Title:  Streptococcus gordonii strains resistant to fluorodeoxyuridine

United States Patent:  6,312,955

Assignee:  Siga Pharmaceuticals (New York, NY)

Filed:  April 29, 1999

The present invention features gram-positive bacteria resistant to 5-fluorodeoxyuridine (FUdR). Such bacteria will preferably be commensal, and will not be resistant to antibiotics. Bacteria according to the present invention may also be transformed with DNA encoding an antigenic protein. Such transformed bacteria may be used to formulate a vaccine, in order to stimulate an immune response to the antigenic protein in a patient. The present invention further provides a method for isolating gram-positive bacteria resistant to FUdR.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

More particularly, the present invention relates to a method for isolating a strain of gram-positive bacteria resistant to 5-fluorodeoxyuridine ("FUdR") comprising (1) culturing the bacteria on culture medium containing FUdR; and (2) selecting those bacteria that grow in the presence of FUdR.

The culture medium for use in the method of the present invention is preferably selected from the group consisting of brain-heart infusion and tryptic soy broth. The culture medium for use in the method of the present invention may comprise from about 0.5 to about 50 .mu.g/ml FUdR. In a preferred embodiment, the culture medium for use in the method of the present invention comprises from about 1 to about 10 .mu.g/ml FUdR. The culture medium preferably further comprises about 12.5 .mu.g/ml uridine and about 2 .mu.g/ml thymidine.

Any gram-positive bacteria may be used in the method of the present invention. Preferably, the bacteria used in the present invention are gram-positive commensal bacteria, and are not resistant to antibiotics. A particularly preferred species of bacteria for use in the present invention is Streptococcus gordonii.

The present invention further provides isolated gram-positive bacteria resistant to FUdR. Such bacteria may be isolated by the method of the present invention, or may be produced by recombinant means. Preferably, bacteria according to the present invention will be commensal bacteria, such as Streptococcus gordonii, and will not be resistant to antibiotics.

A bacterium according to the present invention will be resistant to FUdR due to a point mutation in the tdk gene that results in the introduction of a translational termination codon in the tdk ORF. Such a mutation results in the expression of a prematurely terminated TK polypeptide. In a preferred embodiment of the present invention, said prematurely terminated TK polypeptide lacks domain VII.

FUdR-resistant bacteria according to the present invention may be further transformed with DNA encoding antigenic proteins in order to stimulate an immune response in a patient. In a preferred embodiment, bacteria according to the present invention for use in vaccine compositions will be transformed with DNA encoding a surface protein of a disease-causing bacteria, in order to stimulate an immune response to that disease in a patient. As used in the context of the present invention, the term "patient" will be understood to refer to any animal, including but not limited to humans, in which an immune response is desired to be generated. In a particularly preferred embodiment of the present invention, the DNA will encode the streptococcal M6 protein, or a fragment thereof, as described in U.S. Pat. Nos. 4,784,948 and 5,840,314, the contents of which are incorporated herein by reference. Gram-positive bacteria may be transformed to express heterologous proteins on their surface using the methods described, for example, in U.S. Pat. Nos. 5,616,686, 5,786,205, and 5,821,088, the contents of which are incorporated herein by reference.

Claim 1 of 13 Claims

What is claimed is:

1. A method for isolating a strain of Streptococcus gordonii resistant to 5-fluorodeoxyuridine ("FUdR") comprising

culturing S. gordonii on culture medium containing FUdR; and

selecting those S. gordonii that grow in the presence of FUdR.

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