Poly-N-acetyl-β-(1-6)-glucosamine is a target for protective immunity against Acinetobacter baumannii infections

Abstract

Acinetobacter baumannii has emerged as a highly troublesome, global pathogen. Treatment is complicated by high levels of antibiotic resistance, necessitating alternative means to prevent or treat A. baumannii infections. We evaluated an immunotherapeutic approach against A. baumannii, focusing on the surface polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (PNAG). We used a synthetic oligosaccharide of 9 monosaccharide units (9Glc-NH(2)) conjugated to tetanus toxoid (TT) to induce antibodies in rabbits. In the presence of complement and polymorphonuclear cells, antisera to 9Glc-NH(2)-TT mediated the killing of A. baumannii S1, a high-PNAG-producing strain, but not its isogenic PNAG-negative, in-frame deletion mutant strain, S1 Δpga. Complementing the pgaABCD locus in trans in the shuttle vector pBAD18kan-ori, plasmid Δpga-c, restored the high levels of killing mediated by antibody to PNAG observed with the wild-type S1 strain. No killing was observed when normal rabbit serum (NRS) or heat-inactivated complement was used. Antiserum to 9Glc-NH(2)-TT was highly opsonic against an additional four unrelated multidrug-resistant clinical isolates of A. baumannii that synthesize various levels of surface PNAG. Using two clinically relevant models of A. baumannii infection in mice, pneumonia and bacteremia, antisera to 9Glc-NH(2)-TT significantly reduced levels of A. baumannii in the lungs or blood 2 and 24 h postinfection, respectively, compared to levels of control groups receiving NRS. This was true for all four A. baumannii strains tested. Overall, these results highlight the potential of PNAG as a vaccine component for active immunization or as a target for passive antibody immunotherapy.

Fig 1

Opsonophagocytic killing of A. baumannii strains S1, S1 Δpga, and S1 Δpga-c mediated by a 1:10 dilution of rabbit sera raised to 9Glc-NH₂-TT in the presence of human PMNs and rabbit complement. For each strain, control tubes containing either NRS instead of the immune sera or antibody to 9Glc-NH₂-TT, PMNs, and heat-inactivated complement HI C (56°C for 30 min) (labeled 9Glc-NH₂-TT+HI C) were included in each assay. NRS and 9Glc-NH₂-TT+HI C control samples, which resulted in growth after 90 min of incubation, are represented in the graph with bars with an arbitrary value of 1 for visibility. Bars represent the averages from three independent experiments, and error bars indicate the standard errors of the means (SEM).

Fig 2

Opsonophagocytic killing mediated by rabbit serum raised to the 9Glc-NH₂-TT vaccine at 1:10 and 1:20 dilutions against four MDR A. baumannii clinical isolates, S13, S26, S28, and S29, in the presence of human PMNs and rabbit complement. Control tubes containing bacteria, complement, and PMNs were included in every assay, and growth occurred in all instances. Bars represent the averages from three independent experiments, and error bars represent the SEM.

Fig 3

Passive immunization of mice with rabbit antiserum raised to 9Glc-NH₂-TT led to significantly reduced levels of A. baumannii strains in lung tissues compared to levels of mice subjected to NRS. Mice (n = 7 to 26 per group) were infected i.n. with A. baumannii S1 (2.3 × 10⁵ CFU/mouse), S13 (3.7 × 10⁴ CFU/mouse), S26 (1.5 × 10⁵ CFU/mouse), or S29 (5.9 × 10⁵ CFU/mouse). A. baumannii S1 Δpga (1.2 × 10⁵ CFU/mouse) was used as a specificity control. Bars indicate the mean CFU per g of lung tissue, and error bars indicate the SEM. t tests were used for protection studies with the strains S1, S26, and S1 Δpga. For protections studies with the strains S13 and S29, where two and three replicate experiments, respectively, were carried out, comparisons between control and immune groups were made by t test with the Bonferroni correction. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (P > 0.05).

Fig 4

Passive immunization with rabbit antiserum raised to 9Glc-NH₂-TT significantly reduced levels of A. baumannii surviving in the blood 2 h after i.v. injection compared to levels for mice subjected to NRS. Mice (n = 7 to 15) were injected i.v. with A. baumannii strain S1 (2.3 × 10⁵ CFU/mouse), S13 (3.7 × 10⁴ CFU/mouse), S26 (1.5 × 10⁵ CFU/mouse), or S29 (5.9 × 10⁵ CFU/mouse). A. baumannii S1 Δpga (1.2 × 10⁵ CFU/mouse) was used as a specificity control. Bars indicate the mean CFU per ml of blood, and error bars indicate the SEM. t tests were used for comparisons between control and immune groups for all strains except for strain S1 in the protection studies, where two replicate experiments were carried out and comparisons made by t test with the Bonferroni correction. *, P < 0.05; ns, not significant (P > 0.05).