WO2008133645A2

WO2008133645A2 - Combination vaccine for prevention of tularemia

Info

Publication number: WO2008133645A2
Application number: PCT/US2007/022940
Authority: WO
Inventors: Dennis L. Kasper; Gerald A. Beltz; Shite Sebastian
Original assignee: President And Fellows Of Harvard College
Priority date: 2006-10-31
Filing date: 2007-10-30
Publication date: 2008-11-06
Also published as: WO2008133645A3

Abstract

The invention provides a combination vaccine comprising a glycoconjugate and a live attenuated avirulent strain of Francisella tularensis, as well as methods for their preparation and use in protecting a mammal against tularemia and against infection by Francisella tularensis.

Description

COMBINATION VACCINE FOR PREVENTION OF TULAREMIA

BACKGROUND OF THE INVENTION

Francisella tularensis (F. tularensis) is a pleomorphic Gram-negative facultative intracellular pathogen that is the etiological agent of the potentially fatal human disease, tularemia. Ellis J et al. (2002) Clin Microbiol Rev 15:631-46. The high virulence, low infectious dose, and aerosolized nature of transmission of F. tularensis have raised serious concerns for the exploitation of this microbe as a biowarfare agent. Dennis DT et al. (2001) JAMA 285:2763-73. Two major biovars of F. tularensis exist, namely, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B), both of which remain highly virulent and infectious against a wide range of mammalian species including humans. Ellis J et al. (2002) supra. An empirically derived vaccine strain of F. tularensis referred to as Live Vaccine Strain (LVS; F. tularensis LVS; Ft. LVS) exists and is currently being administered to at risk individuals. Sandstrom G (1994) J Chem Technol Biotechnol 59:315-20. However, the lack of complete understanding for the attenuation of the strain, unwanted side-effects, incomplete immunity to the vaccines, and the scarcity of information regarding the virulence factors of this bacterium have hindered the licensing of this strain to be used as a generalized vaccine in the United States. Sjostedt A (2003) Curr Opin Microbiol 6:66-71 ; Burke DS (1977) J Infect Dis 135:55-60. There is clear unmet need for development of an effective vaccine for protection against tularemia.

SUMMARY OF THE INVENTION

In one aspect of the invention a microbial or pathogen-specific antigen:polysaccharide chemical conjugate is provided, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide. In one embodiment the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D- GaIpNAcAN-(I -4)-α-D-Gal/?N Ac AN-(I -3)-β-D-QuipNAc-(l -2)-β-D-Quip4NFo-l ); 4- (α-D-GalpNAc AN-(I -3)-β-D-Qui/?NAc-( 1 -2)-β-D-Qui/?4NFo-( 1 -4)-α-D-Gal^NAcAN- l); 3-(β-D-QuipNAc-(l-2)-β-D-Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D- GalpNAcAN-1); and 2-(β-D-Quip4NFo-(l-4)-α-D-GalpN Ac AN-(I -4)-α-D- GaIpNAcAN-(I -3)-β-D-QuipNAc-l). In one embodiment the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-GalpN Ac AN-(I -4)-α-D- Gal/7NAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Qui^4NFo-l); 4-(α-D-Gal/?NAcAN-(l-3)- β-D-QuipNAc-(l-2)-β-D-Quip4NFo-(l-4)-α-D-Gal^NAcAN-l); 3-(β-D-QuφNAc-(l-2)- β-D-Qui/?4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-l); and 2-(β-D- Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment a pathogen-specific antigen :poly saccharide chemical conjugate is provided, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide. In one embodiment the pathogen-specific antigen is tetanus toxoid. In one aspect of the invention the chemical conjugate is tetanus toxoid:4-(α-D-Gal;?NAcAN-(l-4)-α-D- GaIpNAcAN-(I -3)-β-D-Qui^NAc-(l -2)-β-D-Quip4NFo-l ). In one aspect of the invention a vaccine composition is provided that comprises an effective amount of a microbial or pathogen-specific antigen:polysaccharide conjugate and a pharmaceutically acceptable carrier. In one embodiment the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D- GaIpNAc AN-( 1 -4)-α-D-Gal/?NAc AN-( 1 -3)-β-D-QuipNAc-( 1 -2)-β-D-Quφ4NFo- 1 ); 4- (α-D-GalpNAcAN-(l -3)-β-D-QuipNAc-(l -2)-β-D-Quip4NFo-(l -4)-α-D-GalpNAcAN- l); 3-(β-D-QuipNAc-(l-2)-β-D-Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D- GaIpNAcAN- 1); and 2-(β-D-Quip4NFo-(l-4)-α-D-GalpN Ac AN-(I -4)-α-D- GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-Gal/>NAcAN-(l-4)-α-D- GaI^NAcAN-(I -3)-β-D-Qui/?NAc-( 1 -2)-β-D-Qui/?4NFo- 1 ); 4-(α-D-Gal/?NAcAN-( 1 -3)- β-D-QuipNAc-(l-2)-β-D-Qui/74NFo-(l-4)-α-D-Gal^NAcAN-l); 3-(β-D-QuipNAc-(l-2)- β-D-Qui/?4NFo-(l -4)-α-D-GalpNAc AN-(I -4)-α-D-GalpNAcAN- 1 ); and 2-(β-D- Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the antigen is a microbial antigen. In one embodiment the antigen is a pathogen-specific antigen. In one embodiment the pathogen-specific antigen is tetanus toxoid. In one aspect of the invention the chemical conjugate is tetanus toxoid:4-(α-D- GaIpNAcAN-(I -4)-α-D-Gal/?NAcAN-( 1 -3)-β-D-Qui/?NAc-( 1 -2)-β-D-Quip4NFo- 1 ). In one embodiment the vaccine composition further comprises an effective amount of a live attenuated avirulent strain of Francisella tularensis. In one embodiment the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis. In one embodiment the reduced expression of wild- type O antigen is no expression of wild-type O antigen. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units. In one embodiment the reduced expression results from a mutation of a gene affecting O- antigen biosynthesis. In one embodiment the mutation is a mutation of a gene within an O-antigen biosynthesis locus. In one embodiment the mutation is a mutation of wbtA. In one aspect of the invention a vaccine composition is provided that comprises an effective amount of an antigen :poly saccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide, an effective amount of a live attenuated avirulent strain of Francisella tularensis, and a pharmaceutically acceptable carrier. In one embodiment the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-GalpN Ac AN-(I -4)-α-D- GφNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quip4NFo-l); 4-(o-D-GφNAcAN-(l-3)- β-D-QuipNAc-(l-2)-β-D-Quψ4NFo-(l-4)-α-D-GalpNAcAN-l); 3-(β-D-QuψNAc-(l-2)- β-D-Qui^4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-l); and 2-(β-D- Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?NAcAN-(l -4)-α-D-Gal/?N Ac AN-(I -3)-β-D-Qui/?NAc-(l -2)- β-D-Qui/?4NFo- 1 ); 4-(α-D-Gal/?NAc AN-( 1 -3)-β-D-Qui/?NAc-( 1 -2)-β-D-Quip4NFo-( 1 - 4)-α-D-GalpNAcAN-l); 3-(β-D-Qui/7NAc-(l-2)-β-D-Quip4NFo-(l-4)-α-D- GaIpNAcAN-(M)-Ci-D-GaIpNAcAN-I); and 2-(β-D-Quip4NFo-(l-4)-α-D-

GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the antigen is bovine serum albumin or human serum albumin. In one aspect of the invention the chemical conjugate is bovine serum albumin:4-(α-D-Gal/?N Ac AN-(I -4)-α- D-GalpNAcAN-(l-3)-β-D-Qui^NAc-(l-2)-β-D-Quip4NFo-l). In one embodiment the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis. In one embodiment the reduced expression of wild-type O antigen is no expression of wild-type O antigen. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella - A - tularensis is at least 10⁴ colony forming units. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units. In one embodiment the reduced expression results from a mutation of a gene affecting O-antigen biosynthesis. In one embodiment the mutation is a mutation of a gene within an O-antigen biosynthesis locus. In one embodiment the mutation is a mutation of wbtA.

In one aspect of the invention a method of vaccinating a mammal against a pathogenic strain of Francisella tularensis is provided that comprises administering to the mammal an effective amount of a microbial or pathogen-specific antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide. In one embodiment the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-GalpN Ac AN-(I- 4)-α-D-Gal/?NAcAN-( 1 -3)-β-D-QφNAc-(l -2)-β-D-Quip4NFo- 1 ); 4-(α-D- Gal/7NAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quψ4NFo-(l-4)-α-D-GalpNAcAN-l); 3- (β-D-QuipNAc-(l -2)-β-D-Qui/?4NFo-(l -4)-α-D-GalpN Ac AN-(I -4)-α-D-GalpNAcAN- l); and 2-(β-D-Quip4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D- QuipNAc-1). In one embodiment the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN- (l-3)-β-D-Qui/?NAc-(l-2)-β-D-Qui/?4NFo-l); 4-(α-D-Gal/?NAcAN-(l-3)-β-D-QuipNAc- ( 1 -2)-β-D-Qui/?4NFo-( 1 -4)-α-D-GφNAc AN- 1 ); 3 -(β-D-QιφN Ac-( 1 -2)-β-D-

Qui/?4NFo-( 1 -4)-α-D-GalpN Ac AN-( 1 -4)-α-D-GalpNAc AN- 1 ); and 2-(β-D-Quip4NFo- ( 1 -4)-α-D-GalpN Ac AN-( 1 -4)-α-D-GalpNAc AN-( 1 -3)-β-D-QuipNAc- 1 ). In one embodiment the pathogen-specific antigen is tetanus toxoid. In one aspect of the invention the chemical conjugate is tetanus toxoid:4-(α-D-Gal/?NAcAN-(l-4)-α-D- GalpNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Qui^4NFo-l). In one embodiment the method further comprises administering an effective amount of a live attenuated avirulent strain of Francisella tularensis. In one embodiment the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis. In one embodiment the reduced expression of wild- type O antigen is no expression of wild-type O antigen. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units. In one embodiment the reduced expression results from a mutation of a gene affecting O- antigen biosynthesis. In one embodiment the mutation is a mutation of a gene within an O-antigen biosynthesis locus. In one embodiment the mutation is a mutation of wbtA. In one aspect of the invention, a method is provided for vaccinating a mammal against a pathogenic strain of Francisella tularensis, comprising administering to the mammal an effective amount of an antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide, and an effective amount of a live attenuated avirulent strain of Francisella tularensis. In one embodiment the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?N Ac AN-(I -4)-α-D-Gal/?NAc AN-(I -3)-β-D-Qui^NAc- (l-2)-β-D-Qui/?4NFo-l); 4-(α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D- Qui/?4NFo-(l-4)-α-D-Gak7NAcAN-l); 3-(β-D-QuipNAc-(l-2)-β-D-Quψ4NFo-(l-4)-α- D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-l); and 2-(β-D-Quip4NFo-(l-4)-α-D- GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D- Gak7NAcAN-(l-4)-α-D-Gak7NAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quip4NFo-l); 4- (α-D-Gal/?NAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Qui/74NFo-(l-4)-α-D-GalpNAcAN- 1 ); 3-(β-D-Qui/?NAc-(l -2)-β-D-Qui/?4NFo-(l -4)-α-D-GalpN Ac AN-(I -4)-α-D- GalpNAcAN-1); and 2-(β-D-Quip4NFo-(l-4)-α-D-GalpNAc AN-(I -4)-α-D- GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one embodiment the antigen is bovine serum albumin. In one embodiment the antigen is human serum albumin. In one aspect of the invention the chemical conjugate is bovine serum albumin:4-(α-D-Gal/>NAcAN-(l-4)-α- D-Gal;?NAcAN-(l-3)-β-D-Qui/?NAc-(l-2)-β-D-Qui/?4NFo-l). In one embodiment the method further comprises administering an effective amount of a live attenuated avirulent strain of Francisella tularensis. In one embodiment the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis. In one embodiment the reduced expression of wild- type O antigen is no expression of wild-type O antigen. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units. In one embodiment the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units. In one embodiment the reduced expression results from a mutation of a gene affecting O- antigen biosynthesis. In one embodiment the mutation is a mutation of a gene within an O-antigen biosynthesis locus. In one embodiment the mutation is a mutation of wbtA.

In one aspect of the invention, the administering of the chemical conjugate is subcutaneous, intranasal, or intradermal. In one aspect of the invention the administering of the live attenuated avirulent strain of Francisella tularensis comprises administering intranasally, subcutaneously, or intradermally. In one embodiment the administering of the chemical conjugate and the administering of the live attenuated avirulent strain of Francisella tularensis are simultaneous. In one embodiment the administering of the chemical conjugate and the administering of the live attenuated avirulent strain of Francisella tularensis are by the same mode of administration. In one embodiment the administering of the chemical conjugate and the administering of the live attenuated avirulent strain of Francisella tularensis are not simultaneous. In one embodiment the administering of the chemical conjugate and the administering of the live attenuated avirulent strain of Francisella tularensis are not by the same mode of administration. In one embodiment the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement of the invention disclosed herein. Figure 1 is a panel of NMR spectra and immunoblots showing the development of glycoconjugate (TT:O-PS) vaccine. (A) Proton NMR analysis of the purified Ft. LVS O-PS. A single tetrasaccharide repeat unit of Ft.LVS O-PS with sugar residues and glycosidic linkages is indicated; (B-D) Analysis of GC vaccine: Silver stain (B), Immunoblot analysis using anti-TT polyclonal serum (Q and anti-F/.LVS polyclonal serum (D).

Figure 2 is graph of an immunization and challenge regimen and a survival curve showing that immunization with TT: O-PS conjugate protects against a lethal intradermal challenge of Ft. LVS. (A) Immunization and challenge regimen. Six- week-old BALB/c mice were immunized subcutaneously twice three weeks apart with the glycoconjugate (GC). Complete and incomplete Freund's served as adjuvant during the first and second immunizations respectively. All mice were challenged intradermally after a 4-week rest period with a lethal intradermal dose (15-fold i.d. LD₅₀) of Ft. LVS. Three mice were sacrificed at day 3 post challenge and bacterial load in the reticuloendothelial tissue determined (data not shown). (B) Protection of BALB/c mice following immunization with TT:O-PS vaccine. Survival of mice was monitored over a 21-day period post challenge after which they were humanely sacrificed. Complete protection was observed when mice were immunized with TT:O-PS vaccine.

Figure 3 is graph of an immunization and challenge regimen and a survival curve showing that Ft.LYSwwbtA intranasal immunization protects mice against a subsequent lethal intranasal challenge of the parent LVS strain. (A) Intranasal immunization and challenge regimen. Six-week-old BALB/c mice were immunized intranasally twice two weeks apart with Ft.LVS::wbtA mutant strain (10⁷ cfu/ mouse). All mice were challenged intranasally after a 4- week rest period with either 20, 200 or 2000-fold i.n. LD₅₀ of Ft. LVS strain. (B) Protection of BALB/c mice following immunization with Ft.LVS::wόtA mutant strain. Survival of mice was monitored over a 21 -day period post challenge after which they were humanely sacrificed. Complete protection of the Ft.LVSwwbtA immunized mice was observed when challenged with 20-fold the i.n. LD₅₀. A delay in time to death was observed when a 200-fold challenge was administered while no significant delay in time to death was observed when mutant- immunized mice were challenged with 2000-fold the i.n. LD₅₀ of Ft. LVS when compared to control mice. Figure 4 is graph of an immunization and challenge regimen and a survival curve showing that TT:O-PS glycoconjugate immunization protects mice against a subsequent low but ordinarily lethal intranasal challenge of parent LVS strain. (A) Glycoconjugate (GC) immunization and challenge regimen. Six-week-old BALB/c mice were immunized subcutaneously twice two weeks apart with the GC. Complete Freund's was used as adjuvant in first immunization, while incomplete Freund's served as an adjuvant during the second immunization. All mice were challenged intranasally after a 4- week rest period with either 20, 200 or 2000-fold i.n. LD₅₀ of Ft. LVS strain. (B) Protection of BALB/c mice following immunization with GC. Survival of mice was monitored over a 21 -day period post challenge after which the survivors were humanely sacrificed. A significant delay in the mean time to death (mtd) was observed when GC-immunized mice were challenged with 20-fold the i.n. LD₅₀. No significant delay in mtd was observed when the GC-immunized mice were challenged with a 200- or 2000-fold i.n. LD₅oθfFΛ LVS when compared to the respective control mice groups.

Figure 5 is graph of an immunization and challenge regimen and a survival curve showing enhanced protection against a lethal Ft.LVS following a combination vaccination regimen. For combination regimen, six-week-old BALB/c mice were immunized intranasally twice two weeks apart with Ft.LVS::wbtA mutant strain (10⁷ cfu/ mouse). These mice were also simultaneously immunized subcutaneously twice two weeks apart with the GC. Complete Freund's and incomplete Freund's served as adjuvants during first and second immunizations respectively. All mice were challenged intranasally after a 4-week rest period with either 30, 300 or 3000-fold i.n. LD₅₀ of Ft. LVS strain. (A) Immunization schedule of combination vaccine regimen: GC immunization (subcutaneous) and Ft.LVS ::w£>tA immunization (intranasal). (B) Survival of BALB/c mice following combination vaccine regimen. Survival was monitored over a 21 day period post challenge after which the survivors were humanely sacrificed. Notably complete protection was observed when immunized mice were challenged with either a 30- or 300-fold i.n. LD₅₀. However, no significant delay in time to death was observed when immunized mice were challenged with 3000-fold the i.n. LD₅₀ofFt. LVS when compared to the control mice.

Figure 6 is a histogram showing ELIspot analysis to determine the level of IFN-γ in GC, Ft.LVS ::w6tA and combination regimen immunized mice. In this assay T cell production of IFN-γ in response to immunization was measured. Briefly, CD3+ T cells were isolated from GC, Ft.LVS ::w6tA and combination regimen immunized mice and were co-cultured with naive APCs in the presence of media (negative control), ConA (positive control), heat-killed Ft. LVS, heat-killed Ft.LVS ::w6tA strain, tetanus and LVS O-PS respectively for 16 hours. The number of T cells producing IFN-γ/2.5 x 10⁵ cells was assessed by ELIspot analysis.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods relating to microbial antigen:polysaccharide chemical conjugate as a vaccine for the prevention of tularemia. In one embodiment the invention provides a novel microbial antigen :poly saccharide chemical conjugate made of tetanus toxoid (TT) chemically conjugated to a single tetrasaccharide repeat unit of O-antigen polysaccharide (O-PS) of the lipopolysaccharide (LPS) of Francisella tularensis. Throughout the disclosure the microbial antigen:polysaccharide chemical conjugates of the invention will be interchangeably also referred to as glycoconjugates, GC, and chemical conjugates. The invention also provides a combination vaccine that comprises a GC of the invention and a live attenuated avirulent strain of F. tularensis for the prevention of tularemia. In one aspect of the invention the GC is an antigen:polysaccharide chemical antigen. In one embodiment the antigen is a non-specific, non-microbial or nonpathogenic antigen for example, bovine serum albumin or human serum albumin. In another embodiment the antigen is a microbial or pathogen-specific antigen. In yet another embodiment the antigen is tetanus toxoid. The live attenuated strains of F. tularensis of the invention are avirulent but immunogenic, i.e., suitable for use in a vaccine. As described in detail below, the live attenuated strains of F. tularensis of the invention are characterized by their reduced expression of wild-type O-antigen polysaccharide as compared to pathogenic F. tularensis. In one embodiment the reduced expression is essentially a complete lack of expression. The methods for preparation and use of a live attenuated vaccine strain of F. tularensis have been described in PCT Patent Application No. PCT/US06/36910, the entire contents of which are incorporated herein by reference. It was surprisingly discovered according to the invention that an improved and synergistic protective effect against tularemia and against infection with F. tularensis was obtained using the combination of a GC of the invention, which glycoconjugate comprises O-antigen polysaccharide of the LPS of F. tularensis, and a live attenuated strain of F. tularensis, Ft. LVS::wbtA (also referred to as Ft. LVSΩwbtA), which strain has no expression of wild-type O-antigen polysaccharide.

In one aspect the invention is a vaccine composition including an effective amount of a microbial, pathogen-specific or any antigen:polysaccharide chemical conjugate. In one aspect of the invention the glycoconjugate is tetanus toxoid:F. tularensis O-antigen polysaccharide (TT:O-PS). In one aspect of the invention O-PS comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?N Ac AN-(I- 4)-α-D-Gal/?NAcAN-(l-3)-β-D-QuψNAc-(l-2)-β-D-Quψ4NFo-l); 4-(α-D- GaIpNAcAN-(I -3)-β-D-QuipNAc-(l -2)-β-D-Qui/?4NFo-(l -4)-α-D-GalpNAcAN- 1 ); 3- (β-D-QφNAc-( 1 -2)-β-D-Quip4NFo-( 1 -4)-α-D-Gal/?N Ac AN-( 1 -4)-α-D-Gal/?NAc AN- l); and 2-(β-D-Qui^4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-Gal^NAcAN-(l-3)-β-D- QuipNAc-1). In one aspect of the invention O-PS is a single tetrasaccharide repeat unit selected from: 4-((X-D-GaIpNAc AN-(I -4)-α-D-Gal/?NAc AN-(I -3)-β-D-QuipNAc-(l -2)- β-D-Quip4NFo-l); 4-(α-D-GalpNAcAN-(l-3)-β-D-Qui/7NAc-(l-2)-β-D-Quip4NFo-(l- 4)-α-D-GalpNAcAN-l); 3-(β-D-Qui/jNAc-(l-2)-β-D-Qui/74NFo-(l-4)-α-D- GaIpNAc AN-( 1 -4)-α-D-GalpNAcAN- 1 ); and 2-(β-D-Quip4NFo-( 1 -4)-α-D- GalpNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-l). In one aspect of the invention O-PS is a fragment of the tetrasaccharides listed herein, e.g., a trisaccharide, a disaccharide, or a monosaccharide.

In one aspect the invention is a vaccine composition further comprising an effective amount of a live attenuated avirulent strain of F. tularensis, wherein the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic F. tularensis, and a pharmaceutically acceptable carrier. In one embodiment the vaccine composition, in addition to the chemical conjugate as described herein, includes two or more individual live attenuated avirulent strains of Francisella tularensis bacteria, wherein at least one live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic F. tularensis.

Clinically and experimentally, glycocoηjugate vaccines have proven effective against various pathogens including intracellular bacteria. Boutonnier et al. (2001) Infect Immun 69;3488-93; Cohen D. et al. (1997) Lancet 349:155-9. As used herein, a conjugate refers to two or more entities bound to one another by any physicochemical means, including covalent coupling, hydrophobic interaction, ionic interaction, and any combination thereof. As used herein, a chemical conjugate is an antigen:polysaccharide conjugate (glycoconjugate) in which an antigen and a polysaccharide are chemically attached to one another, i.e. by covalent bonds, by weak interactions, etc. In one embodiment the antigen:polysaccharide conjugate is a covalent conjugate. The methods and protocols for chemical conjugation of two biomolecules is routine for artisans of ordinary skill. Conlan et al. ((2002) Vaccine 20:3465-3471), have reported a study on the efficacy of a vaccine consisting of a chemical conjugate of the 0-polysaccharide of the lipopolysaccharide of F. tularensis and bovine serum albumin (BSA). Mice immunized with this vaccine generated a robust antibody response, predominantly IgGl, against F. tularensis LPS, and were more resistant than mice immunized with BSA alone to intradermal or aerosol challenge with either F. tularensis LVS or more virulent clinical isolates of the pathogen. However, such vaccination failed to protect against an aerosol challenge with a virulent type A strain of the pathogen. The instant invention is based at least in part on the surprising discovery that a combination vaccine comprising a microbial antigen:polysaccharide conjugate and a live attenuated avirulent strain of F. tularensis produces a synergistic effect and provides a superior protection against pathogenic F. tularensis challenge than either agent alone. In one embodiment the compositions of the invention, i.e., glycoconjugate and live attenuated avirulent strain of F. tularensis, confer resistance to a challenge by a F. tularensis type A strain. Not wishing to be bound by theory or mechanism, based on the central role of IFN-γ in conferring protective immunity against F. tularensis, the enhanced protection observed following the combination regimen is most likely due to enhanced production of IFN-γ. As used herein, microbial refers to a microorganism or microbe, i.e., an organism that is microscopic (too small to be visible to the naked eye). Microorganisms are often described as single-celled, or unicellular, organisms and include bacteria, causing diseases such as plague, tuberculosis and anthrax; protozoa, causing diseases such as malaria, sleeping sickness and toxoplasmosis; and also fungi causing diseases such as ringworm, candidiasis or histoplasmosis. As used herein, microbial also refers to viruses and their products.

As used herein, a microbial antigen is a substance derived from a microbe that when introduced into the body stimulates an immune response that is specific for the antigen. Antigens in general include toxins, bacteria, foreign blood cells, and the cells of transplanted organs. In one aspect of the invention, the microbial antigen is a tetanus toxoid. In another aspect of the invention the microbial antigen is a diphtheria toxoid. In yet another aspect of the invention the microbial antigen is a toxoid from a pathogenic microbe. As used herein, a toxoid is a microbial toxin that has been made inactive but can still combine with or stimulate formation of antibodies. In many bacterial diseases, the bacteria produce a toxin that causes the disease manifestations. Heating the toxin or treating it chemically converts it into a harmless toxoid that can be injected into a human or a nonhuman animal to confer immunity from subsequent infection. According to the instant invention toxoids conjugated to the O-antigen polysaccharide derived from the LPS of F. tularensis provide protection form a pathogenic F. tularensis challenge. When administered in combination with a live attenuated avirulent strain of F. tularensis, the tetanus toxoid:O-polysaccharide chemical conjugate confers synergistic resistance to a pathogenic F. tularensis challenge. An example of an antigen that is not microbial is bovine serum albumin (BSA), and other common carrier proteins.

As used herein, a pathogen is any agent capable of causing disease. The term pathogen is usually restricted to living agents, which include viruses, rickettsia, bacteria, fungi, yeasts, protozoa, helminths, and certain insect larval stages. As used herein, pathogen-specific antigen refers to an antigen that is specific for a particular pathogen. For example tetanus toxoid is a pathogen-specific antigen for Clostridium tetani. Examples of antigens that are not pathogen-specific are bovine serum albumin, human serum albumin, and other common carrier proteins. In one embodiment of the invention the pathogen-specific antigen is F. tularensis specific antigen.

Pathogenic strains of F. tularensis are characterized by their virulence, i.e., their ability to infect a host, evade or overcome natural host defenses, and thus cause symptoms of the disease tularemia. Virulence depends in part on the ability of the microorganism to evade detection and elimination by elements of the immune system of an infected host, and such ability in turn can depend on the amount or structure of certain virulence factors, i.e., molecules expressed by the microorganism.

As used herein, a pathogenic strain of F. tularensis can include but is not limited to those described herein. In one embodiment the pathogenic strain of F. tularensis is a strain of F. tularensis subsp. tularensis (also termed type A). In one embodiment the pathogenic strain of F. tularensis is F. tularensis subsp. holarctica LVS (hereinafter F. tularensis LVS and, equivalently, Ft. LVS).

A strain that is live is one that can reproduce when placed in suitable conditions. Such strain can include a strain that is actively dividing in vivo or in vitro, as well as a strain that is suspended in its growth, e.g., by lyophilization or freezing. An example of a live attenuated strain of F. tularensis is F. tularensis LVS. This strain has been described and is currently in limited use as a vaccine strain, although it is not avirulent. As noted herein, this strain has been reported to undergo phase variation of its LPS, whereby in one state it expresses a form of LPS that stimulates proinflammatory cytokines and enhanced nitric oxide (NO) production. Although this strain is poorly characterized in terms of its virulence factors, it has been determined that F. tularensis LVS does express a modified O antigen, antigenically distinct from wild-type O antigen. Nonetheless, F. tularensis LVS is highly virulent in mice. In one aspect of the invention the live attenuated strains of the present invention are not highly virulent. The virulence is substantially reduced to a level where the organism is not considered pathogenic by those of ordinary skill in the art. As used herein, avirulent means, for example, that the strain is non-lethal when administered to mice as described in the examples below. In one aspect of the invention, the live attenuated strains of F. tularensis have a reduced expression of wild-type O antigen as compared to pathogenic F. tularensis. As used herein, wild-type O antigen refers to O-antigen polysaccharide of F. tularensis LPS, the polysaccharide having identical tetrasaccharide repeats of the rare sugars 2- acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GaINAcAN). Vinogradov EV et al. (1991) Carbohydr Res 214:289-97; Conlan JW et al. (2002) Vaccine 20:3465-71. The repeating structure of wild-type O antigen can be represented as

[ (1→2) (D-Qui4NFm) (1→4) (D-GaINAcAN) (1→4) (D-GaINAcAN) (1→3) (D-

QuiNAc) (1→2) ]

wherein (1— »2), (1→3), and (l-→4) represent types of glycosidic linkages. Prior JL (2003) J Med Microbiol 52:845-51. At least one monoclonal antibody specific for this antigen is commercially available (MAb2033, Abeam, Inc., Cambridge, MA).

As used herein, reduced expression of wild-type O antigen refers to any phenotype characterized by a reduced amount of wild-type O antigen expressed by F. tularensis. In one embodiment reduced expression of wild-type O antigen refers simply to expression of wild-type O antigen in a reduced amount compared to pathogenic F. tularensis. Without meaning to be bound to a particular mechanism, such embodiment can be viewed essentially as reflective of a quantitative change in expression of wild- type O antigen relative to pathogenic F. tularensis. In one embodiment reduced expression of wild-type O antigen refers to expression of an O antigen other than wild- type O antigen. Without meaning to be bound to a particular mechanism, this latter embodiment can be viewed essentially as reflective of a qualitative change in expression of O antigen relative to pathogenic F. tularensis. For example, in the latter embodiment O antigen expression still could be high, but the wild-type O antigen is no longer expressed.

The reduced expression is measured relative to expression of wild-type O antigen by pathogenic F. tularensis. In one embodiment the reduced expression is less than 50 percent relative to pathogenic F. tularensis. In one embodiment the reduced expression is less than 25 percent relative to pathogenic F. tularensis. In one embodiment the reduced expression is less than 10 percent relative to pathogenic F. tularensis. In one embodiment the reduced expression is less than 5 percent relative to pathogenic F. tularensis. In one embodiment the reduced expression is less than 1 percent relative to pathogenic F. tularensis. In one embodiment the reduced expression is no expression, i.e., 0 percent relative to pathogenic F. tularensis.

Expression of wild-type O antigen can be measured using any suitable method. In one embodiment a preparation of F. tularensis, whole cell lysate thereof, or membrane fraction thereof is contacted with an antibody that binds wild-type O antigen, under conditions that permit binding of the antibody to the wild-type O antigen. The amount of antibody bound by the F. tularensis, whole cell lysate thereof, or membrane fraction thereof is then measured using an assay sensitive to the presence of the antibody, e.g., enzyme-linked immunosorbent assay (ELISA) or immunoblotting. Expression of wild- type O antigen can alternatively or in addition be measured by chemical analysis of exopolysaccharide expressed by F. tularensis. It is believed that wild-type O antigen expressed by pathogenic F. tularensis acts to conceal immunogenic epitopes, thereby promoting evasion by the pathogenic F. tularensis of immune defense mechanisms of a host. Reduction or elimination of wild- type O antigen thus is believed to unmask immunogenic epitopes of F. tularensis, thereby promoting elimination of the pathogenic F. tularensis by immune defense mechanisms of a host.

In one embodiment the reduced expression of wild-type O antigen results from a mutation of a gene affecting 0-antigen biosynthesis. It was recently reported that an O- antigen biosynthesis locus has been identified in F. tularensis. The O-antigen biosynthesis locus occupies approximately 17 kb on the F. tularensis chromosome and contains fifteen tightly linked genes involved in O-antigen biosynthesis. Prior JL et al. (2003) J Med Microbiol 52:845-51; GenBank accession no. AY217763, the entire contents of which are incorporated by reference herein. The fifteen genes are all transcribed in the same direction and are likely organized as an operon. The genes in this biosynthesis gene cluster include at least the following: wbtA, wbtB, wbtC, wbtO, wbiE, wbtF, wzy, wbtG, wbtH, wbtl, wbtJ, wzx, wbtK, wbtL, and wbtM. The wbtA gene is the first gene in this locus. Putative assignment of specific functions in respect of O antigen subunit synthesis have been made for products encoded by at least the following genes: wbtA, wbtB, wbtC, wbtD, wbtE, wbtF, wbtG, wbtH, wbtl, wbtJ, wbtK, wbtL, and wbtM. Prior JL et al. (2003) supra.

As used herein, mutation of a gene affecting O-antigen biosynthesis refers to any mutation of a gene contained within the genome of F. tularensis, which gene can include but is not limited to a gene within the O-antigen biosynthesis locus, wherein the mutation alters O-antigen biosynthesis in a quantitative, qualitative, or both qualitative and qualitative manner, as compared to O-antigen biosynthesis in absence of such mutation. In one embodiment the mutation is in a gene outside of the O-antigen biosynthesis locus. In one embodiment the mutation is a mutation of a gene within the O-antigen biosynthesis locus.

When the mutation is a mutation of a gene within the O-antigen biosynthesis locus, the mutation can be in any one or more genes within the O-antigen biosynthesis locus. Thus in various embodiments when the mutation is a mutation of a gene within the O-antigen biosynthesis locus, the mutation is a mutation of any one or combination of wbtA, wbtB, wbtC, wbtD, wbtE, wbtF, wzy, wbtG, wbtH, wbtl, wbύ, wzx, wbtK, wbtL, and wbtM.

As mentioned above, the wbtA gene is the first gene in the O-antigen biosynthesis locus. The wbtA gene is reported to have a nucleotide sequence disclosed in GenBank accession no. AY217763. WbtA, the protein encoded by the wbtA gene, is reported to have an amino acid sequence provided as GenBank accession no. AAS60264.1.

The WbtA protein belongs to the subfamily of dehydratases that catalyzes the conversion of UDP-glucose to UDP-4-keto 4,6 dideoxy glucose, a key step in the synthesis of the D-QuiNAc sugar of the F. tularensis O-antigen repeat unit. This family of enzymes is encoded by nearly all bacterial polysaccharide biosynthesis loci and is involved in the synthesis of complex nucleotide-linked monosaccharides from less complex sugars. The WbtA protein has similarities to several members of the epimerase/dehydratase family, with closest identities to proteins encoded by the pglF gene of Campylobacter jejuni, the wbpM gene from Pseudomonas spp., and the ^eM gene from Bacillus subtilis. Belanger M et al. (1999) Microbiology 145(Pt 12):3505-21. Of the epimerase/dehydratase subfamily, the WbpM protein of P. aeruginosa is the best characterized. In Pseudomonas, this dehydratase is reported to be highly conserved and essential in all serotypes that contain D-QuiNAc or its derivative in its O-antigen repeat unit. The WbtA protein was also found to be highly conserved among the sequenced type A and type B strains of F. tularensis, consistent with a key role of this dehydratase in virulence of F. tularensis.

In one embodiment the mutation is a mutation of wbtA. Mutations useful according to the invention can be made by insertion, deletion, or substitution of nucleotide sequence in the F. tularensis chromosome such that the mutation reduces or abolishes expression of wild-type O antigen. In one embodiment the mutation includes an insertion into the wbtA. gene.

Since it has been identified that the O antigen is a virulence factor of F. tularensis and having available the complete genomic sequence of F. tularensis, including in particular the O-antigen biosynthesis locus (GenBank accession no. AY217763), mutations can be introduced in a site-directed or shotgun manner, followed by screening and verification on the basis of reduced or absent O-antigen expression compared to control F. tularensis. Mutations useful according to the invention include, in one embodiment, any mutation directed to one or more genes involved in the biosynthesis of O antigen. In one embodiment a site-directed mutation is introduced into any one or more genes in the O-antigen biosynthesis locus. In one embodiment a site-directed mutation is introduced into wbtA. Methods for introducing site-directed mutations, i.e., site-directed mutagenesis, are well known in the art and include homologous recombination.

Deletion mutants can be constructed using any of a number of techniques well known and routinely practiced in the art. In one example, a strategy using counterselectable markers can be employed which has commonly been utilized to delete genes in many bacteria. For a review, see, for example, Reyrat JM et al. (1998) Infect Immun 66:4011-7, incorporated herein by reference. In this technique, a double selection strategy is often employed wherein a plasmid is constructed encoding both a selectable and counterselectable marker, with flanking DNA sequences derived from both sides of the desired deletion. The selectable marker is used to select for bacteria in which the plasmid has integrated into the genome in the appropriate location and manner. The counterselectable marker is used to select for the very small percentage of bacteria that have spontaneously eliminated the integrated plasmid. A fraction of these bacteria will then contain only the desired deletion with no other foreign DNA present. The key to the use of this technique is the availability of a suitable counterselectable marker.

In another technique, the cre-lox system is used for site-specific recombination of DNA. The system consists of 34-base pair lox sequences that are recognized by the bacterial ere recombinase gene. If the lox sites are present in the DNA in an appropriate orientation, DNA flanked by the lox sites will be excised by the ere recombinase, resulting in the deletion of all sequences except for one remaining copy of the lox sequence. Using standard recombination techniques, it is possible to delete the targeted gene of interest in the Francisella genome and to replace it with a selectable marker (e.g., a gene coding for kanamycin resistance) that is flanked by the lox sites. Transient expression (by electroporation of a suicide plasmid containing the ere gene under control of a promoter that functions in Francisella) of the ere recombinase should result in efficient elimination of the /ox-flanked marker. This process would result in a mutant containing the desired deletion mutation and one copy of the lox sequences.

Shotgun mutation involves insertion, deletion, or substitution of nucleotide sequence at random sites within the genome. Such mutations can be made, for example, using a transposon-mediated insertion method routinely practiced by artisans of ordinary skill.

For example, a mini mariner transposon has been constructed and used to randomly mutagenize the Francisella chromosome in F. tularensis LVS (PCT/US06/36910). These studies have led to the identification of a mutant with altered surface polysaccharide expression, the insertion of which was mapped to the wbtA gene of the 0-antigen cluster of Ft. LVS. The Ft. LWS::wbtA mutant was shown to exhibit similar doubling times when compared to the wild type LVS strain in in vitro growth experiments. Remarkably, the Ft. LW S:\wbtA strain is severely attenuated in a murine tularemia model, even at the highest challenge dose administered. Furthermore, this mutant is significantly reduced in its ability to disseminate when compared to the wild type LVS strain. The inability of the mutant strain to cause disease and disseminate correlated with essentially complete abrogation of the surface wild-type polysaccharide expression in the Ft. LVS::w6tA strain.

Although mutation of F. tularensis LVS is useful according to the invention, mutation of other strains of F. tularensis, including in particular highly virulent strains of F. tularensis, are contemplated by the invention. In one embodiment the strain is type A F. tularensis. In one embodiment the strain is type B F. tularensis. As used herein, an effective amount refers to an amount effective to induce a desired biological effect. In one embodiment an effective amount refers to an amount of a GC of the invention, either alone or in conjunction with a live attenuated avirulent strain of F. tularensis, effective to induce an immune response to the GC and to a pathogenic strain of F. tularensis. In one embodiment an effective amount refers to an amount of a live attenuated avirulent strain of F. tularensis, either alone or in conjunction with a GC of the invention, effective to induce an immune response to the live attenuated avirulent strain of F. tularensis and to a pathogenic strain of F. tularensis. In one embodiment an effective amount refers to an amount effective to induce an immune response to a live attenuated strain of F. tularensis of the invention in a mammal following exposure to or administration of F. tularensis to the mammal. In one embodiment the effective amount is an amount that is effective to induce protective immunity against typical exposure to a pathogenic strain of F. tularensis. As used herein, typical exposure to a pathogenic strain of F. tularensis refers to exposure by aerosol exposure (e.g., inhalation) or by intradermal or transcutaneous introduction of a clinically relevant inoculum of pathogenic F. tularensis. As used herein, protective immunity refers to an immune response effective to prevent or ameliorate signs or symptoms of disease caused by F. tularensis, including dissemination of F. tularensis in tissues of the reticuloendothelial system. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of a mammal to a vaccine composition may be evaluated., e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, lymphadenopathy, fever, skin ulceration, respiration, physical condition and overall health and performance of the subject.

In one embodiment the effective amount of the microbial antigen:polysaccharide chemical conjugate is at least 0.8 μg. In one embodiment the effective amount of the microbial antigen:polysaccharide chemical conjugate is at least 8.0 μg. In one embodiment the effective amount of the microbial antigen:polysaccharide chemical conjugate is at least 80 μg. In one embodiment the effective amount of the microbial antigen:polysaccharide chemical conjugate is at least 800 μg. In one embodiment the effective amount of the microbial antigen:polysaccharide chemical conjugate is at least 8000 μg.

In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10³ colony forming units (cfu). In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁴ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁵ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁶ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁷ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁸ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10⁹ cfu. In one embodiment the effective amount of the live attenuated avirulent strain of F. tularensis is at least 10¹⁰ cfu.

As used herein, a pharmaceutically acceptable carrier refers to any suitable nontoxic liquid, semisolid, or solid diluent or encapsulating substance that is compatible with the active ingredient and suitable for administration to a human or other mammal. The particular choice of carrier can vary depending on the intended route of administration or formulation. In one embodiment the carrier is sterile. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions of the invention also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable liquid carriers can include, without limitation, physiologic saline, water, and phosphate buffered saline. In one aspect the invention is a method of vaccinating a mammal against a pathogenic F. tularensis bacteria, comprising administering to the mammal an effective amount of a microbial antigen:polysaccharide chemical conjugate and an effective amount of a live attenuated strain of F. tularensis bacteria, wherein the live attenuated strain has reduced expression of wild-type O-antigen as compared to pathogenic F. tularensis. The administration can be carried out simultaneously and/or by the same route of administration. Alternatively the glycocoηjugate can be administered before or after the administration of the a live attenuated strain of Francisella tularensis bacteria, and/or by a different route of administration. As used herein, administering refers to any suitable method of dispensing and delivering a treatment agent to the body of a subject. Suitable routes of administration include but are not limited to intradermal, intranasal, inhalation, intramuscular, intravenous, subcutaneous, mucosal, and enteral. Inhalation specifically includes aerosol administration to lung. Intranasal administration includes liquid (e.g., nose drops) and aerosol to nasal mucosa. Enteral specifically includes but is not limited to oral. As used herein, a mammal refers to a human or a non-human mammal. Formulations for clinical use include the microbial antigen:polysaccharide chemical conjugate, the live attenuated avirulent strain of F. tularensis, alone or in combination with another agent. The other agent in one embodiment is an agent that enhances the immune response to the combination formulation. In one embodiment the other agent is an adjuvant. As used herein, an adjuvant is an agent that stimulates the innate immune system, i.e., stimulates the immune system in a non-specific manner. Adjuvants enhance T-cell activation by promoting the accumulation and activation of other leukocytes at a site of antigen exposure. Adjuvants enhance accessory cell expression of T-cell-activating costimulators and cytokines.

In one embodiment the adjuvant is a cytokine. In one embodiment the adjuvant is interleukin 12 (IL-12). In one embodiment the adjuvant is cholera toxin subunit. In one embodiment the adjuvant is QS21. In one embodiment the adjuvant is an immunostimulatory CpG oligonucleotide. In one embodiment the adjuvant is complete Freund's adjuvant. In one embodiment the adjuvant is incomplete Freund's adjuvant.

Formulations for clinical use can also include combination with another vaccine antigen. The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally adjuvants and other therapeutic ingredients. For use in therapy, an effective amount of the microbial antigen:polysaccharide chemical conjugate and live attenuated avirulent strain of F. tularensis can be administered to a subject by any mode that delivers the microbial antigenipolysaccharide chemical conjugate and live attenuated avirulent strain of F. tularensis to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intranasal, subcutaneous, intradermal, oral, parenteral, intramuscular, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. Preferred routes are by injection or by inhalation. Vaccine compositions are well known in the pharmaceutical arts. For oral administration, the compounds (i.e., glycoconjugates, live attenuated avirulent strain of F. tularensis and optionally other therapeutic agents) can be formulated readily by combining the glycoconjugates, the live attenuated avirulent strain of F. tularensis with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Nasal (i.e., intranasal) delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the nasal mucosa, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. The compounds (i.e., glycoconjugates, live attenuated avirulent strain of F. tularensis and other therapeutic agents), when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the vaccine in water-soluble form. Additionally, suspensions may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004- 0.02% w/v).

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entirety.

EXAMPLES The following Examples describe the methods for preparation and use of glycocoηjugates and combination vaccines comprising glycoconjugates for the prevention of tularemia.

Example 1 Preparation of antigen: polysaccharide chemical conjugates

A purified glucan-free LPS obtained by differential ultracentrifugation of the dialyzed phenol phase of a hot 50% aqueous phenol extraction of F. tularensis LVS (ATCC29864) strain cells, on hydrolysis with 6% (v/v) acetic acid (100 °C, 2 h) afforded an insoluble Lipid A (removed by low speed centrifugation) and a water soluble product containing essentially O-polysaccharide (O-PS). Superdex 200 size exclusion column chromatography (2.5 cm x 80 cm) of the O-PS preparation using a 0.05M aqueous pyridium acetate buffer (pH 4.6) and collection of the major component (K_av 0.02-0.04) eluting close to the void volume of the system gave on lyophilization, the purified O-PS antigen (64% yield from LPS). The O-PS had [α]_D +217° (c 0.2, water) and gave a ¹H-¹³C NMR correlation spectrum consistent with the O-PS being a polymer of a repeating tetrasaccharide units having the structure: 4-(α-D-Gal/?NAcAN-(l-4)-α-D- GaIpNAcAN-(I -3)-β-D-Qui/?NAc-(l-2)-β-D-Qui/?4NFo-l), and was free from glucan, protein and nucleic acid. A proton NMR analysis of the purified Ft. LVS O-PS is shown in FIG. l(A). A single tetrasaccharide repeat unit (QuiN4Fm, 4,6-dideoxy-4- formamido-D-glucose; GaINAcAN, 2-acetamido-2-deoxy-D-galacturonamide; QuiNAc, 2-acetamido-2,6-dideoxy-D-glucose) of FΛLVS O-PS with sugar residues and glycosidic linkages is indicated. O-PS (70 mg) dissolved in water (13 ml) at ice bath temperature was adjusted to pH 10.5-11.0 with 0.1M NaOH and, following the addition of cyanogen bromide (70 mg) in acetonitrile (0.1 ml), the solution was kept at 0 ⁰C (2 min) and then treated with a solution of adipic acid dihydrazide (70 mg) in 0.5M NaHCO₃ (1 ml), and the pH was adjusted to 8.5. The mixture was kept for 12 h at 4 ⁰C and, following dialysis against distilled water, the retentate was lyophilized to yield the activated O-PS. Tetanus toxoid (70 mg) was dissolved in a solution of the activated O-PS (70 mg) in saline (13 ml) and following adjustment to pH 5.1-5.5 with 0.1 M HCl, the solution was treated with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (0.2 g), and stirring on ice at pH 5.5 was continued for 5 h. The reaction mixture was dialyzed against changes of 0.2 M NaCl over 40 h to remove low molecular mass materials and the retentate, after concentration through a 10,000 MW cutoff filter, was fractionated by Biogel A column chromatography (90 cm x 2.5 cm) using a saline eluant and the fraction (/-av 0.08-0.27), eluting before the tetanus toxoid eluting region (K_m 0.30-0.38), which tested positive for protein and carbohydrate, was collected, concentrated by the above described concentration system to ~5 ml and was stored at -20 ⁰C. The conjugate from analysis was further characterized as shown in FIG. 1 (B-D; Silver stain (B), Immunoblot analysis using anti-TT polyclonal serum (Q and anti-.FV.LVS polyclonal serum (D)). Silver stain demonstrated the presence of TT protein in the tetanus toxoid lane and in the GC fraction. Immunoblot analyses using anti-TT (panel C) and anti-

F/.LVS (panel D) polyclonal serums demonstrated the presence of both TT and the O-PS in the GC fraction providing confirmation for the TT: O-PS glycoconjugate.

Example 2 Immunization with TT: O-PS conjugate protects against a lethal intradermal challenge of Ft. LVS

Immunization and challenge regimen (FIG. 2(A)). Six-week-old BALB/c mice were immunized subcutaneously twice three weeks apart with the glycoconjugate (GC). Complete and incomplete Freund's served as adjuvant during the first and second immunizations respectively. All mice were challenged intradermally after a 4-week rest period with a lethal intradermal dose (15-fold i.d. LD₅₀) of Ft. LVS. Three mice were sacrificed at day 3 post challenge and bacterial load in the reticuloendothelial tissue determined (data not shown).

Protection of BALB/c mice following immunization with TT:O-PS vaccine (FIG. 2(B)). Survival of mice was monitored over a 21 -day period post challenge after which they were humanely sacrificed. Complete protection was observed when mice were immunized with TTO-PS vaccine. GC-immunized mice were protected completely when challenged with a 15-fold i.d. LDs₀.

Example 3 Ft.L\S::wbtA intranasal immunization protects mice against a subsequent lethal intranasal challenge of the parent LVS strain

Intranasal immunization and challenge regimen (FIG. 3(A)). Six-week-old BALB/c mice were immunized intranasally twice two weeks apart with Ft.LVSy.wbtA mutant strain (10⁷ cfu/ mouse). All mice were challenged intranasally after a 4- week rest period with either 20, 200 or 2000-fold i.n. LD₅₀ of Ft. LVS strain.

Protection of BALB/c mice following immunization with Ft.LVSy.wbtA mutant strain. Survival of mice was monitored over a 21 -day period post challenge after which they were humanely sacrificed. Complete protection of the Ft.LVSy.wbtA immunized mice was observed when challenged with 20-fold the i.n. LD₅₀. A delay in time to death was observed when a 200-fold challenge was administered while no significant delay in time to death was observed when the mutant-immunized mice were challenged with 2000-fold the i.n. LD₅oof Ft. LVS when compared to the control mice, as shown in FIG. 3(B).

Example 4

TT:O-PS glycocon jugate immunization protects mice against a subsequent low but ordinarily lethal intranasal challenge of parent LVS strain

Glycoconjugate (GC) immunization and challenge regimen (FIG. 4(A)). Six- week-old BALB/c mice were immunized subcutaneously twice two weeks apart with the GC. Complete Freund's was used as adjuvant in first immunization, while incomplete Freund's served as an adjuvant during the second immunization. AU mice were challenged intranasally after a 4-week rest period with either 20, 200 or 2000-fold i.n. LD₅₀ of Ft. LVS strain.

Protection of BALB/c mice following immunization with GC. Survival of mice was monitored over a 21 -day period post challenge after which the survivors were humanely sacrificed (FIG. 4(B)). A significant delay in the mean time to death (mtd) was observed when GC-immunized mice were challenged with 20-fold the i.n. LD₅₀. No significant delay in mtd was observed when the GC-immunized mice were challenged with a 200- or 2000-fold i.n. LD₅₀ of Ft. LVS when compared to the respective control mice groups. GC-immunized mice only demonstrated an increased mtd when challenged intranasally with 20-fold the i.n. LD₅₀ in contrast to earlier studies where complete protection was observed when GC-immunized mice were challenged with a 15 -fold LD₅₀ via the intradermal route.

Example 5

Enhanced protection against a lethal F/.LVS following a combination vaccination regimen

For combination regimen, six-week-old BALB/c mice were immunized intranasally twice two weeks apart with Ft.LVS::wbtA mutant strain (10⁷ cfu/ mouse). These mice were also simultaneously immunized subcutaneously twice two weeks apart with the GC. Complete Freund's and incomplete Freund's served as adjuvants during first and second immunizations, respectively. All mice were challenged intranasally after a 4-week rest period with either 30, 300 or 3000-fold i.n. LD₅₀ of Ft. LVS strain. (A) Immunization schedule of combination vaccine regimen (FIG. 5(A)): GC immunization (subcutaneous) and Ft.LVSv.wbtA immunization (intranasal).

(B) Survival of BALB/c mice following combination vaccine regimen. Survival was monitored over a 21 -day period post challenge after which the survivors were humanely sacrificed (FIG. 5(B)). Notably complete protection was observed when immunized mice were challenged with either a 30- or 300-fold i.n. LD₅₀. However, no significant delay in time to death was observed when immunized were challenged with 3000-fold the i.n. LD₅₀ of Ft. LVS when compared to the control mice. A 300-fold protection was observed when a combination regimen was used which is approximately 15-fold better protection in comparison to immunization with either the GC or the Ft. LVS::wføA mutant strain alone when using similar immunization schedule.

Example 6 ELIspot analysis to determine the level of IFN-γ in GC, Ff. L VS: '.wbtA and combination regimen immunized mice

In this assay T-cell production of IFN-γ in response to immunization was measured (FIG. 6). Briefly, CD3+ T cells were isolated from GC, Ft.LVS::wbtA and combination regimen immunized mice and are co-cultured with naϊve APCs in the presence of media (negative control), ConA (positive control), heat-killed Ft. LVS, heat- killed Ft. LV S::wbt A strain, tetanus, and LVS O-PS, respectively, for 16 hours. The number of T cells producing IFN-γ/2.5 x 10⁵ cells was assessed by ELIspot analysis.

As expected a high level of IFN-γ was observed in the ConA group, which served as a positive control for this assay. An increase in the level of IFN-γ was also observed when CD3+ T cells from the Ft.LVS::wbtA immunized group was co-cultured with naive APCs in the presence of either heat-killed Ft. LWS and heat-killed Ft.LVSwwbtA strain when compared to the media control. Notably a significantly increased level of IFN-γ production was documented when CD3+ T cells from the combination immunized mice was used in this assay. The combination immunization schedule demonstrated a 15-fold better protection (FIG. 5) over the immunization with either GC (FIG. 4) or Ft.LVSv.wbtA (FIG. 3) alone. ELIspot analysis as shown here demonstrated a heightened level of IFN-γ production following a combination immunization regimen.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

We claim:

Claims

1. A microbial or pathogen-specific antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide.

2. The conjugate of claim 1, wherein the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?N Ac AN-(I -4)-α-D- GalpNAcAN-(l-3)-β-D-Qui/7NAc-(l-2)-β-D-Quψ4NFo-l); 4-(α-D-Gal/?NAcAN-(l-3)- β-D-Qui/?NAc-(l -2)-β-D-Qui/?4NFo-(l -4)-α-D-GφNAcAN-l ); 3-(β-D-Qui/?NAc-(l -2)- β-D-Qui/4NFo-(l-4)-α-D-GφNAcAN-(l-4)-α-D-GφNAcAN-l); and 2-(β-D- Qui/?4NFo-( 1 -4)-α-D-GalpNAcAN-( 1 -4)-α-D-GalpNAc AN-(I -3)-β-D-QuipNAc- 1 ).

3. The conjugate of claim 2, wherein the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-GalpNAcAN-(l-4)-α-D-Gal/?NAcAN- (l-3)-β-D-QuψNAc-(l-2)-β-D-Quip4NFo-l); 4-(α-D-GalpNAcAN-(l-3)-β-D-QuψNAc- (l-2)-β-D-Qui^4NFo-(l-4)-α-D-Gal/?NAcAN-l); 3-(β-D-QuipNAc-(l-2)-β-D- Quip4NFo-( 1 -4)-α-D-GalpN Ac AN-( 1 -4)-α-D-GφNAc AN- 1 ); and 2-(β-D-Quip4NFo- ( 1 -4)-α-D-GalpNAcAN-(l -4)-α-D-GφNAc AN-( 1 -3)-β-D-Qui/?NAc- 1 ).

4. The conjugate of claim 1, wherein the antigen is a pathogen-specific antigen.

5. The conjugate of claim 4, wherein the pathogen-specific antigen is tetanus toxoid.

6. A vaccine composition comprising an effective amount of the chemical conjugate of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. The vaccine composition of claim 6, wherein the chemical conjugate is tetanus toxoid:4-(α-D-Galj!?NAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-Qui^NAc-(l-2)-β-D- Qui/?4NFo-l).

8. The vaccine composition of claim 6, further comprising an effective amount of a live attenuated avirulent strain of Francisella tularensis.

9. The vaccine composition of claim 8, wherein the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis.

10. The vaccine composition of claim 9, wherein the reduced expression of wild-type O antigen is no expression of wild-type O antigen.

11. The vaccine composition of claim 9, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units.

12. The vaccine composition of claim 9, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10 to 10 colony forming units.

13. The vaccine composition of claim 9, wherein the reduced expression results from a mutation of a gene affecting O-antigen biosynthesis.

14. The vaccine composition of claim 13, wherein the mutation is a mutation of a gene within an O-antigen biosynthesis locus.

15. The vaccine composition of claim 14, wherein the mutation is a mutation of wbtA.

16. A vaccine composition comprising an effective amount of antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide, an effective amount of a live attenuated avirulent strain of Francisella tularensis, and a pharmaceutically acceptable carrier.

17. The vaccine composition of claim 16, wherein the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?NAc AN-(I- 4)-α-D-Gal/?NAcAN-(l-3)-β-D-Qui/?NAc-(l-2)-β-D-Quip4NFo-l); 4-(α-D-

GalpNAcAN-(l-3)-β-D-Qui^NAc-(l-2)-β-D-Quip4NFo-(l-4)-α-D-GalpNAcAN-l); 3- (β-D-QuipNAc-(l -2)-β-D-Qui/?4NFo-(l -4)-α-D-GalpNAc AN-(I -4)-α-D-Gal/?NAcAN- l); and 2-(β-D-Qui/?4NFo-(l-4)-α-D-GφNAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D- QuipNAc-1).

18. The vaccine composition of claim 17, wherein the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-GalpNAc AN-(I -4)-α-D-

Gal/?NAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quip4NFo-l); 4-(α-D-Gal^NAcAN-(l-3)- β-D-QuψN Ac-( 1 -2)-β-D-Quip4NFo-( 1 -4)-α-D-Gal/?N Ac AN- 1 ); 3 -(β-D-Qui/?N Ac-( 1 -2)- β-D-Quψ4NFo-(l-4)-α-D-Gal/?NAcAN-(l-4)-α-D-Gal^NAcAN-l); and 2-(β-D- Qui/?4NFo-( 1 -4)-α-D-Gal/?NAc AN-( 1 -4)-α-D-Gal/?NAc AN-( 1 -3)-β-D-QuipNAc- 1 ).

19. The vaccine composition of claim 16, wherein the antigen is bovine serum albumin or human serum albumin.

20. The vaccine composition of claim 16, wherein the antigen is human serum albumin.

21. The vaccine composition of claim 16, wherein the antigen is tetanus toxoid.

22. The vaccine composition of claim 16, wherein the chemical conjugate is bovine serum albumin:4-(α-D-Gal^NAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuψNAc-(l-2)- β-D-Qui^4NFo-l).

23. The vaccine composition of claim 16, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units.

24. The vaccine composition of claim 16, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units.

25. The vaccine composition of claim 16, wherein the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis.

26. The vaccine composition of claim 25, wherein the reduced expression of wild- type O antigen is no expression of wild-type O antigen.

27. The vaccine composition of claim 25, wherein the reduced expression results from a mutation of a gene affecting O-antigen biosynthesis.

28. The vaccine composition of claim 27, wherein the mutation is a mutation of a gene within an O-antigen biosynthesis locus.

29. The vaccine composition of claim 28, wherein the mutation is a mutation of wbtA.

30. A method of vaccinating a mammal against a pathogenic strain of Francisella tularensis, comprising administering to the mammal an effective amount of a microbial or pathogen-specific antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide.

31. The method of claim 30, wherein the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-Gal/?NAcAN-(l-4)-α-D- GφN Ac AN-( 1 -3)-β-D-Qui/?NAc-( 1 -2)-β-D-Qui/?4NFo- 1 ); 4-(α-D-GalpN Ac AN-( 1 -3)- β-D-QuipN Ac-( 1 -2)-β-D-Qui/?4NFo-( 1 -4)-α-D-Gal/?NAc AN- 1 ); 3 -(β-D-Qui/?NAc-( 1 -2)- β-D-Qui/?4NFo-( 1 -4)-α-D-Gal/?N Ac AN-( 1 -4)-α-D-Gal/?NAc AN- 1 ); and 2-(β-D- Quip4NFo-( 1 -4)-α-D-Gal/?NAcAN-( 1 -4)-α-D-Gal/?NAc AN-( 1 -3)-β-D-Qui/?NAc- 1 ).

32. The method of claim 31 , wherein the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-((X-D-GaIpNAc AN-(I -4)-α-D-Gal/?N Ac AN- (l-3)-β-D-Qui/?NAc-(l-2)-β-D-Quip4NFo-l); 4-(α-D-GalpNAcAN-(l-3)-β-D-QuipNAc- (l-2)-β-D-Qui^4NFo-(l-4)-α-D-GalpNAcAN-l); 3-(β-D-QuipNAc-(l-2)-β-D- Qui^4NFo-(l-4)-α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN-l); and 2-(β-D-Quip4NFo- ( 1 -4)-α-D-GalpNAcAN-( 1 -4)-α-D-Gal/?NAc AN-( 1 -3)-β-D-Qui/?NAc- 1 ).

33. The method of claim 30, wherein the antigen is a pathogen-specific antigen.

34. The method of claim 33, wherein the pathogen-specific antigen is tetanus toxoid.

35. The method of claim 30, wherein the chemical conjugate is tetanus toxoid:4-(α- D-GalpNAcAN-(l-4)-o-D-GalpNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quip4NFo-l).

5

36. The method of any one of claims 30-34, further comprising administering to the mammal an effective amount of a live attenuated avirulent strain of Francisella tularensis.

W 37. The method of claim 36, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units.

38. The method of claim 36, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units.

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39. The method of claim 36, wherein the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis.

40. The method of claim 39, wherein the reduced expression of wild-type O antigen 20 is no expression of wild-type O antigen.

41. The method of claim 39, wherein the reduced expression results from a mutation of a gene affecting O-antigen biosynthesis.

25 42. The method of claim 41 , wherein the mutation is a mutation of a gene within an O-antigen biosynthesis locus.

43. The method of claim 42, wherein the mutation is a mutation of wbtA.

30 44. A method of vaccinating a mammal against a pathogenic strain of Francisella tularensis, comprising administering to the mammal an effective amount of an antigen:polysaccharide chemical conjugate, wherein the polysaccharide is Francisella tularensis O-antigen polysaccharide; and administering to the mammal an effective amount of a live attenuated avirulent strain of Francisella tularensis.

45. The method of claim 44, wherein the O-antigen polysaccharide comprises at least one tetrasaccharide repeat unit selected from: 4-(α-D-GalpNAcAN-(l-4)-α-D- GφNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D-Quip4NFo-l); 4-(α-D-GφNAcAN-(l-3)- β-D-Qui/?NAc-(l-2)-β-D-Qui/?4NFo-(l-4)-α-D-GalpNAcAN-l); 3-(β-D-Qui^NAc-(l-2)- β-D-Qui/?4NFo-( 1 -4)-α-D-Gal/?NAc AN-( 1 -4)-α-D-Gal/?NAcAN- 1 ); and 2-(β-D- Qui/?4NFo-( 1 -4)-o-D-GφNAcAN-( 1 -4)-α-D-GalpNAc AN-(I -3)-β-D-Qui/?NAc- 1 ).

46. The method of claim 45, wherein the O-antigen polysaccharide is a single tetrasaccharide repeat unit selected from: 4-(α-D-GalpNAcAN-(l-4)-α-D-GalpNAcAN- (l-3)-β-D-QuipNAc-(l-2)-β-D-Qui/?4NFo-l); 4-(α-D-GalpNAcAN-(l-3)-β-D-Qui/7NAc- (l-2)-β-D-Quip4NFo-(l-4)-α-D-GalpNAcAN-l); 3-(β-D-QuψNAc-(l-2)-β-D- Quip4NFo-( 1 -4)-α-D-GalpN Ac AN-( 1 -4)-α-D-Gal/?N Ac AN- 1 ); and 2-(β-D-Qui/?4NFo- ( 1 -4)-α-D-Gal/?NAcAN-( 1 -4)-α-D-Gal/?NAcAN-( 1 -3)-β-D-Qui/?NAc- 1 ).

Al. The method of claim 44, wherein the antigen is bovine serum albumin or human serum albumin.

48. The method of claim 44, wherein the chemical conjugate is bovine serum albumin:4-(α-D-Gak7NAcAN-(l-4)-α-D-GalpNAcAN-(l-3)-β-D-QuipNAc-(l-2)-β-D- Quip4NFo-l).

49. The method of claim 44, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is at least 10⁴ colony forming units.

50. The method of claim 44, wherein the effective amount of the live attenuated avirulent strain of Francisella tularensis is 10⁴ to 10⁸ colony forming units.

51. The method of claim 44, wherein the live attenuated avirulent strain has reduced expression of wild-type O antigen as compared to pathogenic Francisella tularensis.

52. The method of claim 51, wherein the reduced expression of wild-type O antigen is no expression of wild-type O antigen.

53. The method of claim 51 , wherein the reduced expression results from a mutation of a gene affecting O-antigen biosynthesis.

54. The method of claim 53, wherein the mutation is a mutation of a gene within an O-antigen biosynthesis locus.

55. The method of claim 54, wherein the mutation is a mutation of wbtA.

56. The method of claim 30 or claim 44, wherein the administering of the chemical conjugate comprises administering subcutaneously.

57. The method of claim 30 or claim 44, wherein the administering of the chemical conjugate comprises administering intranasally.

58. The method of claim 30 or claim 44, wherein the administering of the chemical conjugate comprises administering intradermally.

59. The method of claim 36 or claim 44, wherein the administering of the live attenuated avirulent strain of Francisella tularensis comprises administering intranasally.

60. The method of claim 36 or claim 44, wherein the administering of the live attenuated avirulent strain of Francisella tularensis comprises administering subcutaneously.

61. The method of claim 36 or claim 44, wherein the administering of the live attenuated avirulent strain of Francisella tularensis comprises administering intradermally.

62. The method of claim 36 or claim 44, wherein the administering the chemical conjugate and the administering the live attenuated avirulent strain of Francisella tularensis are simultaneous.

63. The method of claim 36 or claim 44, wherein the administering the chemical conjugate and the administering the live attenuated avirulent strain of Francisella tularensis are by the same mode of administration.

64. The method of claim 36 or claim 44, wherein the administering the chemical conjugate and the administering the live attenuated avirulent strain of Francisella tularensis are not simultaneous.

65. The method of claim 36 or claim 44, wherein the administering the chemical conjugate and the administering the live attenuated avirulent strain of Francisella tularensis are not by the same mode of administration.

66. The method of any one of claims 30-34 and 44-55, wherein the mammal is a human.

67. A tetanus toxoid:4-(α-D-GalpNAcAN-(l -4)-α-D-Gal/?N Ac AN-(I -3)-β-D- QuipNAc-(l-2)-β-D-Quip4NFo-l) chemical conjugate.