The aim of this study was to improve the potency of the currently used
influenza subunit vaccines, which are of relatively low efficiency in high-risk groups. Influenza A virus (Shangdong/9/93) haemagglutinin/
neuraminidase (H3N2),
granulocyte-macrophage colony-stimulating factor (
GM-CSF) and
interleukin-2 (IL-2) were encapsulated, each separately or combined, in multilamellar vesicles composed of dimyristoyl
phosphatidylcholine. BALB/c mice were immunized once, i.p. or s.c., with 0.05-2.0 microg HN administered either as free
antigen (F-HN), adsorbed to
aluminum hydroxide (Al-HN), or encapsulated in
liposomes (Lip-HN), separately or together with 1 x 10(2)-4.5 x 10(4) units of free or encapsulated
cytokines. Serum
antibodies were assayed on days 11-360 by the haemagglutination-inhibition (HI) test and ELISA. Protective immunity against intranasal virus challenge was determined at 9-14 months post-vaccination. The following results were obtained: (1) The efficiency of encapsulation in
liposomes was 95, 90 and 38% for HN,
IL-2 and
GM-CSF, respectively, and the liposomal preparations were highly stable as an aqueous dispersion for > 2 months at 4 degrees C. (2) Following immunization with 0.5 microg Lip-HN, there was an earlier, up to 50-fold stronger, and 3-5 times longer response than that obtained with nonliposomal HN. (3) Coimmunization with free
cytokines further increased the response 2-20 times and the two
cytokines had an additive effect. (4) Liposomal
cytokines were 2-20 times more effective than the free
cytokines and their stimulatory effect was more durable. (5) A 100% seroconversion (HI titer > or = 40) was achieved with only 10-25% of the routinely used
antigen dose, by encapsulating either
antigen or
cytokine. (6) The level of protection following vaccination with the combined liposomal
vaccines was 70-100% versus 0-25% in mice immunized with Al-HN alone, and no toxicity was observed. In conclusion, our animal experiments show that the liposomal
vaccines are superior to the currently used
influenza vaccines, increasing the response by 2-3 orders of magnitude in mice. This approach may also prove valuable for
subunit vaccines against other microorganisms.