Vaccination with outer membrane complexes elicits rapid protective immunity to multidrug-resistant Acinetobacter baumannii

Abstract

Acinetobacter baumannii causes pneumonias, bacteremias, and skin and soft tissue infections, primarily in the hospitalized setting. The incidence of infections caused by A. baumannii has increased dramatically over the last 30 years, while at the same time the treatment of these infections has been complicated by the emergence of antibiotic-resistant strains. Despite these trends, no vaccines or antibody-based therapies have been developed for the prevention of A. baumannii infection. In this study, an outer membrane complex vaccine consisting of multiple surface antigens from the bacterial membrane of A. baumannii was developed and tested in a murine sepsis model. Immunization elicited humoral and cellular responses that were able to reduce postinfection bacterial loads, reduce postinfection proinflammatory cytokine levels in serum, and protect mice from infection with human clinical isolates of A. baumannii. A single administration of the vaccine was able to elicit protective immunity in as few as 6 days postimmunization. In addition, vaccine antiserum was used successfully to therapeutically rescue naïve mice with established infection. These results indicate that prophylactic vaccination and antibody-based therapies based on an outer membrane complex vaccine may be viable approaches to preventing the morbidity and mortality caused by this pathogen.

FIG. 1.

Preparation and characterization of A. baumannii OMCs. (A) Coomassie blue-stained gel of whole bacterial lysates and OMCs prepared from A. baumannii ATCC 19606. (B) Silver-stained gel of OMCs prepared from A. baumannii ATCC 19606 before (untreated) and after (5% SDS) extraction with detergent. (C) Predicted localization of the 61 proteins identified by LC-MS/MS analysis of A. baumannii OMCs, using PSORTb, version 2, software. (D) Coomassie blue-stained gel of three independent preparations of OMCs prepared from A. baumannii ATCC 19606.

FIG. 2.

Immune response after vaccination with an A. baumannii OMC vaccine. (A) IgG titers in vaccinated and control mice (n = 10/group) 0, 2, and 4 weeks after vaccination (at 0 and 3 weeks). Data points are mean values, with error bars representing the standard deviations. *, P < 0.001 for vaccinated versus control mice (Student's t test). (B) IgG1 and IgG2c titers in vaccinated and control mice (n = 10/group) at 4 weeks. Data are presented as mean values, with error bars representing the standard deviations. *, P < 0.001 for vaccinated versus control mice (Student's t test). (C) Western blot of A. baumannii whole bacterial lysates (WBL) and OMP, obtained with preimmune and 4-week sera from vaccinated mice, showing that there are antibodies against multiple bacterial antigens. (D) IgG titers against membrane antigens from diverse A. baumannii strains in vaccinated and control mice (n = 10/group). Data points are mean values, with error bars representing the standard deviations. *, P < 0.001 for vaccinated versus control mice (Student's t test). (E) IFN-γ release from splenocytes from vaccinated and control mice (n = 5/group) after stimulation with outer membrane proteins. Bars represent median values. **, P < 0.01 for vaccinated versus control mice (Mann-Whitney U test). The dashed horizontal line represents the assay limit of detection in panels A, B, and D.

FIG. 3.

A. baumannii sepsis model. (A) Bacterial loads in spleens, kidneys, and lungs of C57BL/6 mice after intraperitoneal injection of 1.3 × 10⁶ CFU of the ATCC 19606 strain (n = 3 mice/group). (B) Survival of mice after intraperitoneal injection of the indicated inocula of the A. baumannii ATCC 19606 strain (n = 8 mice/group).

FIG. 4.

Immunization with the OMC vaccine decreases postinfection tissue bacterial loads and serum proinflammatory cytokine levels. Twelve hours after infection with 1.0 × 10⁶ CFU of A. baumannii ATCC 19606, tissues and sera were collected from vaccinated and control mice (n = 10/group) for determination of tissue bacterial loads and serum cytokine levels. (A) Bacterial loads in spleens, kidneys, and lungs from vaccinated and control mice at 12 h postinfection. *, P < 0.001 for all tissues in vaccinated versus control mice (Mann-Whitney U test). (B) Serum levels of IL-1β, IL-6, and TNF-α in vaccinated and control mice at 12 h postinfection. *, P < 0.001 for IL-1β, IL-6, and TNF-α in vaccinated versus control mice (Mann-Whitney U test).

FIG. 5.

Immunization with the OMC vaccine protects against diverse A. baumannii strains. Mice were vaccinated with the OMC vaccine or mock vaccinated (n = 10/group) at 0 and 3 weeks and then challenged 2 weeks after the second immunization with 1.0 × 10⁶ CFU (151.3× LD₅₀) of the ATCC 19606 strain (A), 1.0 × 10⁷ CFU (20.9× LD₅₀) of the clinical isolate Ab-154 (B), or 1.5 × 10⁶ CFU (27.3× LD₅₀) of the clinical isolate 113-16 (C). *, P < 0.001 for all panels (log rank test).

FIG. 6.

Role of antibodies against LPS in protection. (A) Purified LPS from Escherichia coli strain O55:B5 and whole bacterial lysates from three A. baumannii strains, ATCC 19606, Ab-154, and 113-16, stained with the LPS/glycoprotein-specific stain ProQ Emerald 300. *, differing banding patterns seen at lower molecular masses. (B) Western blot using 4-week sera from vaccinated mice of whole bacterial lysates from the ATCC 19606, Ab-154, and 113-16 strains before and after proteinase K digestion.

FIG. 7.

Immunization with the OMC vaccine elicits rapid protective immunity against A. baumannii infection. (A) Serum levels of anti-OMC IgG and IgM were determined in mice every 3 days (n = 5/time point) after a single immunization. Data are presented as mean values, with error bars representing standard deviations. The horizontal dashed line represents the limit of detection of the assay. Six days after a single immunization with the OMC vaccine or after mock vaccination (n = 10/group), mice were challenged with 1.3 × 10⁶ CFU (196.8× LD₅₀) of the ATCC 19606 strain (B) or 1.0 × 10⁶ CFU (18.2× LD₅₀) of the clinical isolate 113-16 (C). *, P < 0.001 for vaccinated versus control mice (log rank test).

FIG. 8.

Vaccine antisera can therapeutically rescue mice infected with A. baumannii. Naïve mice were infected with 2 × 10⁵ CFU (30.2× LD₅₀) of the ATCC 19606 strain (A) or 1.9 × 10⁶ CFU (34.6× LD₅₀) of the clinical isolate 113-16 (B). At 1 h postinfection, mice were treated by intravenous administration of either anti-OMC antisera or naïve sera (n = 7/group). *, P < 0.001 for mice treated with anti-OMC serum versus naïve serum (log rank test).