Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-06-09
2004-07-20
Graser, Jennifer E. (Department: 1641)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S241100, C424S234100, C424S200100, C435S243000, C435S252300
Reexamination Certificate
active
06764687
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to live attenuated bacteria for use in a medicament, to vaccines based upon such bacteria useful for the prevention of microbial pathogenesis, to live attenuated bacteria carrying a heterologous gene and to methods for the preparation of such vaccines and bacteria.
BACKGROUND OF THE INVENTION
The means by which a warm blooded animal overcomes microbial pathogenesis is a complex process. Immunity to microbial pathogenesis is one means by which a warm blooded animal avoids pathogenesis, or suffers a less intense pathogenic state. Incomplete immunity to a given pathogen results in morbidity and mortality in a population exposed to a pathogen. It is generally agreed that vaccines based on live but attenuated micro-organisms (live attenuated vaccines) induce a highly effective type of immune response. Such vaccines have the advantage that, once the animal host has been vaccinated, entry of the microbial pathogen into the host induces an accelerated recall of earlier, cell-mediated or humoral immunity which is able to control the further growth of the organism before the infection can assume clinically significant proportions. Vaccines based on a killed pathogen (killed vaccine) are generally conceded to be unable to achieve this type of response. However, vaccines that contain a live pathogen present, depending on the level of attenuation, the danger that the vaccinated host upon vaccination may contract the disease against which protection is being sought. Therefore, it would be desirable to have a vaccine that possesses the immunising attributes of a live micro-organism but that is not capable of causing undesirable side effects upon vaccination.
The general approach for attenuating bacteria is the removal of one or more virulence factors. In most cases however, virulence factors also play a role in inducing immunity. In those cases, deletion of virulence factors unavoidably impairs the immunogenic capacities of the bacterium. This is of course an unwanted situation. A live vaccine should preferably retain the antigenic complement of the wild-type strain. Moreover, the live vaccine should be sufficiently a-virulent to avoid unacceptable pathological effects, but on the other hand it must cause a sufficient level of immunity in the host. Finally, the live attenuated vaccine strain should preferably have substantially no probability for reverting to a virulent wild type strain.
SUMMARY OF THE INVENTION
It was now surprisingly found that a gene encoding a protein known to play a role in the central carbohydrate metabolism in many bacterial genera can be deleted, causing attenuated behaviour in vivo without impairing the viability of such bacteria in vivo. Bacteria from which this gene is deleted do unexpectedly show an attenuated character. Moreover, since the encoded protein plays no role in the induction of immunity, the antigenic load of bacteria from which this gene is deleted, is identical to that of the wild-type. Therefore, such bacteria could unexpectedly be advantageously used in the field of preparation of medicaments, more specifically for the preparation of live attenuated vaccines. The gene that according to the present invention can be deleted and leads to an attenuated in vivo behaviour of the deletion mutants is a gene formerly known as the fruR gene, currently however called the cra gene. It was known that mutants lacking this gene could be grown in vitro, but only if the deficiencies due to lack of Cra activity are compensated for in the growth medium. This means that nutrients on which the Cra-deficient mutant can grow must be present in the growth medium. Generally spoken, pathogenic bacteria are self-supporting in the sense that they adapt their metabolism to the nutrients that are available. The cra gene plays such an adaptive role in many main metabolic pathways (see below). Mutants from which the cra gene has been deleted can however grow perfectly well on glucose and many other sugars as carbon source. In the host animal, such sugars are available and therefore one would not expect the cra gene to be functional under in vivo conditions. And thus, one would not expect Cra-negative mutants to show attenuated characteristics in the host. That explains why, although such mutants were known in the art, they have never been suggested to be potential live attenuated vaccine candidates.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the invention relates to live attenuated bacteria that are no longer capable to express a functional Cra protein as a result of a mutation in the cra gene, for use in a vaccine.
The gene product, (formerly known as FruR; the Fructose Repressor Protein), now also known as Cra (the Catabolite Repressor/Activator Protein), is a regulatory protein in many main pathways of the carbohydrate metabolism.
The cra-gene product Cra regulates the central carbon metabolism. More specifically, Cra positively regulates transcription of genes encoding biosynthetic and oxidative enzymes (e.g. key enzymes in the TCA cycle, the glyoxalate shunt, the gluconeogenic pathway and electron transport) by binding upstream of the promoters of these genes and negatively regulates transcription of genes encoding glycolytic enzymes (e.g. key enzymes in the Embden-Meyerhof and Entner-Doudoroff pathways). Due to its key position in carbohydrate metabolism, the cra gene and its gene product Cra are widespread in the bacterial realm. The Cra protein is a highly conserved protein. It can be found in e.g.
Escherichia coli
, in
Salmonella enterica
species, such as serotype Typhimurium, Enteritidis and Dublin, in Actinobacillus species such as
A. pleuropneumoniae
, in Haemophilus species such as
H. paragallinarum
, in
Aeromonas salmonicidae
, in Pasteurella species such as
P. piscida
and
P. multocida
, in Streptococcus species such as
S. equi
and
S.
suis and in Yersinia species such as
Y. pestis.
The gene itself and its complete nucleotide sequence in Salmonella and Escherichia have been elucidated already in 1991 by Jahreis, K. et al. (Mol. Gen. Genet. 226: 332-336 (1991)). Jahreis showed that the Cra protein in
Salmonella enterica
, serotype Typhimurium and
Escherichia coli
differed only in 4 positions, of which two were merely conservative exchanges. This is of course in line with what could be expected for a protein playing a role in so many universal pathways in the bacterial carbohydrate metabolism, especially where
E. coli
and Salmonella diverged not that far during evolution. The mechanism of binding of the Cra protein has been at least partially elucidated by Ramseier, T. M. et al. (J. Mol. Biol. 234:28-44 (1993)). The role and function of the Cra protein (the Catabolite Repressor/Activator Protein) have been regularly described in the literature, e.g. in a recent mini-review by Saier, M. H. and Ramseier, T. M. (Journ. Bacteriol. 178: 3411-3417 (1996)).
Such a mutation can be an insertion, a deletion, a substitution or a combination thereof, provided that the mutation leads to the failure to express a functional Cra protein. A functional Cra protein is understood to be a protein having the regulating characteristics of the wild-type protein. Therefore, a Cra protein that is defective in at least one of its functions is considered to be a non-functional Cra protein.
Live attenuated bacteria for use according to the invention can be obtained in several ways. One possible way of obtaining such bacteria is by means of classical methods such as the treatment of wild-type bacteria having the cra gene with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment. Strains that do not produce a functional Cra protein can easily be picked up. They grow on minimal medium exclusively in the presence of glucose and other sugars as carbon sources (which differentiates them from cya and crp mutants) but they are not able to grow with gluconeogenic substrates as sole carbon source. (Chin et al., J. Bacteriol. 169: 897-899 (1987)) They can therefore very easily
Cohen Paul S.
Laux David C.
Nuijten Petrus J. M.
Akzo Nobel N.V.
Blackstone William M.
Graser Jennifer E.
LandOfFree
Live attenuated bacteria for use in a vaccine does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Live attenuated bacteria for use in a vaccine, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Live attenuated bacteria for use in a vaccine will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3237073