Adjuvant combinations of liposomes and mycobacteriaial lipids for immunization compositions and vaccines

Abstract

The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldioctadecylammonium-bromide / chloride (DDA) and a lipid extract from The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from <The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from Mycobacterium bovis. The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from Mycobacterium bovis<The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from Mycobacterium bovis BCG The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from Mycobacterium bovis BCG <The present invention provides a vaccin adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from Mycobacterium bovis BCG. The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from <i>Mycobacterium bovis BCG<i>. <The present invention provides a vaccine adjuvant consisting of a combination of a surfactant i.e. dimethyldeoctadecylammonium-bromide / chloride (DDA) and a lipid extract from <i>Mycobacterium bovis BCG<i>. The total lipid extract contains both apolar lipids, polar lipids, and lipids of intermediate polarity of which the apolar lipids were found to induce the most powerful immune responses. The total lipids may be extracted with chloroform / methanol and re-dissolved in water before the addition of surfactant. This preparation may be used to induce prominent cell-mediated immune responses in a mammal in order to combat pathogens, or as a treatment for cancer.

Description

FIELD OF INVENTION [0001] The present invention discloses an adjuvant comprising a surfactant and the total lipid extract of the BCG mycobacterium as well as the apolar fraction thereof and a vaccine comprising said adjuvant and an antigenic substance. BACKGROUND OF THE INVENTION [0002] The first vaccines consisted of live, attenuated pathogens. The attenuated forms were either naturally occurring closely related organisms or obtained through serial passages in culture. For example, tuberculosis (TB) in man has for many years been combated by vaccination with an attenuated strain of Mycobacterium bovis, the M. bovis BCG vaccine developed more than 80 years ago. However, although more than 3 billion doses of BCG have been administered (more than any other vaccine) (Bloom and Fine, 1994), it does not always provide satisfactory resistance to humans TB in every population. [0003] Today, a more up-to-date approach is to use highly purified substances, e.g. purified recombinant proteins....

AI Technical Summary

Benefits of technology

[0012] In the present invention it is demonstrated that a surprisingly high protective immune response is obtained when lipids extracted from M. bovis BCG are used as adjuvant together with the cationic surfactant. A greatly increased protective immune response is obtained when DDA is added to a total lipid extract of M. bovis BCG, compared to that obtained using either of the components alone, showing a synergistic adjuvant effect of DDA and the total lipid extract. The combination of DDA and total lipids extracted from M. bovis BCG gives an even higher protective immune response than DDA / TDB, another combination previously shown to have unexpectedly high efficacy (Holten-Andersen et al, 2004). Furthermore, the total lipid extract does not require extensive purification rendering it cheap to produce and therefore appropriate for inclusion in future vaccines suitable for use throughout the world. It is further shown that the apolar fraction of the total lipid extract has a stronger adjuvant effect than the polar fraction when they are mixed with DDA.

[0053] Loading of antigen to antigen-presenting cells, such as dendritic cells, have shown to be an effective method for generating active T-cells with a role in antitumor immunity.

[0101] The protective efficacy of Ag85B-ESAT6 / DDA / total lipids after an aerosol challenge was tested 3 and 6 months post first immunization and compared to that of two different doses of the standard BCG vaccine. At the early time point (3 months), the high dose of BCG and the subunit vaccine gave rise to significant levels of protection in both organs (FIG. 8, panel A). Although the standard BCG vaccine is a live vaccine, which should provide superior long-term protection, vaccination with the Ag85B-ESAT6 / DDA / total lipids resulted in sustained immune responses giving rise to protective levels comparable to the high dose of BCG at this late time point (FIG. 8, panel B). Compared to the low dose of BCG, Ag85B-ESAT6 / DDA / total lipids gives significantly higher levels of protection in the lungs (p<0.05). These data demonstrates that Ag85B-ESAT6 / DDA / total lipids is inducing stable immunological memory superior to that induced by the low dose of the current human vaccine. Induction of Humoral Activity with M. bovis BCG Total Lipids

[0117] That total lipids / DDA can enhance immune responses not only to a TB antigen but also to antigens from other sources was tested by immunizing with 5 micrograms of the Ag85B-ESAT6 fusion as well as a hybrid molecule consisting of the GLURP and the MSP-3 of Plasmodium falciparum and 5 micrograms of ovalbumin. In addition, MOMP, tetanus toxoid, and diphtheria toxoid were also included for immunisation. All antigens were emulsified in 100 micrograms of total lipids / 250 micrograms of DDA and administered three times by the s.c. route. Immune responses were monitored 5 weeks after the first immunization (Table 6 and 6A). TABLE 6The ability of total lipids / DDA to enhance immune responses ofantigens from various sources, expt. 1AntigenIgG1aIgG2abImmune responsecAg85B-ESAT6667025010116 ± 109 Glurp-MSP3670<1001112 ± 365OVA2500<100793 ± 81No antigend119 ± 16aAntigen-specific IgG1 levels (midpoint titres) 5 weeks after the first immunization as measured by ELISA. bAntigen-specific IgG2a levels (midpoint titres) 5 weeks after the first immunization as measured by ELISA. cRelease of IFN-gamma from blood lymphocytes isolated 5 weeks after the first immunization and re-stimulated in vitro with the relevant antigen in a dose of 5 microgram / millilitres. dBlood lymphocytes from un-immunized mice stimulated with the no antigen.

Problems solved by technology

However, although more than 3 billion doses of BCG have been administered (more than any other vaccine) (Bloom and Fine, 1994), it does not always provide satisfactory resistance to humans TB in every population.

Unfortunately, many highly purified substances are not very immunogenic and do not induce a sufficient immune response to confer protection.

A large number of adjuvants exist but most of these suffer from numerous problems that preclude their use in humans.

The only adjuvants accepted for human use are aluminum-based adjuvants (AlOH-salts) and MF-59, but they both induce Th2-biased responses, which makes them unsuitable for a TB vaccine (Lindblad et al, 1997).

Lack of ease of production and a high price may be an important issue for QS-21 and other novel, promising adjuvant compounds.

Despite the fact that many adjuvant systems have been developed, the need for new adjuvant systems is still recognized (Moingeon et al, 2001) and is evident in the paucity of choices available for clinical use.

Taken together, several immunostimulating lipid compounds have been isolated from mycobacteria, but the laborious and thereby expensive purification schemes required makes them unlikely to be included in a future TB vaccine.

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