Vibriobactin antibodies: a vaccine strategy

Abstract

A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane, was synthesized and linked to ovalbumin (OVA) and bovine serum albumin (BSA). The antigenicity of the VIB microbial iron chelator conjugates and their iron complexes was evaluated. When mice were immunized with the resulting OVA-VIB conjugate, a selective and unequivocal antigenic response to the VIB hapten was observed; IgG monoclonal antibodies specific to the vibriobactin fragment of the BSA and OVA conjugates were isolated. The results are consistent with the idea that the isolated adducts of siderophores covalently linked to their bacterial outer membrane receptors represent a credible target for vaccine development.

Figure 1

Naturally occurring iron chelators (siderophores): hydroxamates (1) and catecholamides (2 and 3).

Figure 2

Retrosynthetic analysis of vibriobactin-OVA (4) and vibriobactin–BSA (5) conjugates from vibriobactin thiol (6).

Figure 3

Competitive binding ELISA of diluted polyclonal sera (1:10000) from mice immunized with the OVA-VIB conjugate (4). Animal M1 (A) was immunized with 4 twice s.c. at 50 μg/injection, while mouse M2 (B) was immunized with 4 twice s.c. at 100 μg/injection. The sera were incubated with varying concentrations of antigen 4, prior to being tested via ELISA against antigen 5. The dotted line indicates the amount of 4 (~ 0.05 μg/mL) needed to reduce the optical density of the p-nitrophenol product to 50% of that of the sera not incubated with any 4. The closed circles indicate the optical density of unconjugated OVA (27) (10 μg/mL).

Figure 4

Competitive binding ELISA of purified mAb from 5A6–2D5 (A) and 2F10–1A9 (B). The mAb (0.11 μg protein/mL) were incubated with varying concentrations of antigen 4, prior to being tested via ELISA against antigen 5. The dotted line indicates the amount of 4 (~ 0.05–0.06 μg/mL) needed to reduce the optical density of the p-nitrophenol product to 50% of that of the mAb not incubated with any 4. The closed circles indicate the optical density of 27 (10 μg/mL).

Scheme 1

Synthesis of 17^{a. a}Reagents: (a) 4-chloro-1-butanol, K₂CO₃, KI, 1-butanol, 125 °C, 1 d; (b) di-tert-butyl dicarbonate, THF, 42%; (c) 92%; (d) potassium phthalimide, DMF, 90 °C, 48 h, 71%; (e) TFA, CH₂Cl₂, 0 °C→room temp, 2 h, quantitative; (f) 2,3-dimethoxybenzoic acid, CDI, CH₂Cl₂, NEt₃, 70%; (g) N-(BOC)-L-threonine N-hydroxysuccinimide ester (3 equiv), DMF, 55%; (h) hydrazine hydrate, EtOH, 65%; (i) 3,3 ′-dithiopropionic acid (0.4 equiv), CDI, CH₂Cl₂, 40%; (j) BBr₃, CH₂Cl₂, 73%; (k) ethyl 2,3-dihydroxybenzimidate, failed.

Scheme 1

Synthesis of 17^{a. a}Reagents: (a) 4-chloro-1-butanol, K₂CO₃, KI, 1-butanol, 125 °C, 1 d; (b) di-tert-butyl dicarbonate, THF, 42%; (c) 92%; (d) potassium phthalimide, DMF, 90 °C, 48 h, 71%; (e) TFA, CH₂Cl₂, 0 °C→room temp, 2 h, quantitative; (f) 2,3-dimethoxybenzoic acid, CDI, CH₂Cl₂, NEt₃, 70%; (g) N-(BOC)-L-threonine N-hydroxysuccinimide ester (3 equiv), DMF, 55%; (h) hydrazine hydrate, EtOH, 65%; (i) 3,3 ′-dithiopropionic acid (0.4 equiv), CDI, CH₂Cl₂, 40%; (j) BBr₃, CH₂Cl₂, 73%; (k) ethyl 2,3-dihydroxybenzimidate, failed.

Scheme 2

Synthesis of 20^{a. a}Reagents: (a) 60% NaH, 4-methoxybenzyl bromide (3.5 equiv), DMF, 62%; (b) 2 N NaOH, dioxane, 90%.

Scheme 3

Synthesis of vibriobactin thiol 6^{a. a}Reagents: (a) 20, CDI, CH₂Cl₂, NEt₃, 57%; (b) N-(BOC)-L-threonine N-hydroxysuccinimide ester (2.5 equiv), DMF, 40 °C, 60%; (c) hydrazine hydrate, EtOH, 90%; (d) 3,3 ′-dithiopropionic acid, CDI, CH₂Cl₂, NEt₃, 60%; (e) TFA, anisole, CH₂Cl₂, 0 °C→room temp, 2 h, 60%; (f) ethyl 2,3-dihydroxy-benzimidate, EtOH, reflux, 36 h, 20%; (g) H₂, 3 atm, Pd black, EtOH, 1 d, 60%.

Scheme 3

Synthesis of vibriobactin thiol 6^{a. a}Reagents: (a) 20, CDI, CH₂Cl₂, NEt₃, 57%; (b) N-(BOC)-L-threonine N-hydroxysuccinimide ester (2.5 equiv), DMF, 40 °C, 60%; (c) hydrazine hydrate, EtOH, 90%; (d) 3,3 ′-dithiopropionic acid, CDI, CH₂Cl₂, NEt₃, 60%; (e) TFA, anisole, CH₂Cl₂, 0 °C→room temp, 2 h, 60%; (f) ethyl 2,3-dihydroxy-benzimidate, EtOH, reflux, 36 h, 20%; (g) H₂, 3 atm, Pd black, EtOH, 1 d, 60%.

Scheme 4

Coupling of vibriobactin thiol 6 to carrier proteins^{a. a}Reagents: (a) 50% DMSO, 0.1 M phosphate buffer (pH 7.4), 8 h; (b) cysteine.