Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1999-04-16
2002-05-28
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S193100, C424S194100, C424S203100, C424S234100, C424S150100, C424S184100, C424S236100, C424S260100, C549S321000, C530S806000, C530S808000, C548S530000, C548S230000, C548S184000, C548S185000
Reexamination Certificate
active
06395282
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to an immunogenic conjugate comprising an autoinducer molecule of a Gram negative bacteria or a synthetic analogue thereof linked to a carrier molecule. The present invention also relates to antibodies and binding portions thereof capable of binding the immunogenic conjugate, and vaccines for the treatment or prevention of infection by autoinducer producing bacteria.
2. BACKGROUND OF THE INVENTION
Some pathogenic Gram negative bacteria produce chemical moieties known as bacterial autoinducers (BAIs). BAIs are produced by Gram negative bacteria as a mechanism for communicating with other bacteria when they have grown to a high cell density. This mechanism is known as quorum sensing.
BAIs assist in the transcriptional control of genes involved in a wide range of metabolic activities. When the bacterial population reaches a critical threshold, the concentration of BAIs also reach a concentration sufficient to enable the BAIs to bind a group of transcription factors, known as R-proteins. Binding of the BAIs to the R-proteins triggers binding of the newly formed BAI/R-protein complex to DNA, which then induces transcription of a group of genes. In the gram negative pathogenic bacteria, a subgroup of the activated genes are pathogenic determinants.
BAIs are small, non-immunogenic, lipid-soluble molecules which are capable of diffusing out of the bacteria, and into the environment where they enter host cells. BAIs share structural characteristics, in particular, they have a homoserine lactone ring with an N-acyl side chain. Variability between different BAIs resides primarily in the structure of the acyl side chain.
It has been proposed that in addition to regulating transcription in bacteria, the BAIs also regulate transcription in cells of an infected mammalian host. A list of presently known bacterial autoinducers (BAIs) and the Gram negative bacteria which produce them are identified in Table 1 below:
TABLE 1
Gram negative bacteria:
Bacterial autoinducer (BAI):
Aeromonas hydrophila
AHAI
Agrobacterium tumefaciens
N-(3-oxo)-octanoyl-L-homoserine lactone
(OOHL)
Burkholderia cepacia
N-octanoylhomoserine lactone
Chromobacterium violaceum
N-hexanoyl-L-homoserine lactone (HHL)
Enterobacter agglomerans
N-(3-oxo)-hexanoyl-L-homoserine lactone
(OHHL)
Erwinia stewarti
OHHL
Erwinia carotovora
OHHL
Escherichia coli
Structure not yet determined
Nitrosomas europea
OHHL
Photobacterium fischeri
OHHL, OOHL; OHL
Pseudomonas aeruginosa
N-(3-oxododecanoyl)-L-homoserine
lactone (PAI-1);
N-(butanoyl)-L-homoserine lactone (PAI-
2)
Pseudomonas aureofaciens
Structure not yet determined
Rhizobium leguminosarum
N-(3-hydroxy)-tetradecanoyl-L-
homoserine lactone (HtDeHL)
Serratia liquefaciens
PAI-2
Vibrio fischeri
OHHL
Vibrio harveyi
N-(3-hydroxy)-butanoyl-L-homoserine
lactone (HBHL)
Yersinia enterocolitica
OHHL, HHL
The Gram negative bacterium
Pseudomonas aeruginosa
is an opportunistic human pathogen that causes infections in immunocompromised hosts. PAI-1 has been shown to inhibit the proliferation of lymphocytes in vivo and downregulates expression of tumor necrosis factor and interleukin-12 (Telford et al., 1998, Infect Immun. 66(1):36-42).
Pseudomonas aeruginosa
frequently colonizes the lungs of individuals with cystic fibrosis (Hoiby, N., 1974, Acta Pathologica Microbiolo. Scand. Sect. B. 82: 551-558; Reynolds et al., 1975, Ann. Intern. Med. 82:819-832). This bacterium produces a number of extracellular virulence factors including exotoxin A, which is encoded by the toxA gene (Iglewski, B. H. and Kabat, D., 1975, Proc. Natl. Acad. Sci. USA. 72:2284-2288; Iglewski et al., 1978, Proc. Natl. Acad. Sci. USA. 75:3211-3215); an elastolytic protease encoded by the lasA gene; an elastolytic protease encoded by the lasB gene; and an alkaline protease encoded by the aprA gene (Morihara, K. and Homma, J. Y., 1985, Bacterial Enzymes and Virulence, ed. Holder, I. A. (CRC Press, Boca Raton, Fla.) pp. 41-79; Bever, R. A. and Iglewski, B. H., 1988, J. Bacteriol. 170:4309-4313; Kessler, E. and Saffrin, M., 1988, J. Bacteriol. 170:5241-5247).
Pseudomonas aeruginosa
utilizes a partially redundant quorum sensing mechanism which includes two autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) and N-(butanoyl)-L-homoserine lactone (PAI-2) (see Table 1). These autoinducers control expression of a number of virulence factors, including the elastolytic proteases lasA and lasB, autoinducer synthase, alkaline protease, exotoxin A and rhamnolipid synthase. (Gambello, et al., 1993, Infection & Immunity 61:1180-84; Latifi, et al., 1996, Mol. Microbiol. 21:1137-46; Passador, et al.,1993, Science 260:1127-30; Pesci, et al., 1997, J. Bact. 179:3127-32; Seed, et. al., 1995, J. Bact. 177:654-59; and Toder, et al., 1994, Infection & Immunity 62:1320-27.) It is the production of these virulence factors which enable
Pseudomonas aeruginosa
to invade and induce disease in humans.
Current treatments for Gram negative bacterial infections typically target surface antigens of the bacteria to make antibodies. Development of vaccines and diagnostic antibodies to autoinducers are hindered by the fact that autoinducers are not only non-immunogenic, but are also freely diffusible through the lipid bilayer and are not covalently attached to the bacteria. Several studies have demonstrated that a non-immunogenic bacterial capsular polysaccharide may be conjugated to an immunogenic compound to generate antibodies to the capsular polysaccharide (Anderson, U.S. Pat. No. 4,673,574 (conjugation of a fragment of a bacterial capsular polymer to a diphtheria or tetanus toxin or toxoid); Wessels et al., 1990, J. Clin. Invest. 86:1428-1433 (conjugation of a polysaccharide of type III group B Streptococcus to tetanus toxoid); and Schneerson et al., 1980, J. Exp. Med. 152:361-376 (conjugation of
H. influenzae
type b capsular polysaccharide to tetanus toxoid and other carriers.)) However, in contrast to autoinducers which are lipid diffusible, these anti-polysaccharide treatments are designed for production of antibodies specific to surface antigens covalently attached to the bacteria, resulting in lysis of the bacteria.
While synthetic autoinducer analogs limit bacterial growth in vitro, this approach fails to harness the capabilities of an active immune response that is a potentially long-lasting and effective therapeutic or prophylactic treatment (Pearson et al., U.S. Pat. No. 5,591,872). Furthermore, although autoinducer molecules themselves can be used in diagnostic bioassays, including bioluminescence, antibiotic production, or bacterial growth, these diagnostic assays fail to provide a prophylactic or therapeutic benefit to individuals exposed to autoinducer-producing Gram negative bacteria (Bycroft et al., U.S. Pat. No. 5,593,827).
3. SUMMARY OF THE INVENTION
The present invention relates to immunogenic conjugates comprising a carrier molecule covalently conjugated or otherwise bound to an autoinducer of a Gram negative bacteria of a compound of Formula (I):
where X is O, S, N—(C
1
-C
6
) alkyl, NR
2
, N-phenyl; Y is C
1
-C
6
straight or branched alkyl, C
1
-C
6
straight or branched alkenyl, C
1
-C
6
straight or branched alkynyl; Z is C═O, C═S, CHOH, C═N—NR
1
, C═N—OH, C
1
-C
8
straight or branched alkyl, C
1
-C
8
straight or branched alkenyl, C
1
-C
8
straight or branched alkynyl; L is C
1
-C
18
straight or branched alkyl, C
1
-C
18
straight or branched alkenyl, C
1
-C
18
straight branched alkynyl, or —CO
2
H, —CO
2
R
1
, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR
1
, —CH═CH—CH
2
Br, —CH═CH—CH
2
Cl, —SAc or SH, where R
1
is C
1
-C
6
straight or branched alkyl, m is 0 or 1; z is 0 or 1; R
2
is H, C
1
-C
6
straight or branched alkyl, C
1
-C
6
straight or branched alkenyl or C
1
-C
6
straight or branched alkynyl, or CO
2
H; and Q is CH or N; and n is 0-3 with the proviso that when n is 0, X is N—(C
1
-
6
alkyl) or N-phenyl. In a specific embodiment, the carrier molecule comprises a lysine-containing protein, preferably,
Iglewski Barbara H.
Kende Andrew S.
Pearson James P.
Phipps Richard P.
Smith Roger
Devi S.
Nixon & Peabody LLP
University of Rochester
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