A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice

Abstract

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

Keywords: bacterial protein expression; cell-free (CF) protein synthesis; conjugate vaccine; oligosaccharyl transferase; protein glycan coupling technology; protein glycosylation; synthetic glycobiology; vaccine carrier protein.

FIGURE 1

Biosynthesis of glycoconjugate vaccine candidates against ETEC bacteria. (A) Biosynthesis of ETEC O148 O-PS from plasmid pMW07-O148, which encodes the entire O-PS locus from ETEC strain B7A (serotype O148:H28) between galF and gnd. (B) Glycoconjugate biosynthesis is enabled by combining engineered O-antigen biosynthesis with N-linked protein glycosylation by C. jejuni PglB (CjPglB). Several plasmid-encoded glycosyltransferases sequentially add the repeat-unit sugars to the lipid carrier undecaprenol pyrophosphate in the cytoplasmic membrane. The lipid-linked oligosaccharide is flipped by Wzx and polymerized by the Wzy polymerase, and subsequently transferred to “DQNAT” acceptor sites in the carrier protein by PglB. Deletion of waaL in the host strain is used to eliminate transfer of the O-PS to lipid A-core and ensure efficient transfer to the acceptor protein, while deletion of lpxM results in pentaacylated lipid A structure having significantly reduced toxicity. (C) Immunoblot analysis of purified carrier proteins derived from Escherichia coli CLM24 cells carrying a plasmid encoding either CRM₁₉₇ ^4xDQNAT or PD^4xDQNAT along with plasmid pMW07-O148 encoding the ETEC O148 O-PS biosynthetic pathway and plasmid pMAF10 encoding wild-type CjPglB (wt) or an inactive mutant of CjPglB (mut) as indicated. Blots were probed with anti-His antibody to detect acceptor proteins and anti-ETEC O148 antibody to detect O-PS. Images depict aglycosylated and multiply glycosylated forms of CRM₁₉₇ ^4xDQNAT or PD^4xDQNAT. Molecular weight (M _W) markers are indicated on the left. Results are representative of three biological replicates.

FIGURE 2

Glycoconjugates are immunogenic. (A) Schematic of the prime-boost immunization schedule. Mice received an initial injection on day 0 and identically formulated booster injections on days 21 and 42. Blood was drawn on days 0, 35, 49, and at study termination on day 56. (B) O-PS-specific IgG titers in day 56 serum of individual mice (black circles) and mean titers of each group (red lines) as determined by ELISA with LPS derived from ETEC strain B7A (serotype O148:H28) as immobilized antigen. Groups of eight BALB/c mice were immunized s. c. with 100 μL PBS alone or PBS containing 50 μg of either aglycosylated PD carrier protein or glycosylated ETEC O148 conjugate (PD-O148) adjuvanted with aluminium phosphate adjuvant. Mice were boosted on days 21 and 42 with identical doses of each immunogen. Data were analyzed for statistical significance using the Mann-Whitney test (*p < 0.05; **p < 0.01; ns, not significant). (C) Bactericidal killing activity of antibodies in the serum of mice immunized with PBS, unmodified PD carrier protein, or ETEC O148 conjugate (PD-O148). Survival data were derived from a standard SBA where dilutions of serum from immunized mice were tested against ETEC strain B7A (serotype O148:H28) in the presence of human complement. SBA curves were generated with pooled sera from each group. Data are the mean of three biological replicates ±SEM.

FIGURE 3

Glycoconjugates are functional and protective. (A) Schematic of the immunization and challenge schedule. Mice received an initial immunogen injection on day 0 and identically formulated booster injections on days 21 and 42. On day 49, mice received antibiotics in the drinking water to eradicate normal flora and fecal pellets were collected. On day 51, mice were subsequently infected by gavage and checked daily for 3 days. On day 54, fecal pellets were collected, and mice were sacrificed. (B) Vaccine effects on stool shedding of ETEC B7A in infected mice 3 days post-infection. Groups of eight BALB/c mice receiving PBS, unmodified PD carrier protein, or PD-O148 were infected with ∼1 × 10⁴ CFUs of ETEC B7A. Fecal samples were collected for DNA extraction and analyzed by qPCR to detect pathogen. ETEC burden in fecal pellets was measured by qPCR of the eltA gene, which encodes heat-labile endotoxin (LT). Data are the mean (red bars) of individual mice (black circles). (C) O-PS-specific SIgA titers in day 49 fecal pellet extracts pooled from pairs of mice within the same group (black circles) and mean titers of each group (red lines) as determined by ELISA with LPS derived from ETEC strain B7A (serotype O148:H28) as immobilized antigen. Data in (B) and (C) were analyzed for statistical significance using the Mann-Whitney test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).

FIGURE 4

Glycoconjugates are functional and protective. (A) The iVAX platform provides a rapid means to develop and distribute conjugate vaccines against bacterial pathogens. (B) Immunoblot analysis of PD^4xDQNAT generated using iVAX lysate. Specifically, 5-mL reactions with glyco-enriched S12 extract derived from CLM24 ΔlpxM cells carrying pMW07-O78 and pSF-CjPglB-LpxE were primed with plasmid pJL1-PD^4xDQNAT. Blots were probed with anti-His antibody to detect acceptor proteins and anti-ETEC O78 antibody to detect O-PS antigens. Images depict aglycosylated and multiply glycosylated forms of PD^4xDQNAT. Molecular weight (M _W) markers are indicated on the left. Results are representative of three biological replicates. (C) O-PS-specific IgG titers in day 56 serum of individual mice (black circles) and mean titers of each group (red lines) as determined by ELISA with LPS derived from ETEC strain H10407 (serotype O78:H11) as immobilized antigen. Groups of eight BALB/c mice were immunized s. c. with 100 μL PBS alone or PBS containing 24 μg of either aglycosylated PD carrier protein or glycosylated ETEC O78 conjugate adjuvanted with aluminium phosphate adjuvant. Mice were boosted on days 21 and 42 with identical doses of each immunogen. Data were analyzed for statistical significance using the Mann-Whitney test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). (D) Bactericidal killing activity of serum antibodies from mice immunized with same immunogens as in (C). Survival data were derived from a standard SBA where serum dilutions were tested against ETEC strain H10407 in the presence of human complement. SBA curves were generated with pooled sera from each group. Data are the mean of three biological replicates ±SEM.