Claims for Patent: 9,750,793

Summary for Patent: 9,750,793

Title:	Multifunctional oral vaccine based on chromosome recombineering
Abstract:	A recombineered Salmonella typhi Ty21a, compositions and vaccines comprising such a Ty21a, and a method for recombineering comprising inserting a large antigenic region into a bacterial chromosome for the purpose of making multivalent vaccines to protect against one or more disease agents are described herein.
Inventor(s):	Dharmasena; Madushini Nirosha (Germantown, MD), Kopecko; Dennis J. (Silver Spring, MD)
Assignee:	The United States of America, as Represented by the Secretary, Department of Health and Human Services (Washington, DC)
Application Number:	14/428,870
Patent Claims:	1. A Salmonella Typhi Ty21a comprising a Shigella sonnei form 1 O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein: a) heterologous Shigella sonnei form 1 O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen; b) immune protection is elicited against virulent Shigella sonnei challenge; and c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella sonnei form 1 O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen; and d) the region comprises a DNA sequence, wherein the DNA sequence comprises: 1) the DNA sequence as set out in SEQ ID NO:2, or 2) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:2. 2. The Salmonella Typhi Ty21a of claim 1, further comprising an O-antigen biosynthetic gene region from a bacterial strain selected from the group consisting of: Shigella species, Escherichia coli serotypes, Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacter species, Yersinia species, Plesiomonas species, and Pseudomonas species. 3. A Salmonella Typhi Ty21a comprising a Shigella dysenteriae 1 O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein: a) heterologous Shigella dysenteriae serotype 1 O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen; b) immune protection is elicited against virulent Shigella dysenteriae challenge; and c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella dysenteriae serotype 1 O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen; and d) the region comprises a DNA sequence, wherein the DNA sequence comprises: 1) the DNA sequence as set out in SEQ ID NO:33, or 2) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:33. 4. The Salmonella Typhi Ty21a of claim 3, further comprising an O-antigen biosynthetic gene region from a bacterial strain selected from the group consisting of: Shigella species, Escherichia coli serotypes, Salmonella enterica serovars, Vibrio cholerae serotypes, Enterobacter species, Yersinia species, Plesiomonas species, and Pseudomonas species. 5. A plasmid construct having i) a DNA sequence as set out in SEQ ID NO: 1 or ii) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:1. 6. The plasmid construct of claim 5, further comprising a Shigella sonnei O-antigen biosynthetic gene region or a Shigella dysenteriae 1 O-antigen biosynthetic gene region. 7. A method of recombineering a large antigenic gene region into a bacterial chromosome, comprising: i) cloning the region into a vector containing: ia) a genetically selectable marker flanked 5' and 3' by an FRT site, respectively; ib) a multiple cloning site downstream of the 3' FRT site; and ic) two sites of chromosome homology, one of the two located upstream of the 5' FRT site, and one of the two located downstream of the multiple cloning site; ii) integrating the region into the bacterial chromosome using X, red recombination; iii) selecting for the genetically selectable marker; and iv) removing the selectable marker, thus recombineering the large antigenic gene region into the chromosome. 8. The method of claim 7, wherein the large antigenic gene region is about 5 to about 20 kb long. 9. The method of claim 7, wherein the vector is selected from the group consisting of a plasmid, phage, phasmid, and cosmid construct. 10. The method of claim 9, wherein the plasmid construct has i) a DNA sequence as set out in SEQ ID NO: 1 or ii) a DNA sequence that shares at least about 90% sequence identity with the DNA sequence set out in SEQ ID NO:1. 11. The method of claim 7, wherein the bacterial chromosome is from Salmonella Typhi Ty21a. 12. The method of claim 7, wherein the genetically selectable marker is an antibiotic resistance marker. 13. The method of claim 12, wherein the antibiotic resistance marker is kanamycin. 14. The method of claim 7, wherein the large antigenic gene region is selected from the group consisting of a Shigella sonnei O-antigen biosynthetic gene region, a Shigella dysenteriae 1 O-antigen biosynthetic gene region, a Shigella flexneri 2a 0-antigen biosynthetic gene region, and a Shigella flexneri 3a O-antigen biosynthetic gene region. 15. The method of claim 7, wherein the large antigenic gene region is engineered between the two sites of chromosome homology, each site comprising between about 500 to about 1000 bp regions of bacterial chromosome homology before step ii. 16. The method of claim 13, wherein the kanamycin resistance gene is removed via recombination induced following transformation of chromosomal integrants of step ii with pCP20. 17. A vaccine comprising a pharmaceutically acceptable solid carrier and Salmonella Typhi Ty21a comprising a Shigella flexneri 2a O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein: a) heterologous Shigella flexneri 2a O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen; b) immune protection is elicited against virulent Shigella flexneri 2a challenge; and c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella flexneri 2a O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen. 18. A vaccine comprising a pharmaceutically acceptable solid carrier and Salmonella Typhi Ty21a comprising a Shigella flexneri 3a O-antigen biosynthetic gene region inserted into the Salmonella Typhi Ty21a chromosome, wherein: a) heterologous Shigella flexneri 3a O-antigen is stably expressed together with or without homologous Salmonella Typhi O-antigen; b) immune protection is elicited against virulent Shigella flexneri 3a challenge; and c) immune protection is elicited against virulent Salmonella Typhi challenge when heterologous Shigella flexneri 3a O-antigen is stably expressed together with homologous Salmonella Typhi O-antigen. 19. A vaccine comprising the Salmonella Typhi Ty21a of claim 1 in combination with a physiologically acceptable carrier. 20. A method of treating at least one bacterial infection comprising administering a prophylactically or therapeutically effective amount of the Salmonella Typhi Ty21a of claim 1 to a subject, thus treating the at least one bacterial infection. 21. The Salmonella Typhi Ty21a of claim 1, wherein the Shigella sonnei form 1 O-antigen biosynthetic gene region is partially or wholly chemically synthesized. 22. The plasmid construct of claim 5, wherein the DNA sequence is partially or wholly chemically synthesized.

Details for Patent 9,750,793

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Emergent Travel Health, Inc.	VIVOTIF	typhoid vaccine live oral ty21a	Capsule	103123	12/15/1989		2032-09-17