1
22 22 51
27 24 53
200 173 270
105 99 133
54 51 102
48 43 69
104
M. Minutello et al. / Vaccine 17 (1999) 99±104
to evaluate better the potential advantages oered by this new in¯uenza vaccine. [9]
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