A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates

Abstract

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM₁₉₇ lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Keywords: Escherichia coli; FimH; UPEC; UTI; cystitis; non-human primate.

Fig 1

Cynomolgus macaque model of UTI challenge. (A) Timeline of blood draws and urine sampling by catheterization following transurethral challenge with E. coli ST131 O25b bacteria. (B) Time course of bacteriuria after administration of 1 × 10⁸ bacteria via intravesical catheterization on day 0. Thin lines represent individual animals, and thick lines indicate geometric means within each group. Dotted lines indicate the LLOQ. (C) Resolution of induced bacteriuria in cynomolgus macaques infected with different challenge doses: 1 × 10⁸ (A, n = 8; blue bars), 1 × 10⁷ (B, n = 3, green bars), or 1 × 10⁶ (C, n = 3, purple bars) viable E. coli. (D and E) No systemic signs of infection were observed in NHPs inoculated with O25b E. coli. WBC count and serum CRP. Dotted lines represent the reference range of values in healthy animals (CRP: 0.2–5.1 µg/mL); thin lines represent individual animals, and thick lines indicate geometric means within each group. (F and G) Recruitment of PMN cells and detectable levels of IL-8 and MPO in urine confirms bladder inflammation in response to the inoculation of E. coli. Light microscopy micrographs of urine sediments collected at days 2 and 7 post-challenge. Urine samples collected via catheterization were cytospun into slides and stained with Giemsa. Representative images show the presence of PMN cells in urine sediment of infected animals. Concentration of IL-8 and MPO in urine over a period of 28 days. Thin lines represent individual animals, and thick lines indicate geometric means within each group. Dotted lines represent the LLOQ.

Fig 2

Vaccine immunogenicity. (A) Schedule of NHP vaccinations, bleeds prior to UPEC challenge (n = 1 experiment with nine NHPs/group). NHPs were immunized with placebo (gray), adjuvanted FimH-DSG alone or in combination with a tetravalent O-antigen conjugate mixture at weeks 0, 4, and 14. (B) Graph shows serum anti-FimH IgG titers measured at post-doses 2 and 3. Each symbol represents an individual animal. Error bars indicate geometric mean with 95% CI. Dotted line indicates the LLOQ. (C) Live cell ligand binding inhibition titers (log IC₅₀) in yeast mannan assay at pre-vaccination and at post-dose 2 and post-dose 3 time points. (D) Anti-O1a, O2, O6, and O25b IgG titers in the sera of NHPs immunized with FimH-DSG in combination with a tetravalent O-antigen conjugate mixture at pre-vaccination (week 0) and at post-doses 2 (week 6) and 3 (week 16).

Fig 3

Bacteriuria measured by qPCR over 28 days. NHPs were immunized with adjuvanted FimH-DSG alone or in combination with a tetravalent O-antigen conjugate mixture at weeks 0, 4, and 14 (Fig. 2). On week 19, 10⁸ viable UPEC (n = 9) or PBS (n = 2; sentinels, open symbols) were administered via intravesical catheterization. On indicated days post-challenge, urine samples were collected via bladder catheterization. Each symbol represents an individual animal. Ratios indicate the numbers of NHPs with urine sample presenting >100 bacteria/mL over the total number of challenged animals. Error bars indicate geometric mean with 95% CI. Dotted line indicates the LLOQ.

Fig 4

Vaccine efficacy. NHPs (n = 9) were immunized with placebo, adjuvanted FimH-DSG antigen alone or in combination with a tetravalent O-antigen conjugate mixture at weeks 0, 4, and 14. On week 19, 1 × 10⁸ viable UPEC isolate was administered via intravesical catheterization. (A) Concentration of IL-8 in urine of individual NHPs over a period of 7 days post-challenge. Error bars indicates geometric mean with 95% CI. Dotted lines represent the LLOQ. (B) Percentage of NHPs with PMN cell increase in urine over a period of 28 days post-challenge. On indicated days post-challenge, urine samples collected via bladder catheterization were cytospun into slides and stained with Giemsa and May-Grunwald stain. Slides were evaluated by light microscopy for the presence or absence of increased PMN cells. (C) Concentration of MPO in urine during the post-challenge period (as in panel A).