Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease

Abstract

A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM₁₉₇ lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM₁₉₇ lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections.

Importance: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM₁₉₇ carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Keywords: Escherichia coli; K1 capsule; O-antigen; glycoconjugate vaccine.

Fig 1

Genotypic characterization of 41 invasive serotype O1 Antimicrobial Testing Leadership and Surveillance (ATLAS) BSI isolates. (A) Sequence type (ST) distribution, (B) phylogenomic analysis of the E. coli O1 blood isolates. The phylogenetic tree of the O1 isolates was constructed using the average nucleotide identity calculated from whole genome sequence alignment using the Neighbor-Joining method. Each branch represents an isolate. The ST of each phylogenetic group is labeled on each branch. The O25b:K5 ST131 isolate PFEEC0066 is included as an outgroup control. ST59 isolate PFEEC0007 is a UTI isolate used for long-chain O-antigen production (see Materials and Methods). Asterisks highlight isolates from which the serotype O1a O-antigen structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy (Fig. 2; Fig. S2 to S4). Blue boxes highlight isolates investigated for their susceptibility to immune serum in vitro and/or for their virulence in vivo.

Fig 2

Structural analysis of serotype O1 O-antigens purified from three BSI isolates. (A) Structure of the E. coli O1a polysaccharide antigen repeating unit consisting of five monosaccharide units. (B) The chemical difference plot between O1a long-chain (LC) polysaccharide purified from strain PFEEC0007 (ST59) wzzB/fepE and reference values previously reported (28) reveals a few miss-assignments of NMR signals. (C) ¹H-NMR spectra of E. coli O-antigens extracted from blood isolates PFEEC0074 (ST38) (brown), PFEEC0497 (ST95) (green), and PFEEC0007 (ST59) wzzB/fepE (blue). The left panel shows the anomeric and ring resonances and the right panel shows the methyl resonances. Some resonances are annotated.

Fig 3

E. coli O1:K1 strains PFEEC0085 (A) and PFEEC0435 (B) are susceptible to OPA killing by rabbit O1a glycoconjugate immune serum in the presence of HL60 effector cells and baby rabbit complement. The activity of immune serum samples after three 10 µg antigen doses with 50 µg AlPO₄ adjuvant (week 15 timepoint) was compared to preimmune control sera (week 0 timepoint). T0, input CFUs; BG, background CFUs from no-serum control incubation. 50% BG, half maximal growth inhibition CFU value for determining EC₅₀ titers. Plotted data are the average of duplicate titrations with StDev error bars. Optimized OPAs developed for strains PFEEC0085 and PFEEC0435 required bacteria harvested at a mid-log phase in Todd-Hewitt broth (THB) and Dulbecco’s modified Eagle’s medium (DMEM) media, respectively. Despite comparable input CFUs, final CFUs were higher for strain PFEEC0085 due to growth during the assay incubation period.

Fig 4

Distinct virulence properties of two ST95 O1:K1:H7 isolates. (A and B) Susceptibility of strains PFEEC0085 and PFEEC0435 and derived K1 mutants to i.p. challenge. Each symbol represents a group of 10 mice challenged with the indicated inoculum of bacteria (CFU/animal).

Fig 5

Immunogenicity and efficacy of the O1a CRM₁₉₇ glycoconjugate. (A) Dosing and challenge schedule (20 mice per group). (B and C) Antigen-specific IgG titers and OPA titers were generated with strain PFEE0085 at week 7 (PD2) and week 15 (PD3) time points. Dotted lines represent GMT titers of mouse naïve serum baseline controls. Corresponding IgG and OPA GMT values are summarized in Table S2. (D) Survival of mice (n = 10) following lethal challenge at week 16 with strains PFEEC0085 and PFEEC0089 after three vaccine doses. Asterisks indicate P values between bracketed groups. Statistical survival differences were determined using a log-rank test (Mantel-Cox). ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.