Whole-cell vaccine candidates induce a protective response against virulent Acinetobacter baumannii

Abstract

Acinetobacter baumannii causes multi-system diseases in both nosocomial settings and a pre-disposed general population. The bacterium is not only desiccation-resistant but also notoriously resistant to multiple antibiotics and drugs of last resort including carbapenem, colistin, and sulbactam. The World Health Organization has categorized carbapenem-resistant A. baumannii at the top of its critical pathogen list in a bid to direct urgent countermeasure development. Several early-stage vaccines have shown a range of efficacies in healthy mice, but no vaccine candidates have advanced into clinical trials. Herein, we report our findings that both an ionizing γ-radiation-inactivated and a non-ionizing ultraviolet C-inactivated whole-cell vaccine candidate protects neutropenic mice from pulmonary challenge with virulent AB5075, a particularly pathogenic isolate. In addition, we demonstrate that a humoral response is sufficient for this protection via the passive immunization of neutropenic mice.

Keywords: A. baumannii; MDP; UVC; humoral; protection; pulmonary; vaccine; whole-cell.

Figure 1

UVC inactivation of A. baumannii vaccine candidates B1 and P1. 0.1 mL volumes of bacterial cultures at approximately 1 x 10⁹ CFU/mL were compounded with the MDP complex and exposed to UVC irradiation (4.5 mW/cm²) for increasing times. Energy is shown in millijoules (mJ/cm²). The treated samples were serially diluted and plated to determine the concentration of live, colony-forming, bacteria. The experiment was done in triplicate and error bars show the standard error of the mean.

Figure 2

Analysis of immunogen protein profiles. B1 and P1 cultures of AB5075 were inactivated with UVC with (+) and without (–) the protective MDP complex. 1 x 10⁸ bacterial cells were denatured in SDS-PAGE sample buffer and electrophoresed in a 4-20% polyacrylamide gel. Samples were then stained with Coomassie Brilliant Blue to visualize the total protein profiles of the four samples. Migration of molecular weight markers is shown to the right of the figure. Arrows indicate prominent differences between the B1 and P1 preparations. An asterisk highlights the one prominent exception at 40 kDa in samples prepared without MDP.

Figure 3

 (A) Analysis of UVC-irradiated immunogens with and without MDP. Western analysis of epitopes. P1 samples irradiated in the presence and absence of MDP were loaded at the indicated concentration (approx. 2.5 x 10⁷ CFU in lane 1) in replicate. (B) Carbonylation analysis. B1 and P1 samples were UVC inactivated with and without the inclusion of the MDP Mn-antioxidant complex. After reacting the proteins with DNPH, the samples were denatured, electrophoresed, and transferred to nitrocellulose. The membranes were probed with anti-DNP antibody and detected with HRP-conjugated secondary antibody.

Figure 4

Pulmonary challenge in neutropenic mice. (A) Vaccination schedule. BALB/c mice, two independent groups of five (10 total/per group) were vaccinated and boosted at weeks 4 and 6 post vaccination. Cyclophosphamide (CP) was given at days -4 and -1 pre-challenge. In one group, immune serum (IS) was given 1 day before pre-challenge via i.p. injection with cyclophosphamide treatment. Mice were challenged at 8 weeks via droplet exposure and observed for one week. (B) Gamma-Irradiated P1B1. Survival analysis of challenged mice vaccinated with immunogens prepared via gamma irradiation in the presence or absence of MDP. (C) UVC-Irradiated-P1B1. Survival analysis of challenged mice vaccinated with immunogens prepared via UVC-irradiation in the presence or absence of MDP (D) Passive immunization. Survival analysis of mice given sera from P1 and B1 vaccinated mice. Log-rank (Mantle–Cox) test p values were calculated and include the Holm-Bonferroni correction for multiple comparisons. Values are indicated by ns, not significant (p ≥ 0.05), and **p ≥ 0.01). (E) Sera reactivity against A. baumannii. ELISA data reactivity of sera raised against P1 and a pool of P1B1 using MDP inactivated P1 as a target. (F) Sera reactivity against A. baumannii. ELISA data reactivity of sera raised against B1 and a pool of P1B1 using MDP inactivated B1 as a target.