Brucellosis: Improved Diagnostics and Vaccine Insights from Synthetic Glycans

Abstract

Brucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven "neglected zoonoses" that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500 000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide-protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals.

Figure 1

OPS structure of B. abortus, B. melitensis, and B. suis (except biovar 2) showing the M and A elements of the block copolymer. The capping tetrasaccharide containing a single 1,3 linkage defines the M epitope. The relative length of the M and A segments defined by the number of repeats, x and z, varies between strains. In solution, the Z rotamer of the formamido residue is the most abundant rotamer.

Figure 2

Glycoconjugate synthesis involved the reaction of esters 1 and 2 with ethylenediamine to give amides 3 and 4. Reaction of these with diethyl or dibutyl squarate gave stable squarate half-esters, which were reacted with lysine amino acids of bovine serum albumin (BSA) to give glycoconjugates 5 and 6.

Figure 3

Synthetic targets used to probe antibodies present in infected cattle and in mice immunized with glycoconjugate vaccine 12d. M tetrasaccharide 7a, disaccharide 8a, and the two-component trisaccharides 9a and 10a found in the M repeating sequence. M and A hexasaccharides 11a and 12a represent larger epitopes. Trisaccharide 13a represents a small A-type epitope. Oligosaccharide amides 7b–13b were conjugated to BSA to provide glycoconjugates for use in ELISA. Hexasaccharide 12a was derivatized, conjugated to tetanus toxoid, and then used to immunize mice.

Scheme 1

Reagents and conditions: TMSOTf, 3 Å MS, CH₂Cl₂.

Scheme 2

Reagents and conditions: (a) TMSOTf, 3 Å MS, PhMe, 95 °C, 1 h; (b) NaOCH₃, CH₃OH, rt, 4 h; (c) MeOTf, 3 Å MS, CH₂Cl₂, rt, 48 h; (d) H₂S, Py/H₂O, 40 °C, 16 h; (e) (HCO)₂O, MeOH, −20 °C, 3 h; (f) H₂, Pd(OH)₂, MeOH/H₂O, rt, 16 h.

Scheme 3

Reagents and conditions: (a) MeOTf, 3 Å MS, CH₂Cl₂, rt, 48 h; (b) NaOCH₃, CH₃OH, rt, 4 h; (c) TMSOTf, 3 Å MS, CH₂Cl₂, rt, 1 h; (d) H₂S, Py/H₂O, 40 °C, 16 h; (e) (HCO)₂O, MeOH, −20 °C, 3 h; (f) H₂, Pd(OH)₂, MeOH/H₂O, rt, 16 h.

Figure 4

Heptasaccharide 27 was synthesized as the amine and conjugated to tetanus toxoid using disuccinimidyl glutarate (DSG) to obtain 28 and to BSA using diethyl squarate to obtain 29. Conjugate 28 was used to immunize mice and conjugate 29 to monitor antibody responses by ELISA.

Scheme 4

Reagents and conditions: (a) TMSOTf, NIS, 4 Å MS, CH₂Cl₂, −20 °C to rt, 3 h; (b) NaOMe, MeOH, rt, 6 h: (c) H₂S, Py/H₂O, 40 °C, 16 h; (d) (HCO)₂O, MeOH, −20 °C, 3 h; (e) H₂, Pd(OH)₂, MeOH/H₂O, rt, 16 h.

Figure 5

Comparison of the glycoconjugate and Brucella LPS binding profiles with antibodies generated in mice immunized with glycoconjugates 12d and 28.

Scheme 5. Conjugation of B. abortus Strain S99 OPS to Tetanus Toxoid To Yield the Vaccine Conjugate OPS-TTS99

Reagents and conditions: (a) 10 mM NaIO₄, 50 mM NaOAc, pH 5.5, 4 °C, 1 h; (b) 0.5 M NH₄Cl, 0.1 M NaCNBH₃, 37 °C; (c) aminated OPS (5 mg/mL) in PBS containing 10% DMSO and 5 mg/mL disuccinimidal glutarate (dsg), 45 min, rt; (d) activated OPS, tetanus toxoid (2.5 mg/mL in PBS).

Figure 6

Final bleed titers from eight CD1 mice immunized with the modified B. abortus S99 conjugate OPS-TTS99. The antigens used for antibody detection are shown on the x axis.