Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2000-05-09
2004-06-29
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C435S252100
Reexamination Certificate
active
06756042
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to attenuated microorganisms that can be used in vaccine compositions for the prevention or treatment of bacterial or viral infections.
BACKGROUND TO THE INVENTION
It is well established that live attenuated micro-organisms are highly effective vaccines; immune responses elicited by such vaccines are often of greater magnitude and of longer duration than those produced by non-replicating immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection. In addition, unlike killed preparations, live vaccines are able to induce potent cell-mediated responses which may be connected with their ability to replicate in antigen-presenting cells, such as macrophages.
There has been a long history of the use of live attenuated Salmonella vaccines as safe and effective vaccines for the prevention of salmonellosis in animals and humans. Indeed, the live attenuated oral typhoid vaccine, Ty21a (Vivotif), manufactured by the Swiss Serum Vaccine Institute, has proved to be a very successful vaccine for the prevention of typhoid fever and has been licensed in many countries including the US and Europe.
However, the attenuation of this strain was achieved using chemical mutagenesis techniques and the basis of attenuation of the strain is not fully understood. Because of this, the vaccine is not ideal in terms of the number of doses (currently four) and the number of live organisms that have to be given at each dose.
Modern molecular biology techniques, coupled with the increasing knowledge of Salmonella pathogenesis, has led to the identification of several genes that are essential for the in vivo growth and survival of the organisms. This has provided new gene targets for attenuation, leading to the concept that future vaccine strains can be ‘rationally’ attenuated by introducing defined non-reverting mutations into selected genes known to be involved in virulence. This will facilitate the development of improved vaccines, particularly in terms of the immunogenicity and therefore the number of doses that have to be given.
Although many attenuated strains of Salmonella are now known, few have qualified as potential vaccine candidates for use in humans. This may be due in part to the need to balance the immunogenicity of the vaccine with the possibility of the Salmonella microorganism becoming reactive.
It is clear that the selection of appropriate targets for attenuation which will result in a suitable vaccine candidate, is not straightforward and cannot easily be predicted. Many factors may influence the suitability of the attenuated strain as an appropriate vaccine, and there is much research being carried out to identify suitable strains. For example, many attenuated strains tested as vaccine candidates lead to vaccinemia or abscesses in the patient.
It is therefore desirable to develop a vaccine having a high degree of immunogenicity with reduced possibility of the microorganism strain reverting to an reactive form and which exhibits a good safety profile with limited side effects.
SUMMARY OF THE INVENTION
The present invention is based on the finding that two specific attenuating mutations introduced into a Salmonella microorganism can produce a vaccine having a high degree of immunogenicity and a low risk of the microorganism reverting to a reactive form. The resulting vaccine strains exhibit a good side-effect profile.
The first mutation is contained within a region of the Salmonella pathogenicity island two (Spi2), the second is an auxotrophic mutation, i.e. a mutation to disrupt the expression of a gene that encodes a protein required in a biosynthetic pathway.
According to a first aspect of the invention, a Salmonella microorganism has an attenuating mutation which disrupts the expression of a gene located within the Spi2 pathogenicity island, and an independent auxotrophic mutation. The preferred attenuating mutation is within the apparatus gene ssaV, and the preferred auxotrophic mutation is within aroC.
The microorganism preferably further comprises one or more heterologous antigens or therapeutic proteins, for example antigens for pathogenic
E. coli
, Shigella, hepatitis A, B or C, Herpes Simplex Virus and Human papilloma virus. Therefore, the microorganism may act as a delivery vehicle to immunise against infections other than Salmonella.
The Salmonella microorganisms may be used to manufacture a vaccine composition which may be administered to a patient via the intravenous or oral route, in a method for the treatment of a bacterial or viral infection, e.g. for the treatment of typhoid.
The attenuated Salmonella microorganisms of the present invention form vaccines which surprisingly stimulate mucosal as well as systemic immunity. Further, the microorganisms do not cause spleen abscesses in an animal model, whereas mutants with single mutations do. This is a particular advantage of the double mutants as defined herein.
DESCRIPTION OF THE INVENTION
The microorganisms and vaccine compositions of the present invention may be prepared by known techniques.
The choice of particular Salmonella microorganism and the selection of the appropriate mutation, can be made by the skilled person without undue experimentation. A preferred microorganism is
Salmonella typhimurium.
A first mutation may be introduced into a gene located within the region of the Salmonella pathogenicity island 2, this region being disclosed in WO-A-9617951.
The Salmonella pathogenicity island two (Spi2) is one of two classical pathogenicity islands located on the Salmonella chromosome. Spi2 comprises several genes that encode a type III secretion system involved in transporting Spi2 encoded virulence-associated proteins (so-called effector proteins) outside of the Salmonella bacteria and potentially directly into target host cells such as macrophages. Part of Spi2 (the apparatus genes) encodes the secretion apparatus of the type III system. Spi2 is absolutely essential for the pathogenesis and virulence of Salmonella in the mouse, an observation now documented by several different groups around the world.
S. typhimurium
Spi2 mutants are highly attenuated in mice challenged by the oral, intravenous and intraperitoneal routes of administration.
The Spi2 gene may be either an apparatus gene or an effector gene. Preferably, the gene is an apparatus gene. The apparatus genes located within Spi2 are now well characterised; see for example Hensel et al, Molecular Microbiology (1997); 24(1): 155-167 Genes suitable for use in the present invention include ssaV, ssaJ, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaU and ssaH genes.
The mutation in the Spi2 region does not necessarily have to be within a gene to disrupt the function. For example, a mutation in an upstream regulatory region may also disrupt gene expression, leading to attenuation. Mutations in an intergenic region may also be sufficient to disrupt gene function.
In a preferred embodiment of the invention, the apparatus gene is ssaV. In a separate preferred embodiment, the mutation lies within an intergenic region between ssaJ and ssaK.
The second mutation is termed an “auxotrophic mutation” as it disrupts a gene which is essential in a biosynthetic pathway. The biosynthetic pathway is one present in Salmonella, but not present in mammals. Therefore, the mutants cannot depend on metabolites found in the treated patient to circumvent the effect of the mutation. Suitable genes for the auxotrophic mutation, include any aro gene, e.g. aroA, aroC, aroD and aroE.
In a preferred embodiment of the invention, the vaccine composition comprises a Salmonella microorganism having attenuating mutations in ssaV and aroC.
The mutations may be introduced into the microorganism using any known technique. Preferably, the mutation is a deletion mutation, where disruption of the gene is caused by the excision of nucleic acids. Alternatively, mutations may be introduced by the insertion of nucleic acids or by point mutations. Met
Dougan Gordon
Feldman Robert Graham
Hindle Zoe
Holden David William
Santangelo Joseph David
Microscience, Ltd.
Navarro Mark
Saliwanchik Lloyd & Saliwanchik
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