A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. I. Vaccine characterization and efficacy studies in mice

The aim of this study was to improve the potency of the currently used infl uenza subunit vaccines, which are of relatively low efficiency in high-risk groups. Influenza A virus (Shangdong/9/93) haemagglutinin/neuraminidase (H 3N2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interle ukin-2 (IL-2) were encapsulated, each separately or combined, in multilamel lar vesicles composed of dimyristoyl phosphatidylcholine. BALB/c mice were immunized once, i.p. or s.c., with 0.05-2.0 mu g HN administered either as free antigen (F-HN), adsorbed to aluminum hydroxide (Al-HN), or encapsulate d 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 d ays 11-360 by the haemagglutination-inhibition (HI) test and ELISA. Protect ive immunity against intranasal virus challenge was determined at 9-14 mont hs post-vaccination. The following results were obtained: (1) The efficienc y of encapsulation in liposomes was 95, 90 and 38% for MN, IL-2 and GM-CSF, respectively, and the liposomal preparations were highly stable as an aque ous dispersion for >2 months at 4 degrees C. (2) Following immunization wit h 0.5 mu g Lip-MN, there was an earlier, up to 50-fold stronger, and 3-5 ti mes longer response than that obtained with nonliposomal HN. (3) Coimmuniza tion with free cytokines further increased the response 2-20 times and the two cytokines had an additive effect. (4) Liposomal cytokines were 2-20 tim es more effective than the free cytokines and their stimulatory effect was more durable. (5) A 100% seroconversion (HT titer greater than or equal to 40) was achieved with only 10-25% of the routinely used antigen dose, by en capsulating either antigen or cytokine. (6) The level of protection followi ng 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 conc lusion, our animal experiments show that the liposomal vaccines are superio r to the currently used influenza vaccines, increasing the response by 2-3 orders of magnitude in mice. This approach may also prove valuable for subu nit vaccines against other microorganisms. (C) 1999 Elsevier Science Ltd. A ll rights reserved.