Adjuvants for influenza vaccine

Adjuvants for influenza vaccine

0 INSTITUT PASTEURIELSEVIER Res. Immunol. Paris 1998 1998, 149, 19-23 Adjuvants N.N. Bouveret for influenza vaccine Le Cam (*), J. Ronco, A. Fr...

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Res. Immunol.

Paris 1998

1998, 149, 19-23

Adjuvants N.N. Bouveret

for influenza vaccine

Le Cam (*), J. Ronco,

A. Francon,

C. Blondeau

and B. Fanget

Pasteur M&ieux Connaught, 3, avenue Pasteur, P.O. Box 10, 92430 Mames-la-Coquette (France)

Every year, the influenza virus infects a large part of the world’s population. While far greater during pandemic years, at least 3,000 deaths are caused each year by influenza and its complications. Elderly subjects and subjects with an underlying pathology, such as cardiac disease, respiratory disease, renal insufficiency, etc., are those most at risk. Several types of vaccines have been developed which contain either purified inactivated whole virus, split or subunit virions, or which are live attenuated strains. For more than 20 years, such a purified formaldehydeinactivated Triton X-100 split vaccine has been produced by Pasteur MCrieux Connaught. As long as the circulating virus resembles that in the vaccine, efficacy is in the range of 70 to 90% in the adult population [ 11. However, in elderly subjects, a lower immunological response is often observed after injection of influenza vaccine, which may be related to ageing [2]. While the elderly generally retain immunity against many previously encountered pathogens, evidence suggests that immunological memory may be impaired as one ages. Furthermore, ageassociated alterations in immune function may be linked to changes in the composition of the CD4 compartment, and as a consequence thereof, in the balance of cytokines produced by T cells [3]. These changes affect the cytokine profile of IFNy and IL4 production, and therefore, the cooperation of T cells and B cells, which in turn influences the levels and isotype distribution of

Received January 22, 1998. (*) Corresponding


immunoglobulins. An improvement in the efficacy of the influenza vaccine is thus greatly needed, since the elderly population has both the highest risk of influenza complications and a poor response to vaccination. In order to overcome these problems, vaccine research is progressing in two directions: development of live-attenuated strains and improvement of the current vaccines by addition of adjuvants. The desirable immune-enhancing effects of adjuvants for influenza vaccines are the following : - to optimize the antigen presentation, promoting better T helper cell stimulation in order to increase systemic neutralizing antibody titres against the virus, - to enhance neutralizing antibodies in mucosal secretions (IgA), which are the first barrier against influenza infection, - to induce the CTL response, which may help in virus clearance, and - to elicit a more prolonged immune response to the virus (i.e., long-lasting protection). The adjuvants QS-21 (from Quillaja Saponaria Molina fraction 21) [4] and PCPP (poly[di(carboxylatophenoxy)phosphazene]) [5] have already been described in the literature as strong adjuvants for different vaccines in the corresponding animal models tested. In order to evaluate their tolerance and efficacy in humans, both adjuvants,



when added to the classical currently marketed influenza vaccine, have been tested in Phase I clinical trials.


25 pg and 50 p,g doses, and a third study comparing 50 and 100 pg. The most interesting results came from the dose-effect trial (10, 25, 50 carried out in 271 elderly subjects (mean age = 85.5 years) living in hospitals and retirement homes. The methodology was randomized and double-blind, controlled with the currently marketed, non-adjuvanted influenza vaccine (Vaxigrip”). Each subject gave a blood sample before vaccination and then another 21 days after vaccination, with a follow-up of antibody levels at day 90 and day 360. The evaluation criterion was the HA antibody response against each vaccine strain 21 days after injection. The method of titration was haemagglutination inhibition (HAI) following the WHO method.

QS-21 QS-21, extracted from the bark of a South American tree, belongs to the Saponin group, which is known for its adjuvant properties, but also for its side-effects when used as a crude preparation. Aquila Biopharmaceuticals, Inc. (USA) has developed a process of liquid chromatography that separates the fractions of QuilA that have variable toxicity and adjuvanticity. QS-21, one of these fractions, has been demonstrated to be safe and effective in animals [4].

Whatever the strain, there was no dose-effect, although an adjuvant effect was found for the A/H3N2 strain, as shown in table I. In contrast, no adjuvant effect was observed against the A/HlNl strain or against the B strain.

Results using QS-21 with flu vaccine in mice are shown in figure 1, and indicate a dose-effect response with a major HA antibody increase for the A/H3N2 strain, a mild effect for the A/HlNl strain and no effect for the B strain.

A similar conclusion could be made at day 90, but not at day 360 when titres dropped in all vaccine groups.

This product has been tested in three distinct trials in humans: two studies with the 10 pg,



80 60




Fig. 1. HA antibody response by QS-21 doses in mice.


Table I. Strain-specific


[email protected]




in relation to tested vaccine: A/Shangdong/9/93

(H3N2) results.


0 [email protected]

10 Pg

25 Pg

50 I4

Day 0 (n=250)

Subjects (N) : Seroprotection : GMT :



11% 10

3.3% 7.5

59 12% 9.7

65 15% 9.4

Day 21 (n=250)

Subjects (N) : Seroprotection : Seroconversion : GMT:



61 38% 49% 26.3 (x3.5)

59 39% 46% 27.2 (x2.8)

65 52% 49% 33.6 (x3.6)


26% 19.6 (x2)

mean the.

A subset of the study population was revaccinated the following year with either the yearly vaccine for the previous control group or with the yearly vaccine adjuvated with 50 yg of QS-21 for the adjuvant group. A booster effect was observed against the A/I-I3N2 strain and against the B strain, respectively: strain GMT (with QS-21) =2.94 (Wilcoxon’s GMT (without QS-21)


andBsrrain GMT (with QS-21) GMT (without QS-21)


- 1.52 (Wilcoxon’s test,

p =

p =



In conclusion, a limited adjuvant effect, not dose-related, was observed for the A/H3N2 strain after one injection of QS-21 adjuvanted flu vaccine. One year later, after revaccination with a QS-2 1-adjuvanted flu vaccine of the recommended updated strain composition, a significant booster effect was observed for both the A/H3N2 and B strains.

PCPP tremendously increases the HA response to all three strains. The adjuvant effect is strongest for the 500 pg dose, as shown in figure 2. A Phase I study using PCPP was carried out in Paris on both young and elderly subjects. The study was randomized and double-blind at each level; three doses of PCPP were tested: 100, 200 and 500 pg. The control vaccine was the nonadjuvanted influenza vaccine. Each subject received one injection of the product and was followed for 12 months. Blood samples were taken before vaccination, at day 2 1, at 6 months and at 12 months. A total of 96 subjects (48 young subjects and 41 subjects over 60 years of age) were enrolled. No serious adverse event related to the vaccine was observed. In young subjects, there was a tendency to have better results with the adjuvanted vaccine ; this trend was confirmed in the elderly subjects against all three strains and particularly for the A/H3N2 strain (table II), which had better results whatever the serological parameter considered.

PCPP Conclusion PCPP is a polymer produced by Virus Research Institute (USA); the adjuvant properties have been demonstrated in animals for various models such as tetanus toxoid, influenza, and HIV [5,6, 7, 81. Added to the influenza vaccine,

The aim of this presentation has been to show clinical data in humans that compare the abilities of two different adjuvants to improve the protective anti-HA antibody response.

100 mcg

200 mcg PCPP


500 mcg


Fig. 2. HA antibody response by PCPP doses in mice.

Table II. Strain-specific


in relation to tested vaccine: A/Johannesburg/33/94

(H3N2) results.

PCPP Day 0 (n=4%)

Subjects (N) : Semprotection: GMT:

Day 21 (n=41)

Subjects (N) : Seroprotection: Seroconversion : GMT :

0 IQ

100 Pi?

200 l-4

500 ‘4%


6 0% 5

8 0% 6.3

10 0% 6.6

5 20% 40%

8 63% 75%

80% 80%

29% 14

15 60% 36%


(x5311) GMT=geometric

mean titre.

- QS-21 enhanced anti-HA titres only for the AkI3N2 strain when compared to the vaccine without adjuvant. Furthermore, this effect did not depend on the dose of adjuvant inoculated. Revaccination with the aaiuvanted vaccine &kif&$ -r. biT,,r- CbI -ALLCIILbtiC 6 fk I ew” W&C Gf &Ke. virus strains contained in the vaccine. These results suggest that QS-21 may have interest far other vaccines wit’tt tiffereat schemes

of administration schedule).


at least

a two-dose

- PCPP showed an adjuvant effect after a single Inieciion on ziI1 three vrrus strains ‘in e’rireiry sdo$z”&, ;zlr;z w& mapx ?!cYL4t fk he dw-l2N2 strain. These results appear promising, and are being extended in a phase II study (large-scale trid).



References [l] LaMontagne, J.R. et al. (1983), Summary of clinical trials of inactivated influenza vaccine. Rev. Infect. Dis., 5, 723-36.

[2] Powers,D.C. ef al. (1993),Effect of ageon cytotoxic lymphocyte memoryaswell asserumandlocal antibody responses elicited by inactivated influenza vaccine. J. Infect. Dis., 167, 584-92.

[3] Nijhuis, E.W.P. et al. (1994),Age-relatedincreasein the fraction of CD27CD4+ T cellsand IL-4 production as a feature of CD4+ T cell differentiation in vivo. Clin. Exp. Zmmunol., 96, 528-534. [4] Kens& C.R. et al. (1991), Separationand characterization of Saponinswith adjuvantactivity from Quil-




laja saponaria Molina Cortex. J. Zmmunol., 146, 431-37. [5] Payne,L.G. et al. (1995), Water-solublephosphazene polymersfor parenteraland mucosalvaccine delivery, in “Vaccine Design: The subunit and adjuvant approach”.(PowelI, MP, Newman,MJ). (pp. 473-93) Plenum Press, New York.

[6] Lu, Y. et al. (1996), Utility of SHIV for testing HIV-l vaccine candidatesin macaques.J. Acquir. Immune Deft.

Syndr. Hum. Retrovirol.,


[7] Payne,L.G. et al. (1997). PCPPasa parenteraladjuvant for disease antigens.Dev. Biol. Stand, 92,79-87. [8] Payne, L.G. et al. (1998), Poly[di(carboxylatophenoxy)phosphazene](PCPP) is a potent immunoadjuvant for an influenza vaccine.Vaccine, 16, 92-98.