Vaccination with a live attenuated Acinetobacter baumannii deficient in thioredoxin provides protection against systemic Acinetobacter infection

Abstract

Multi-drug resistant Acinetobacter baumannii (MDR-Ab), an opportunistic pathogen associated with nosocomial and combat related infections, has a high mortality due to its virulence and limited treatment options. Deletion of the thioredoxin gene (TrxA) from a clinical isolate of MDR-Ab resulted in a 100-fold increase in 50% lethal dose (LD₅₀) in a systemic challenge murine model. Thus, we investigated the potential use of this attenuated strain as a live vaccine against MDR-Ab. Mice were vaccinated by subcutaneous (s.c.) injection of 2×10⁵ CFU of the ΔtrxA mutant, boosted 14days later with an equivalent inoculum, and then challenged 30days post-vaccination by i.p. injection with 10 LD₅₀ of the wild type (WT) Ci79 strain. Efficacy of vaccination was evaluated by monitoring MDR-Ab specific antibody titers and cytokine production, observing pathology and organ burdens after WT challenge, and measuring levels of serum pentraxin-3, a molecular correlate of A. baumannii infection severity, before and after challenge. Mice vaccinated with ΔtrxA were fully protected against the lethal challenge of WT. However, minimal immunoglobulin class switching was observed with IgM predominating. Spleens harvested from vaccinated mice exhibited negligible levels of IL-4, IFN-γ and IL-17 production when stimulated with UV-inactivated WT Ci79. Importantly, tissues obtained from vaccinated mice displayed reduced pathology and organ burden compared to challenged non-vaccinated mice. Additionally, serum pentraxin-3 concentrations were not increased 24h after challenge in vaccinated mice, correlating with reduction of WT MDR-Ab infection in ΔtrxA immunized mice. Furthermore, passive immunization with ΔtrxA-immune sera provided protection against lethal systemic Ci79 challenge. Collectively, the defined live attenuated ΔtrxA strain is a vaccine candidate against emerging MDR Acinetobacter infection.

Keywords: Acinetobacter baumannii; Pentraxin-3; Thioredoxin; Vaccine.

Fig. 1

ΔtrxA vaccination induces robust humoral responses but minimal splenic cell-mediated immunity. C57BL6 mice (n = 13) were injected i.p. with 2 × 10⁵ CFU of ΔtrxA and received a booster injection 2 weeks later. Mice were then rested for 2 weeks before challenge with A. baumannii WT Ci79. For assessing humoral responses, bloods (n = 10) were collected on days 14 and 28 after initial vaccination and serum antibody reactivity against UV-inactivated ΔtrxA and Ci79 were determined. Endpoint titers were calculated and presented as a box-and-whisker plot for total anti- ΔtrxA and Ci79 antibodies (A) and anti-Ci79 isotypes (B). For assessing cell-mediated immune responses (C), spleens (n = 3) were collected 14 days after the booster and single spleenocytes were made and stimulated with UV inactivated Ci79, ConA (mitogen, positive control), BSA (unrelated antigen control) or unstimulated (media) for 72 hrs. Levels of cytokines IL-4, IFN-γ and IL-17 produced by spleenocytes were measured in culture supernatants by ELISA. *p < 0.05. (Representative of two independent experiments).

Fig. 2

ΔtrxA vaccinated mice are protected against systemic WT challenge. C57BL6 mice (n = 10) received a primary and a booster dose of ΔtrxA (2 × 10⁵ CFU) or PBS alone (mock control) at 2 weeks apart via i.p. injection. Mice were rested for 2 weeks and then challenged (i.p.) with either 2 or 10 LD₅₀ of the WT Ci79 strain. (A) Survival of the mice was monitored for 4 weeks. *p< 0.05 (Mantel-Cox log rank test), comparing WT challenged ΔtrxA vaccinated to PBS treated groups. (Representative of three independent experiments). In addition, (B) spleens, livers, and kidneys were collected from ΔtrxA or mock vaccinated mice (n=3 per group) 24 hrs after i.p. WT Ci79 challenge (10 LD₅₀). Tissues were homogenized and bacteria were enumerated by dilution plating on LB agar containing chloramphenicol. (C) Sera were collected from mice 1 hour before and 24 hrs after Ci79 challenge and PTX 3 concentrations were assessed using a PTX 3 ELISA kit. (Representative of two independent experiments).

Fig. 3

Vaccination with ΔtrxA reduced organ pathology following WT Ci79 challenge. Representative pathology (H&E staining) associated with A. baumannii challenge of mock and ΔtrxA mutant vaccinated mice. A. Liver 100X: Infected mock vaccinated animals show inflammatory infiltrates (arrows) and congestion of vessels (arrowheads), whereas livers from infected ΔtrxA mutant vaccinated animals were similar to naïve mice; inserts show hepatic veins. B. Liver: Marked inflammatory infiltrates (arrows) and congestion (arrowheads) in hepatic veins and microvessels (MV) of infected mock mice (400X). In contrast, similar histological features (100X) without significant pathology were observed with both naïve and infected ΔtrxA vaccinated mice. C. Spleen 40X: Disruption of the white pulp (WP) in infected mock vaccinated animals; similar appearance of naïve and infected ΔtrxA vaccinated mouse spleens. D. Spleen 100X: Greater lymphocyte depletion, or clear areas indicative of cell death, (white arrows) in the WP and hemorrhage in the red pulp (white arrowheads) of infected mock, compared to ΔtrxA vaccinated animals; inserts 200X. (Representative of groups of 3 mice from two independent experiments).

Fig. 4

Subcutaneous immunization with ΔtrxA also induces a robust humoral response and protects mice against a lethal WT Ci79 challenge. C57BL6 mice (n=10 per group) were injected s.c. with 2 × 10⁵ CFU of ΔtrxA and received a booster injection 2 weeks later. Mice were then rested for 2 weeks before challenge with A baumannii Ci79 strain. (A) Bloods were collected on days 14 and 28 after initial vaccination and serum antibody reactivity against UV-inactivated ΔtrxA and WT Ci79 were determined. Endpoint titers were calculated and presented as a box-and-whisker plot for total and IgM anti-Ci79 antibodies. (B) Mice were monitored for mortality for 4 weeks. The difference in survival between WT challenged ΔtrxA and mock vaccinated mice is significanct (p< 0.05, Mantel-Cox log rank test). (Representative of two independent experiments).

Fig. 5

Passive transfer of ΔtrxA-immune serum protects mice against systemic WT A. baumannii Ci79 challenge. (A) Naïve mice (n=6 per group) were i.p. injected with serum (heat-inactivated or untreated) from ΔtrxA or mock vaccinated mice at −25 and −13 hrs prior to and +12 hrs post Ci79 (10 LD₅₀) i.p. challenge. Naïve mice without serum transfer were used as a control. All animals were monitored daily for survival for 4 weeks. *p< 0.05 (Mantel-Cox log rank test), comparing ΔtrxA immune serum transfer group to PBS none-immune serum and no serum transfer treated groups. (B) ΔtrxA antisera cross reacts with various MDR A baumannii clinical isolates. Sera from ΔtrxA immunized mice (n =6) were serially diluted and reacted with various clinical isolates. Endpoint total antibody titers were calculated and presented as individual dots for each serum. (Representative of two independent experiments).