The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

glycan, glycoconjugate vaccine, O-antigen polysaccharide, anti-glycan antibodies, humoral immune response, outer membrane vesicle

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,4,4	Fo
3,4,4	bDQuip4N	106.6	76.6	75.9	58.1	73.3	19.5
3,4,2	Ac
3,4,6	NH2
3,4	aDGalpNA	101.5	52.3	70.5	81.3	73.4	?
3,2	Ac
3,6	NH2
3	aDGalpNA	101.0	52.3	68.8	78.0	73.1	?
2	Ac
	bDGlcpN	?	57.1	82.8	67.4	78.5	63.1


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,4,4	Fo
3,4,4	bDQuip4N	4.50	3.36	3.50	3.62	3.48	1.18
3,4,2	Ac
3,4,6	NH2
3,4	aDGalpNA	5.03	4.26	4.11	4.47	4.85	-
3,2	Ac
3,6	NH2
3	aDGalpNA	5.41	4.24	4.00	4.43	4.25	-
2	Ac
	bDGlcpN	4.57	3.75	3.72	3.63	3.43	3.74-3.90


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,4,4	Fo
3,4,4	bDQuip4N	106.6/4.50	76.6/3.36	75.9/3.50	58.1/3.62	73.3/3.48	19.5/1.18
3,4,2	Ac
3,4,6	NH2
3,4	aDGalpNA	101.5/5.03	52.3/4.26	70.5/4.11	81.3/4.47	73.4/4.85	
3,2	Ac
3,6	NH2
3	aDGalpNA	101.0/5.41	52.3/4.24	68.8/4.00	78.0/4.43	73.1/4.25	
2	Ac
	bDGlcpN	?/4.57	57.1/3.75	82.8/3.72	67.4/3.63	78.5/3.43	63.1/3.74-3.90

The spectrum also has 3 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@@H](O[C@H]2[C@@H](C(N)=O)O[C@H](O[C@H]3[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]3NC(C)=O)[C@H](NC(C)=O)[C@H]2O)O[C@H](C(N)=O)[C@H](O[C@@H]2O[C@H](C)[C@@H](NC=O)[C@H](O)[C@H]2O)[C@@H]1O

826.763 g/mol (C₃₁H₅₀N₆O₂₀)