Radiation-Inactivated Acinetobacter baumannii Vaccine Candidates

Abstract

Acinetobacter baumannii is a bacterial pathogen that is often multidrug-resistant (MDR) and causes a range of life-threatening illnesses, including pneumonia, septicemia, and wound infections. Some antibiotic treatments can reduce mortality if dosed early enough before an infection progresses, but there are few other treatment options when it comes to MDR-infection. Although several prophylactic strategies have been assessed, no vaccine candidates have advanced to clinical trials or have been approved. Herein, we rapidly produced protective whole-cell immunogens from planktonic and biofilm-like cultures of A. baumannii, strain AB5075 grown using a variety of methods. After selecting a panel of five cultures based on distinct protein profiles, replicative activity was extinguished by exposure to 10 kGy gamma radiation in the presence of a Deinococcus antioxidant complex composed of manganous (Mn²⁺) ions, a decapeptide, and orthophosphate. Mn²⁺ antioxidants prevent hydroxylation and carbonylation of irradiated proteins, but do not protect nucleic acids, yielding replication-deficient immunogenic A. baumannii vaccine candidates. Mice were immunized and boosted twice with 1.0 × 10⁷ irradiated bacterial cells and then challenged intranasally with AB5075 using two mouse models. Planktonic cultures grown for 16 h in rich media and biofilm cultures grown in static cultures underneath minimal (M9) media stimulated immunity that led to 80-100% protection.

Keywords: A. baumannii; Deinococcus; MDP; inactivated; irradiated; mouse; protective; pulmonary; vaccine; whole-cell.

Figure 1

Analysis of proteins from A. baumannii cultures grown under varying conditions.

Figure 2

Denaturing gel analysis of proteins from planktonic and biofilm cultures of A. baumannii. Two-dimensional electrophoresis gels are shown of cell lysates from cultures harvested in (A,C) planktonic growth phase (P1) or in (B,D) biofilm phase (B1). The gels in Panels A and B were stained with Coomassie Blue to show all visible proteins. Arrows point to several proteins that are present or increased in that gel, but that are absent or decreased in the accompanying gel. Replicate gels were transferred to membranes and probed with polyclonal rat antisera raised against A. baumannii (panels C and D). Panel C shows proteins detected from planktonic growth phase (P1) and Panel D shows proteins detected from biofilm phase (B1). Panels E and F show Western blot analysis of P1, P2, B1, B2, and B3 lysates before and after irradiation in the presence of MDP probed with polyclonal rat sera raised against either planktonic (E) or biofilm (F) forms of A. baumannii AB5075.

Figure 3

Irradiation inactivation of A. baumannii colony-forming units. A. baumannii samples P1, P2, B1, B2, and B3 were irradiated with or without MDP (as indicated) and analyzed for replicative viability. Serial dilutions of irradiated bacteria were plated to determine CFU/mL.

Figure 4

Stability of unirradiated and irradiated bacterial cultures. (A) Stability of unirradiated A. baumannii at 4 °C. P1, P2, B1, B2, and B3 cultures were prepared with or without the MDP complex and stored at 4 °C for up to 16 weeks at concentrations between 10¹⁰ and 10¹¹ CFU/mL. A portion of each sample was removed for colony counting by plating serial dilutions on agar plates at the indicated time points. (B) Stability of irradiated A. baumannii at 4 °C. P1, P2, B1, B2, and B3 cultures were prepared with or without the MDP complex, exposed to 10 kGy gamma irradiation, and stored at 4 °C for up to 16 weeks at concentrations between 10¹⁰ and 10¹¹ CFU/mL. A portion of each sample was removed and serially diluted for microscopic examination. The concentration of intact cells/mL are reported at the indicated time points.

Figure 5

Immunization and intranasal challenge of mice with A. baumannii. (A) Graphic representation of the vaccination schedule. Mice were immunized at weeks 0, 4, and 6. Mice were challenged intranasally at week 8 and monitored daily until the end of week 9. (B–E) Kaplan–Meier Survival Analysis of vaccinated, or mock vaccinated mice challenged with AB5075 via intranasal inoculation as detailed in A. Panels B and C show groups of 8 mice immunized with candidate vaccines as indicated. B Shows survival in a healthy C57BL/6 mouse model, C shows survival in a neutropenic BALB/c model. Panels D and E show a second round of experiments using groups of 10 mice in the healthy C57BL/6 and neutropenic BALB/c models respectively. Log-rank (Mantle–Cox) test results of significance between survival numbers of groups is indicated by ns (not significant, p > 0.05), * p < 0.05, ** p < 0.01, and *** p < 0.001.