Title:  Attenuated microorganisms for the treatment of infection

United States Patent:  6,756,042

Issued:  June 29, 2004

Inventors:  Feldman; Robert Graham (Berkshire, GB); Dougan; Gordon (Berkshire, GB); Santangelo; Joseph David (Berkshire, GB); Holden; David William (Berkshire, GB); Shea; Jacqueline Elizabeth (Berkshire, GB); Hindle; Zoe (Berkshire, GB)

Assignee:  Microscience, Ltd. (GB)

Appl. No.:  569601

Filed:  May 9, 2000

Abstract

The present invention pertains to a Salmonella microorganism having an attenuating mutation which disrupts the expression of a gene located within the Spi2 pathogenicity island, and an auxotrophic mutation. The microorganism therefore has a double mutation which helps prevent reactivity of the microorganism while maintaining the effectiveness of the microorganism to elicit an immune response. The present invention also pertains to vaccine compositions and methods for treating and preventing a Salmonella infection in a patient.

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. Methods for introducing the mutations into the specific regions will be apparent to the skilled person.

In addition to the two mutations, the Salmonella microorganism may also comprise heterologous antigens. The attenuated microorganism can therefore act as a delivery vehicle for administering antigens against other bacterial or viral infections. Antigens which are suitable for use in this way will be apparent to the skilled person and include:

Pathogenic E. coli antigens, i.e. ETEC

Hepatitis A, B and C antigens

Lime disease antigens

vibrio cholera antigens

Helicobacter antigens

Herpes Simplex virus antigens

Human papilloma virus antigens

This system also has the potential to deliver therapeutic proteins, e.g. cytokines, for the treatment of patients, e.g. patients infected with hepatitis. Methods for the delivery of heterologous antigens or therapeutic proteins using the vaccine compositions will be apparent to the skilled person.

Vaccines made using the microorganisms of the invention have application to the treatment of infections in human patients and in the treatment of veterinary infections.

The double mutation provides an effective means to attenuate the microorganism to provide a safe vaccine candidate.

The vaccine compositions provide effective protection even in immunocompromised patients, and importantly offer a low risk in developing spleen abscesses. Spleen abscesses have been identified using vaccines based on a single mutation, and therefore the present compositions may offer a substantial benefit to patients.

To formulate the vaccine compositions, the mutant microorganisms may be present in a composition together with any suitable pharmaceutically acceptable adjuvant, diluent or excipient. Suitable formulations will be apparent to the skilled person. The formulations may be developed for any suitable means of administration. Preferred administration is via the oral or intravenous routes and the vaccines are live attenuated Salmonella microorganisms. The number of microorganisms that are required to be present in the formulations can be determined and optimised by the skilled person. However, in general, a patient may be administered approximately 10⁷ -10¹⁰ CFUs, preferably approximately 10⁸ -10⁹ CFUs in a single dosage unit.

Claim 1 of 11 Claims

What is claimed is:

1. An isolated Salmonella microorganism comprising an attenuating mutation which disrupts expression of the ssaV gene and an auxotrophic mutation which disrupts expression of the aroC gene.

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