United States Patent:  6,040,427

Assignee:  SmithKline Beecham Biologicals (Rixensart, BE)

Filed:  July 1, 1998

The Bordetella pertussis toxin is genetically modified to express a toxin protein which is deficient in target-cell receptor binding and is used in a vaccine for protection against whooping cough.

SUMMARY OF INVENTION

The present invention relates to a recombinant DNA molecule which encodes a protein specifically reactive with antibodies against the wild-type pertussis toxin but which is defective in pertussis toxin target cell receptor binding.

In related aspects, this invention is a recombinant plasmid which comprises the recombinant DNA molecule of this invention operatively linked to a regulatory region. Said regulatory region contains regulatory sequences necessary for transcription of the protein coding sequence and subsequent translation in a host cell transformed with the recombinant plasmid of the invention.

This invention also relates to a pertussis toxin protein encoded by the recombinant DNA molecule of the invention, which is specifically reactive with antibodies against the wild-type pertussis toxin but which is also defective in pertussis toxin target cell receptor binding.

In another aspect, this invention is a vaccine for stimulating protection against whooping cough wherein such vaccine comprises an immunoprotective and non-toxic quantity of the protein encoded by the recombinant DNA molecule of the invention. For vaccinal purposes, the protein of the invention may be purified away from the host cell or cell culture medium, or alternatively, it may be associated with the outer surface membrane of the host cell.

This invention further relates to a process for preparing the protein of the invention. This process comprises growing a host cell transformed with the recombinant DNA molecule of this invention in a suitable culture medium.

Claim 1 of 20 Claims

1. An isolated DNA molecule comprising a recombinant coding sequence encoding a Bordetella pertussis holotoxin comprising a modified B-oligomer comprising either or both of a modified dimer D1or D2 wherein said modified dimer comprises an unmodified S4 subunit and either a modified S2 subunit or a modified S3 subunit and said modified B-oligomer optionally comprises an unmodified S5 subunit and further optionally comprises an unmodified S1 subunit, wherein said holotoxin comprising a modified B-oligomer is specifically reactive with antisera capable of recognizing a wild-type Bordetella pertussis holotoxin having no modified subunits;

wherein said modified S2 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr¹⁰², Tyr¹⁰³ and Asn¹⁰⁵, an amino acid substitution of either or both of Tyr¹⁰² and Tyr¹⁰³ and a substitution of a negatively-charged amino acid for Asn¹⁰⁵ ; and

wherein said modified S3 subunit comprises one or more amino acid sequence modifications selected from the group consisting of a deletion of one or more of Tyr¹⁰², Tyr¹⁰³ and Lyr¹⁰⁵, an amino acid substitution of either or both of Tyr¹⁰² and Tyr¹⁰³, and a substitution of a negatively-charged amino acid for Lys¹⁰⁵.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.