Active and passive immunization against Acineto bacter baumannii using an inactivated whole cell vaccine
Michael J. McConnell
∗ , Jerónimo Pachón
Abstract
The treatment of infections caused by Acineto bacterbaumannii has become increasingly complicated due to the emergence of highly resistant strains. In the present study we demonstrate that immunization with an inactivated whole cell vaccine elicits a robust antibody response that provides protection against challenge with multiple A. Baumannii strains in a murine model of disseminated sepsis. In addition, we show that passive immunization with serum raised against inactivated cells protects mice from subsequent infection.
These results demonstrate that active and passive immunization using an inactivated whole cell vaccine may be an effective approach for preventing infection by A. baumannii .
Keywords: Acinetobacter; baumannii; Vaccine Passive; immunization.
1.
Introduction
Acinetobacter baumannii has emerged as an important cause of nosocomial infections, most notably in patients receiving mechanical ventilation, patients sustaining burns or trauma, and in surgical-site infections
[1] . A. baumannii has also been implicated in infections in military personal sustaining war-related injuries in Iraq and Afghanistan [2] . More recently, A. baumannii has been identified as a causative agent in community acquired pneumonias and infections in long-term care facilities [3,4] . The treatment of these infections has become increasingly difficult due to the emergence of highly resistant strains. Carbapenems have traditionally been the first-line therapy for A. baumannii infections, however their use has been limited by the increasing prevalence of resistant strains. In 2007 resistance rates to imipenem were between 38 and
71%, whereas in the early 1990s resistance to imipenem was between 0 and 2% [5] . This has necessitated increased use of the cationic peptide antibiotic colistin, although resistance to colistin has now been described [6] . Most worrisome, are recent reports describing pan-resistant strains with resistance to all clinically used antibiotics [7,8] .
Despite these trends, no vaccines or antibody-based treatments have been developed for A. baumannii infections. The development of a vaccine for A. baumannii is complicated by the fact that no protective bacterial antigens have been identified and few virulence factors for this organism have been characterized.
Inactivated and attenuated bacterial cells have been used for the development of a number of vaccines due to their ability to stimulate robust, protective immune responses [9–12] . In addition, vaccines based on whole cells can stimulate a response against multiple antigens, which may be important for providing protection against a broad range of strains within a bacterial species. The goal of this study was to evaluate active and passive immunization against A. baumannii using an inactivated whole cell (IWC) vaccine.
2.
Materials and methods
2.1.
Bacterial strains
The A. baumannii ATCC 19606 strain, as well as the two previously characterized clinical isolates, Ab-154 and 113-16, were used in this study. Ab-154 is an antibiotic sensitive strain isolated during a nationwide surveillance study carried out in Spain in 2000 [13] . The 113-16 strain is a panresistant clinical isolate from a hospital outbreak that occurred in Seville, Spain in 2002 [8].
2.2.
Vaccine preparation
The IWC vaccine was prepared by growing the ATCC 19606 strain in Mueller-Hinton broth to an OD600 of
0.8. Bacteria were washed extensively in PBS before inactivation in 0.05 M formalin for 18 h. Complete inactivation of the bacteria was confirmed by plating on blood agar. The concentration of inactivated cells was adjusted to 1 × 10
9 cells/ml and combined 1:1 (v:v) with the aluminium phosphate adjuvant, Adjuphos
(Brenntag Biosector, Frederikssund, Denmark). For vaccination, 6 to 8-week, female C57BL/6 mice were

immunized by intramuscular injection of 100
μ l of the vaccine into each quadriceps muscle (total dose = 1 ×
10
8 inactivated cells) on days 0 and 21. Control mice were immunized similarly with a mixture of PBS and adjuvant. Experiments involving mice were approved by the University Hospital Virgen del Rocío
Committee on Ethics and Experimentation.
2.3.
ELISAs and Wertern blots
For ELISAs, outer membrane proteins were extracted from the ATCC 19606 strain as described previously
[14] and used to coat 96well plates at 0.3 μg/well. Sera collected on days 0, 14, and 28 were used in ELISAs as described previously [15] . For Western blotting, 3 μg of whole bacterial lysates from A. baumannii ATCC
19606 and 3 μg of outer membrane proteins were resolved on a 12% gel before transfer to nitrocellulose.
Mouse serum collected at 28 days was used at a dilution of 1:2000 to probe the blot before developing with chemiluminescence.
2.4.
Mouse model of A. baumannii infection
A murine model of disseminated sepsis was used for bacterial challenge. A. baumannii strains were grown
◦ for 18 h at 37 C in Mueller-Hinton broth and adjusted to the appropriate concentration in physiologic saline.
Inocula were prepared by mixing the bacterial suspensions 1:1 (v:v) with a 10% solution (w/v) of porcine mucin (Sigma, St. Louis, MO). Mice were injected intraperitoneally with 0.5 ml of the inocula and bacterial concentrations of the inocula were determined by plating dilutions on blood agar. Strain LD50 values were determined by infecting groups of 8 mice with 10-fold dilutions of bacteria and analyzing survival data using the Probit method. For active immunization studies, mice were challenged on day 28 after receiving immunizations on days 0 and 21. For passive immunization studies, 200 μl of naïve serum or serum collected at 28 days from vaccinated mice were administered intravenously 3 h prior to bacterial challenge
2.5.
Tissue bacterial loads and serum cytokine levels
Post-infection tissue bacterial loads were determined by euthanizing mice with thiopental and aseptically removing spleens. Spleens were weighed, homogenized in physiologic saline, and plated on blood agar for bacterial quantification. Serum levels of IL-1β, TNF-α, and IL-6 were determined 12 h post-infection using the BD OptEIA Set Mouse kits (BD Biosciences, San Jose, CA).
2.6.
Statistical analysis
Statistical analyses were performed using SPSS version 15.0 (SPSS Inc., Chicago, IL). Antibody titers, bacterial loads, and cytokine levels were compared using the Mann–Whitney U test. Survival data were compared using the log-rank test
3.
Results
3.1.
Antibody response after vaccination with the IWC vaccine
A vaccine dose of 1 × 10
8 inactivated cells was used after preliminary studies demonstrated that increasing the dose further did not result in an increased antibody response (data not shown Fourteen days after the first immunization, significant levels of antigen-specific IgG and IgM were detectable in the sera of vaccinated mice (P < 0.001 compared to control mice; Mann–Whitney U test; Fig. 1 A). Levels of both IgG and IgM were significantly increased in serum collected 7 days after a second immunization at 21 days (P < 0.001 compared to 14-day IgG levels for IgG; P = 0.04 compared to 14-day IgM levels for IgM; Mann–Whitney U test). Control mice had no detectable antigen-specific antibodies at any timepoint. Levels of IgG1 and IgG2c, the IgG2a homolog in C57BL/6 mice [16] , were determined in 28-day sera. Vaccinated mice had significant levels of both IgG subtypes (P < 0.001 compared to control mice; Mann–Whitney U test), suggesting that both the Th1 and Th2 immune pathways had been stimulated ( Fig. 1 B). A Western blot was performed using
28-day serum from vaccinated mice to probe a blot containing whole bacteria lysates and proteins of the bacterial outer membrane. As shown in Fig. 1 C, the serum reacted with multiple proteins from both samples, indicating that vaccination with the IWC vaccine elicited antibodies against multiple bacterial antigens, many of which are located in the bacterial outer membrane.
3.2.
Effect of vaccination on post-infection bacterial loads and serum cytokine levels
A mouse model of disseminated sepsis was used to characterize the efficacy of the IWC vaccine. This model produces an acute sepsis in which bacteria rapidly disseminate throughout the body (data not shown), resulting in death between 18 and 48 h. Using this model, the LD50 values for the strains used here were

ATCC 1960, 6.6 × 10
3 cfu, Ab-154; 4.8 × 10
5 cfu, and 113-16; 5.5 × 10
4 cfu. The effect of immunization on bacterial loads was determined by quantifying bacteria in the spleens of vaccinated and control mice 12 h after infection with 1.6 × 10
6 cfu (242.4 × LD50) of the ATCC 19606 strain. Spleen bacterial loads were approximately 10
5 times lower in vaccinated mice compared to control mice, indicating that vaccination was able to reduce bacterial burden post-infection (P < 0.001; Mann–Whitney U test; Fig. 2 A). To determine if vaccination with the IWC vaccine could prevent the pro-inflammatory cytokine release produced by bacterial sepsis, serum levels of IL-1
β
, TNF-
α
, and IL-6 were measured in vaccinated and control mice 12 h postinfection with 1.6 × 10
6 cfu (242.4 × LD50) of the ATCC 19606 strain. As shown in Fig. 2 B, serum levels of all three cytokines were significantly lower in vaccinated mice compared to control mice (P < 0.001 for all cytokines; Mann–Whitney U test).
3.3.
Survival against challenge with A. baumannii
In order to determine if vaccination with the IWC vaccine could protect mice from infection, mice immunized on days 0 and 21 were subjected to bacterial challenge 7 days after the final immunization.
Vaccinated mice challenged with 1.6 × 10
6 cfu (242.4 × LD50)of the ATCC 19606 strain were completely protected from challenge, whereas all but one of the control mice succumbed to infection (P < 0.001; logrank test Fig. 2 C). Vaccinated mice were also completely protected from challenge with 3.1 × 10
6 cfu (6.5 ×
LD50)of the clinical isolate Ab-154, whereas all control mice died within 24h (P < 0.001; log-rank test).
Similar results were obtained when mice were challenged with 4.1 × 10
6 cfu (74.5 × LD50) of the panresistant clinical isolate 113-16, with all but one of the vaccinated mice surviving challenge and all control mice succumbing to infection (P < 0.001; log-rank test). These results demonstrate that vaccination with the
IWC vaccine provides protective immunity against infection with diverse A. baumannii strains
3.4.
Passive immunization with vaccine artisera 4. Discussión
We next determined if vaccine serum from mice immunized Infections caused by A. baumannii represent a growing public with the IWC vaccine could be used to passively immunize naïve heath concern due to their increasing prevalence and the increasmice. To determine if vaccine serum could reduce post-infection ing rates of antibiotic resistance. However, unlike pathogens that bacterial loads, vaccine or naïve sera were administered to mice cause widespread disease in the general population, A. baumannii 3 h prior to infection with 2.0 × 10 5 cfu (30.3 × LD50) of the ATCC produces infection in certain groups with well-defined risk fac19606 strain and bacterial loads in the spleen were determined tors such as patients receiving mechanical ventilation, residents 12 h post-infection. As shown in Fig. 3 A, passive immunization with of long-term care facilities, and military personal [1,2,4] . Targeted serum from vaccinated mice reduced post-infection bacterial loads vaccination of these groups may therefore be most effective in more than 1000-fold compared to mice that received naïve serum preventing A. baumannii infection. In the present study, we demon(P =
0.002; Mann–Whitney U test). In order to determine if pas-strate that immunization with aluminumadjuvanted, inactivated sively immunized mice were protected from bacterial challenge, whole cells may represent a viable strategy for vaccinating against vaccine or naïve sera were administered to mice 3 h prior to chal-A. baumannii. Immunization with the IWC vaccine produced a lenge with 3.5 × 10
5 cfu (53.0 ×
LD50) of the ATCC 19606 strain. robustantibodyresponse,andwasabletosignificantlyreducepost-All mice passively immunized with vaccine serum were protected infection bacterial loads and serum proinflammatory cytokine from challenge, whereas all but one of the mice receiving naïve levels. Importantly, immunization was able to protect mice from serum succumbed to infection (P = 0.0011; log-rank test; Fig.
3 B). challenge with A. baumannii.
It has previously been shown that the surface expression of certain membrane proteins is down-regulated in
A. baumannii during the acquisition of antibiotic resistance [17] . This may be problematic if these antigens are targeted by a vaccine composed of a single protein subunit. Here we show that immunization with an
IWC vaccine produces antibodies against multiple bacterial antigens. This may be beneficial in providing protection against diverse strains of A. baumannii with varying surface protein compositions. Importantly, in this study we demonstrate that vaccination with the IWC vaccine provided protection from 3 clonally distinct strains, including a panresistant clinical isolate. An additional potential benefit of using an IWC vaccine is that inactivated whole cells are relatively easy and inexpensive to manufacture and the production process does not require denaturing of the vaccine antigens. Conversely, protein subunit vaccines can require

complicated and expensive purification processes, and may result in antigens that are conformationally distinct from their native form.
In situations in which the completion of a vaccination schedule prior to bacterial exposure is not possible, the use of passive immunization may be beneficial. This approach has been developed for preventing infections caused by other antibiotic resistant bacteria, such as Staphylococcus aureus [18] . Here we show that passive immunization with serum from mice vaccinated with the IWC vaccine is able to reduce post-infection bacterial loads and protect mice from infection when administered 3 h prior to inoculation. These results demonstrate that antibodies alone are sufficient for providing protective immunity against A. baumannii infection, which may have implications for the development of future antibodybased therapies. In summary, these data demonstrate that active and passive immunization can prevent disseminated A. baumannii infection in a murine model, and that an IWC vaccine may provide a viable immunization approach.
Acknowledgements
The authors thank members of the Pachón laboratory for helpful discussions and Pilar Peréz-Romero for providing reagents and critical reading of the manuscript. This work was funded by the Consejería de
Innovación, Ciencia y Empresa de la Junta de Andalucía P08-CTS-03842 awarded to JP and by the
Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III -co-financed by European Development
Regional Fund “A way to achieve Europe” ERDF, Spanish Network for the Research in Infectious Diseases
(REIPI RD06/0008/0000). MJM is supported by the Subprograma Juan de la Cierva from the Ministerio de
Ciencia e Innovación of Spain.
Conflict of interest statement: MJM and JP are listed as inventors on a patent registered by the Foundation for Health Research of Seville (FISEVI) with the Spanish Office of Patents and Trademarks covering the vaccine described in this study.
References
[1] Munoz-Price LS, Weinstein RA. Acinetobacter infection. N Engl J Med 2008;358(March (12)):1271–81.
[2] Jones A, Morgan D, Walsh A, Turton J, Livermore D, Pitt T, et al. Importation of multidrug-resistant
Acinetobacter spp infections with casualties from Iraq. Lancet Infect Dis 2006;6(June (6)):317–8.
[3] Ho PL, Cheng VC, Chu CM. Antibiotic resistance in community-acquired pneumonia caused by
Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii. Chest
2009;136(October (4)):1119–27.
[4] Sengstock DM, Thyagarajan R, Apalara J, Mira A, Chopra T, Kaye KS. Multidrug-Resistant
Acinetobacter baumannii: an emerging pathogen among older adults in community hospitals and nursing homes. Clin Infect Dis 2010;50(June (12)):1617–8.
[5] Vila J, Pachon J. Therapeutic options for Acinetobacter baumannii infections. Expert Opin Pharmacother
2008;9(March (4)):587–99.
[6] Adams MD, Nickel GC, Bajaksouzian S, Lavender H, Murthy AR, Jacobs MR, et al. Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system.
Antimicrob Agents Chemother 2009;53(September (9)):3628–34.
[7] Taccone FS, Rodriguez-Villalobos H, De Backer D, De Moor V, Deviere J, Vincent JL, et al. Successful treatment of septic shock due to pan-resistant Acinetobacter baumannii using combined antimicrobial therapy including tigecycline. Eur J Clin Microbiol Infect Dis 2006;25(April (4)):257–60.
[8] Valencia R, Arroyo LA, Conde M, Aldana JM, Torres MJ, Fernandez-Cuenca F, et al. Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter baumannii in a tertiary care university hospital.
Infect Control Hosp Epidemiol 2009;30(March (3)):257–63.
[9] Billips BK, Yaggie RE, Cashy JP, Schaeffer AJ, Klumpp DJ. A live-attenuated vaccine for the treatment of urinary tract infection by uropathogenic Escherichia coli. J Infect Dis 2009;200(July (2)):263–72.
[10] Cripps AW, Dunkley ML, Clancy RL. Mucosal and systemic immunizations with killed Pseudomonas aeruginosa protect against acute respiratory infection in rats. Infect Immun 1994;62(April (4)):1427–36.

[11] Cripps AW, Peek K, Dunkley M, Vento K, Marjason JK, McIntyre ME, et al. Safety and immunogenicity of an oral inactivated whole-cell Pseudomonas aeruginosa vaccine administered to healthy human subjects. Infect Immun 2006;74(February (2)):968–74.
[12] Zaidi TS, Priebe GP, Pier GB. A live-attenuated Pseudomonas aeruginosa vaccine elicits outer membrane protein-specific active and passive protection against corneal infection. Infect Immun
2006;74(February (2)):975– 83.
[13] Fernandez-Cuenca F, Pascual A, Ribera A, Vila J, Bou G, Cisneros JM, et al. Clonal diversity and antimicrobial susceptibility of Acinetobacter baumannii isolated in Spain. A nationwide multicenter study:
GEIH-Ab project 2000. Enferm Infecc Microbiol Clin 2004;22(May (5)):267–71.
[14] Cuenca FF, Pascual A, Martinez Marinez L, Conejo MC, Perea EJ. Evaluation of SDS-polyacrylamide gel systems for the study of outer membrane protein profiles of clinical strains of Acinetobacter baumannii. J
Basic Microbiol 2003;43(3):194–201.
[15] McConnell MJ, Hanna PC, Imperiale MJ. Cytokine response and survival of mice immunized with an adenovirus expressing Bacillus anthracis protective antigen domain 4. Infect Immun 2006;74(February
(2)):1009–15.
[16] Martin RM, Brady JL, Lew AM. The need for IgG2c specific antiserum when isotyping antibodies from
C57BL/6 and NOD mice. J Immunol Methods 1998;212(March (2)):187–92.
[17] Vila J, Marti S, Sanchez-Cespedes J. Porins, efflux pumps and multidrug resistance in Acinetobacter baumannii. J Antimicrob Chemother 2007;59(June (6)):1210–5.
[18] Schaffer AC, Lee JC. Vaccination and passive immunisation against Staphylococcus aureus. Int J
Antimicrob Agents 2008;32(November (Suppl. 1)):S71–8.
Corresponding author at: Unit of Infectious Disease, Microbiology, and Preventive Medicine, Hospital
Universitario Virgen del Rocío, Edificio Laboratorio Anexo Caracola, Avenida Manuel Siurot s/n, 41013
Sevilla, Spain. Tel.: +34 955013651; fax: +34 955013292.
E-mail addresses: mmcconn@umich.edu
, mmcconne75@hotmail.com (M. J. McConnell).