Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific... – Lipid or oil
Reexamination Certificate
1995-04-07
2001-10-09
Wortman, Donna C. (Department: 1648)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
Lipid or oil
C424S084000
Reexamination Certificate
active
06299884
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates generally to immunological adjuvants for use in increasing efficiency of vaccines and is particularly directed to adjuvants comprising oil-in-water emulsions.
2. Background
The emergence of new subunit vaccines created by recombinant DNA technology has intensified the need for safe and effective adjuvants. Traditional live anti-viral vaccines require no adjuvants. Killed virus vaccines are generally much more immunogenic than subunit vaccines and can be effective with no adjuvant or with adjuvants that have limited ability to stimulate immune responses. The new, recombinant DNA-derived subunit vaccines, while offering significant advantages over the traditional vaccines in terms of safety and cost of production, generally represent isolated proteins or mixtures of proteins that have limited immunogenicity compared to whole viruses. Such materials are referred to generally in this specification as molecular antigens, to distinguish them from the whole organisms (and parts thereof) that were previously used in vaccines. These vaccines will require adjuvants with significant immunostimulatory capabilities to reach their full potential in preventing disease.
Currently, the only adjuvants approved for human use in the United States are aluminum salts (alum). These adjuvants have been useful for some vaccines including hepatitis B, diphtheria, polio, rabies and influenza, but may not be useful for others, especially if stimulation of cell-mediated immunity is required for protection. Reports indicate that alum failed to improve the effectiveness of whooping cough and typhoid vaccines and provided only a slight effect with adenovirus vaccines. Problems with aluminum salts include induction of granulomas at the injection site and lot-to-lot variation of alum preparations.
Complete Freund's adjuvant (CFA) is a powerful immunostimulatory agent that has been used successfully with many antigens on an experimental basis. CFA is comprised of three components: a mineral oil, an emulsifying agent such as Arlacel A, and killed mycobacteria such as
Mycobacterium tuberculosis.
Aqueous antigen solutions are mixed with these components to create a water-in-oil emulsion. CFA causes severe side effects, however, including pain, abscess formation, and fever, which prevent its use in either human or veterinary vaccines. The side effects are primarily due to the host's reactions to the mycobacterial component of CFA. Incomplete Freund's adjuvant (IFA) is similar to CFA without the bacterial component. While not approved for use in the United States, IFA has been useful for several types of vaccines in other countries. IFA has been used successfully in humans with influenza and polio vaccines and with several animal vaccines including rabies, canine distemper, and foot-and-mouth disease. Experiments have shown that both the oil and emulsifier used in IFA can cause tumors in mice, indicating that an alternative adjuvant would be a better choice for human use.
Muramyl dipeptide (MDP) represents the minimal unit of the mycobacterial cell wall complex that generates the adjuvant activity observed with CFA; see Ellouz et al. (1974)
Biochem. Biophys. Res. Comm.,
59:1317. Many synthetic analogues of MDP have been generated that exhibit a wide range of adjuvant potency and side effects (reviewed in Chedid et al. (1978)
Prog. Allergy,
25:63). Three analogues that may be especially useful as vaccine adjuvants are threonyl derivatives of MDP, see Byars et al. (1987)
Vaccine,
5:223; n-butyl derivatives of MDP, see Chedid et al. (1982)
Infect. and Immun.,
35:417; and lipophilic derivative of muramyl tripeptide, see Gisler et al. (1981) in
Immunomodulations of Microbial Products and Related Synthetic Compounds,
Y. Yamamura and S. Kotani, eds., Excerpta Medica, Amsterdam, p. 167. These compounds effectively stimulate humoral and cell-mediated immunity and exhibit low levels of toxicity.
One promising lipophilic derivative of MDP is N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-3(hydroxyphosphoryloxy)]ethylamide (MTP-PE). This muramyl tripeptide has phospholipid tails that allow association of the hydrophobic portion of the molecule with a lipid environment while the muramyl peptide portion associates with the aqueous environment. Thus the MTP-PE itself can act as an emulsifying agent to generate stable oil in water emulsions.
Original mouse experiments in the laboratories of the present inventors with MTP-PE showed that this adjuvant was effective in stimulating anti-HSV gD antibody titers against herpes simplex virus gD anti-gen and that effectiveness was vastly improved if the MTP-PE and gD were delivered in oil (IFA) rather than in aqueous solution. Since IFA is not approved for human use, other oil delivery systems were investigated for MTP-PE and antigen. An emulsion of 4% squalene with 0.008% Tween 80 and HSV gD gave very good immunity in the guinea pig. This formulation, MTP-PE-LO (low oil), was emulsified by passing through a hypodermic needle and was quite unstable. Nevertheless, this formulation gave high antibody titers in the guinea pig and good protection in a HSV challenge of immunized guinea pigs. The formulation was most effective when delivered in the footpad but also gave reasonable antibody titers and protection when delivered intramuscularly. These data have appeared in 2 publications (Sanchez-Pescador et al., J. Immunology 141, 1720-1727, 1988 and Technological Advances in Vaccine Development, Lasky et al., ed., Alan R. Liss, Inc., p. 445-469, 1988). The MTP-PE-LO formulation was also effective in stimulating the immune response to the yeast-produced HIV envelope protein in guinea pigs. Both ELISA antibody titers and virus neutralizing antibody titers were stimulated to a high level with the MTP-PE formulation. However, when the same formulation was tested in large animals, such as goats and baboons, the compositions were not as effective. The desirability of additional adjuvant formulations for use with molecular antigens in humans and other large animals is evident.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide an adjuvant formulation suitable for stimulating immune responses to molecular antigens in large mammals.
Surprisingly, it has been found that a satisfactory adjuvant formulation is provided by a composition comprising a metabolizable oil and an emulsifying agent, wherein the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter and wherein the composition exists in the absence of any polyoxy-proplyene-polyoxyethylene block copolymer. Such block copolymers were previously thought to be essential for the preparation of submicron oil-in-water emulsions. The composition can also contain an immunostimulating agent (which con be the same as the emulsifying agent, if an amphipathic immunostimulating agent is selected).
DESCRIPTION OF SPECIFIC EMBODIMENTS
The present invention provides an adjuvant composition comprising a metabolizable oil and an emulsifying agent, wherein the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter. Investigations in the laboratories of the present inventors, reported in detail in the examples that follow, show a surprising superiority over adjuvant compositions containing oil and emulsifying agents in which the oil droplets are significantly larger than those provided by the present invention.
The individual components of the adjuvant compositions of the present invention are known, although such compositions have not been combined in the same manner and provided in a droplet size of such small diameter. Accordingly, the individual components, although described below both generally and in some-detail for preferred embodiments, are well known in the art, and the terms used he
Barchfeld Gail
Ott Gary
Van Nest Gary
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Robins Roberta L.
Wortman Donna C.
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