LPS with reduced toxicity from genetically modified gram...

Details LPS with reduced toxicity from genetically modified gram... LPS with reduced toxicity from genetically modified gram...

2001-07-02

2002-11-19

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S025000, C536S017200, C536S123130

Reexamination Certificate

active

06482807

ABSTRACT:

This application is a 371 of PCT/NL98/00633 Nov. 3, 1998.
BACKGROUND TO THE INVENTION
The subject invention lies in the field of vaccines and more specifically provides novel compounds that can be used as adjuvants in vaccines. Many adjuvants have been described e.g. Freund type mineral oil emulsions, aluminium salts, saponins, muramyl dipeptide and derivatives MPL, MF59 etc. However only a few have actually been licensed for use in humans. This is generally due to an unfavorable ratio between immunostimmulatory action versus toxicity. A general reference concerning adjuvants can be found in The Theory and Practical Application of Adjuvants (D.E.S. Stewart-Tull ed. John Wiley & Sons 1995) and the information therein is incorporated by reference. The prior art also teaches for a number of organisms that enzymatic treatment of LPS can lead to reduced toxicity. The LPS illustrated as having undergone such treatment are:
Salmonella typhimurium
and
Salmonella minnesota
. The following are also suggested to exhibit such: all Gram negative bacteria and specifically Salmonella, Escherichia, Haemophilus, Moraxella, Campylobacter and Neisseria. Nowhere however are details provided concerning proof of adjuvant activity.
Looking at this prior art in detail shows that Munford et al (in U.S. Pat. No. 4,929,604 issued in 1990) show
S typhimurium
LPS in which 95% of secondary acyl groups have been removed through enzymatic treatment. The Munford treatment cannot specifically remove secondary acyl chains ensuring only partial deacylation. The Munford method cannot provide uniform product at best nearly all secondary acyl groups will be removed.
They suggest adjuvant activity could be present due to B cell mitogenicity testing. B cell mitogenicity testing however is not a reliable test to indicate adjuvant activity. It is probable that such product will not exhibit adjuvant activity. The Munford method in fact only shows removal of secondary acyl chains from the non reducing end of LPS. The resulting product does not contain any secondary acyl group on the reducing end of the LPS. The Munford product lacks both myristoyl and lauroyl secondary side chains. The Munford method cannot specifically remove only myristoyl or only lauroyl. The Munford method cannot remove only secondary acyl chain from one specific location. The Munford method is suggested to also be applicable to Escherichia, Haemophilus and Neisseria.
They show a Salmonella LPS with one phosphate group on the non reducing end and one on the reducing end. The Salmonella LPS has 1 myristoyl and 1 lauroyl group on the non reducing end. The Salmonella LPS has no secondary acyl group on the reducing end.
Myers et al in U.S. Pat. No. 4,912,094 use alkaline hydrolysis under controlled conditions to remove only the beta-hydroxymyristic acyl residue that is ester linked to the reducing end glucosamine at position 3. Thus a product in which one of the primary acyl chains has been chemically removed is described. Nothing is mentioned vis a vis secondary acyl chain removal. The resulting product is stated to be less toxic and maintains antigenic properties. This is merely stated based on reduced mitogenicity of MPL A (acid hydrolyzed) vis a vis B cell proliferation for the deacylated version. B cell mitogenicity testing however is not a reliable test to indicate adjuvant activity.
Escherichia coli
and
Salmonella minnesota
LPS are given as examples. Only biological activity data are however given for the
Salmonella minnesota
LPS. They suggest the method to be applicable to all LPS but offer no support thereof.
The same subject matter is discussed in an article of Erwin et al with Munford as co-author (1991). Quoting from the abstract of the Erwin article itself the following is remarked in the abstract “These studies indicate that the contribution of secondary acyl chains to the bioactivities of a given LPS cannot be predicted with confidence from the reported structure-activity relationships of Lipid A or from the behavior of other deacylated LPS.”
Genes involved in lipid A acyloxyacylation are known in the art. Recently two late functioning acyltransferases of lipid A biosynthesis in
Escherichia coli
were identified as the products of the htrB and msbB genes (Clementz et al., 1996,1997); the hrtB gene was previously described as required for growth on rich media above 33° C., and the msbB gene as a multicopy suppressor of htrB. In the optimal reaction, HtrB transfers laurate to (KDO)2-lipid IVA, after which MsbB can add myristate to complete lipid A acylation (FIG.
1
). The predominant products formed by htrB and msbB mutants are tetra- and penta-acyl species, respectively. The genes display 27.5% identity; a third gene belonging to this family is also present in the
E. coli
chromosome, but its function in lipid A biosynthesis remains to be demonstrated.
The
Haemophilus influenzae
genome sequence contains both htrB and MsbB homologues; mutation in htrB is associated with modification of both phosphorylation and acylation of LPS (Lee et al., 1995), suggesting a pleiotropic effect of the loss of the acyloxyacyl chains on decoration of the oligosaccharide chain. A knockout mutation in the
H. influenzae
htrB gene was shown to reduce LPS-associated toxicity (Nichols et al., 1997).
Apicella (also author of the cited Lee et al document) et al also describe a htrB knockout mutant in WO97/19688. They described a
H. influenzae
tetra acyl mutant obtained via a mutation in htrB said mutant LPS supposedly having substantially reduced toxicity yet with retained antigenicity.
They used homology of
E coli
htrB sequence to find a similar sequence for Haemophilus, This similar sequence had 56% identity and 73% similarity to the
E. coli
htrB sequence. Mutants of
H. influenzae
were made and grown. Analysis of the mutant Haemophilus LPS revealed reduction in phosphoethanolamines, 50% less with two in the inner core. A species being a mono or diphosphoryl pentaacyl Lipid A of
H. influenzae
missing one of the secondary acyl chains (e.g. myrisitic acid moiety) in about 10% is also revealed by Apicella. In addition a tetraacyl was illustrated as having been present in about 90%. Thus the Apicella method produces a mixture of recombinant H. influenzae LPS structures wherein the majority product has no secondary acyl chains. Bactericidal assays of LOS preparations are provided by Apicella as are infant rat model and chinchilla immunisations using the mutant
H. influenzae
strain. The tests use LPS per se as immunogen they do not illustrate or suggest anything concerning adjuvant activity. The immune response against LPS per se is exhibited in the tests of Apicella et al.
A Salmonella mutant is also disclosed. This mutant was achieved following the method analogously to the one for
H. influenzae
The Salmonella mutant provides an LPS in which the 3′substitution on the N linked C14 is a C16 rather than a C12 fatty acid. This embodiment was tenfold less toxic than wild type. No details on antigenicity are provided for this substance.
They suggested the method could also be applicable to Neisseria, Moraxella, and Campylobacter. In Example 6 e.g. Apicella suggested analogous steps to the H. influenzae could be carried out for Neisseria but nothing is illustrated and the method has clearly not been carried out. To date no teaching concerning such gene in lipid A synthesis of Neisseria has been found and no details of tests wherein the gene involved in this stage of lipid A synthesis of Neisseria have been provided.
The Apicella prior art document reveals that mutation in Salmonella appears to induce another acyltransferase rather than resulting in omission of secondary acylation in contrast to the result provided for
H. influenzae
. This illustrates unpredictability in the result when mutating genes associated with lipid A synthesis in various Gram negative organisms and is in line also with the teaching of Erwin and Munford.
The Salmonella product is a hepta or hexaacyl i.e. has the same number of secondary and primary acyl chains as th

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