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Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii
Microbial Pathogenesis 96 (2016) 20e25
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii Xiaojiao Zhang 1, Tianxiang Yang 1, Ju Cao, Jide Sun, Wei Dai, Liping Zhang* Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 10 April 2016 Accepted 28 April 2016 Available online 29 April 2016
Multidrug-resistant Acinetobacter baumannii (A. baumannii) is a rapidly emerging pathogen causing infections with high mortality rates due to inadequate medical treatment. New ways to prevent and treat such infections are of a critical medical need. In this study, intranasal vaccination with A. baumannii outer membrane protein A (OmpA) induced both systemic and mucosal antibodies. After challenge intraperitoneally by clinical strains of multidrug-resistant A. baumannii, mice immunized with OmpA had a significantly higher survival rate than control mice. The OmpA protein level tested positive by western blot in clinical strains of A. baumannii. Furthermore, characterization of human sera for anti-OmpA immunoglobulin G (IgG) antibody levels demonstrated that OmpA protein was immunogenic in healthy individuals and patients with A. baumannii invasive infections. In conclusion, to the best of our knowledge, this is the first study protective efficacy of mucosal immunization with OmpA as a protein antigen against multidrug-resistant A. Baumannii. © 2016 Elsevier Ltd. All rights reserved.
Keywords: A. Baumannii Protein Vaccine
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.1. Bacterial strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.3. Collecting saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4. Collecting blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.5. Cloning, expression, and purification of recombinant OmpA in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.6. Production of hyperimmune mouse sera against OmpA in BALB/c mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.7. Active immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.8. Enzyme-linked immunosorbent assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.9. Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.10. Challenge studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11. Human sera samples and anti-OmpA immunoglobulin G (IgG) characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.12. Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.1. Immune responses elicited by mucosal immunization with recombinant OmpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2. Characterization of OmpA expression in clinical multidrug-resistant A. baumannii strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3. Protection against A. baumannii invasive infection by mucosal immunization with OmpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4. Characterization of specific anti-OmpA antibodies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
* Corresponding author. Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing, China. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2016.04.019 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
4.
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Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1. Introduction Acinetobacter baumannii has become an increasingly important human pathogen that causes various infections, including bacteremia, meningitis, pneumonia, endocarditis, skin and soft tissue infections and urinary tract infections [1,2]. In the last decade, A. baumannii has emerged as one of the most multidrug-resistant pathogens throughout the world [3e5]. In most cases, it is thought that infections are acquired after exposure to A. baumannii that persists on contaminated hospital equipment or by contact with healthcare personnel that have been exposed to the organism through contact with an infected patient. Infections caused by multidrug-resistant A. baumannii are associated with prolonged hospitalization, tremendous health care costs and high rates of death despite treatment [6e10]. Even more concerning is the increasing resistance of A. baumannii to both colistin and tigecycline [11e13]. However, both colistin and tigecycline which are metabolized through the kidney, are toxic especially in children. Innovative therapeutic approaches such as the development of new drugs and vaccines are necessary to treat infectious diseases caused by multidrug-resistant A. baumannii. It is likely that the most effective and rapid intervention for multidrug-resistant resistant A. baumannii will be a therapeutic vaccine. However, a lack of understanding of the protective immunity against A. baumannii has caused little progress in the development of an efficacious vaccine. Vaccines based on surface-exposed and secreted proteins are already commercially available and others are in development [14,15]. Outer membrane protein A (OmpA) has been found to be a predominant target of humoral immunity during bacterial infections [14e20]. In this study, we aimed to evaluate the protective effects of mucosal immunization with OmpA against A. baumannii infections. 2. Materials and methods 2.1. Bacterial strains The A. baumannii strain ATCC 19606 is an antibiotic susceptible reference strain, and clinic strains included in this study are multidrug-resistant clinical isolates previously characterized by pulsed-field gel electrophoresis [20,21]. The clinical isolates were confirmed as A. baumannii by a fully-automatic germ analysis system and amplified intrinsic blaOXA-51, and detailed information on bacterial strains has bee described in our previous study [20]. Escherichia coli (E. coli)DH5a competent Escherichia coli (Invitrogen) was used as the host for performing plasmid cloning. Recombinant proteins were expressed in BL21 competent E. coli (Novagen). 2.2. Animals 150 BABL/c mice, SPF grade, female, 6e8weeks, 18e22 g, bought from the Center of Laboratory of Chongqing Medical University. All animal experiments were approved by the respective ethics committees of ChongQing Medical University. 2.3. Collecting saliva Each
mouse
was
primed
with
2%
Grose
pilocarpine
hydrochloride (0.75 mg/100 g), Saliva was collected from each mouse by the insertion of absorbent sticks (Polyfiltronics) to the mouth. After 5 min, the sticks were transferred to phosphatebuffered saline, pH 7.4 (PBS), containing 10.0 mg of protease inhibitor (Sigma) per ml to prevent the proteolysis of antibodies. The dissolved saliva was pooled for each group of CBA/N mice and was stored at 70 C. 2.4. Collecting blood Pricking Orbital intravenous to collect blood, blood was stored at 70 C. 2.5. Cloning, expression, and purification of recombinant OmpA in E. coli The full-length AbOmpA gene (1317 bp, GenBank accession number AY485227) was amplified by PCR. A chromosomal preparation of A. baumannii (ATCC 19606) was used as the PCR substrate. The upstream primer 50 -ACAGGATCCATGAAATTGAGTCGTATT30 was designed to carry a BamHI restriction site and the downstream primer 50 -ACAAGCTTTTATTGAGCTGCTGCA-30 carried a HindIII restriction site. The PCR products digested with BamHI and HindIII were ligated into the pET28a expression vector. BL21/ pET28a cells harboring the OmpA gene were grown in Luria-Bertani (LB) medium at 37 C, and recombinant proteins were overexpressed with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 25 C, for 4 h. After sonication of bacterial cells, the pellet containing the inclusion bodies was discarded and the supernatant containing the soluble form of AbOmpA was collected by centrifugation. The 6X-His tagged protein was purified over a Ni-agarose affinity column according to the manufacturer's instructions (Qiagen). The column was equilibrated with binding buffer (20 mM TriseHCl, 500 mM NaCl and 5 mM imidazole) and was then combined with sample proteins. His-tagged OmpA was eluted by an elution buffer (20 mM TriseHCl, 500 mM NaCl and 300 mM imidazole). Imidazole and NaCl washed away or diluted through a Centrifugal Filter Devices(Amicon) and the samples were dissolved in phosphate-buffered saline (PBS, pH7.4) to reduce the salt concentrationrAbOmpA was analyzed for purity by SDS-PAGE (10% separation gel, Bio-Rad) after denaturation with the sample buffer at 100 C for 5min.After electrophoresis, the gels were stained with 0.1% (w/v) Coomassie blue R-250 and analyzed using the Gel Imaging System(Bio-Rad). 2.6. Production of hyperimmune mouse sera against OmpA in BALB/ c mice Hyperimmune mouse sera specific for OmpA (anti-OmpA) were generated by intraperitoneal immunization of BALB/c mice with recombinant OmpA protein. Every mouse was primed with 100 ml 1.5%sodium pentobarbital solution to anesthetize. Each mouse was primed with 10 mg of OmpA, emulsified in complete Freund's adjuvant (CFA, 1:1 ratio, v/v) on day zero, and boosted with the same concentration of recombinant protein emulsified in incomplete Freund's adjuvant (IFA, 1:1 ratio, v/v) on day 14. On day 28, each BALB/c mouse received the last dose of 10 mg of antigen in sterile PBS. At the sametime, the control mice were used PBS to
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replace OmpA, operation was same. Pooled sera from blood collected 14 days after the final immunization were stored at 20 C for future assays. 2.7. Active immunization In mucosal immunization studies, Every mouse was primed with 100 ml 1.5% sodium pentobarbital solution to anesthetize. groups of BALB/c mice (10 per group) were immunized three times intranasal vaccination with 10 mg of recombinant antigens in cholera toxin (CT, Sigma). The dose of the antigen solution corresponded to a mixture of 25 ml of antigen and 25 ml (0.4 mg/ml) of CT. The intranasal immunization was carried out with the BALB/c mouse held in a supine position with the head down while 10 ml of the antigen solution was delivered slowly with a micropipette onto the nares. At the sametime, the control mice were used PBS to replace OmpA, operation was same. Mice were immunized and boosted at 3 weeks, and then serum and saliva were harvested 1 week after boosting. 2.8. Enzyme-linked immunosorbent assay (ELISA) Ninety-six-well ELISA plates were coated with recombinant OmpA (10 mg/ml, 100 ml per well in PBS) overnight at 4 C and blocked with 200 ml of 10% fetal calf serum (Sigma) in PBS for 3 h at room temperature. Individual sera or saliva from immunized mice were tested in duplicate. Sera and saliva samples (100 ml) were added and serially diluted in PBS. After 1 h of incubation at 37 C, the plates were washed three times with 250 ml of PBS containing 0.05% Tween 20 (PBS-T) and titers of IgG and IgA were determined by the addition of 100 ml of peroxidase conjugated goat anti-mouse IgG (Sigma) and peroxidase-conjugated goat anti-mouse IgA (Sigma), respectively. Following incubation for an additional hour at 37 C, the plates were washed six times and the color reaction for the ELISA assay was developed by adding 100 ml of tetramethylbenzidine (Sigma). The assay was allowed to react for 15 min at 37 C and stopped with 50 ml 2% H2SO4. The plates were read spectrophotometrically at 450 nm and the endpoint titers were calculated for all samples. 2.9. Western blot analysis Whole-cell lysates were prepared from A. baumannii strains by boiling cell suspensions containing SDS-PAGE sample buffer (Ambresco) for 5 min. After electrophoresis in 10% polyacrylamide gels, the proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes for immunoblotting with BALB/c hyperimmune mouse sera diluted 1:1000. The detection of antigens was performed by an indirect antibody immunoassay using HRP-labeled anti-mouse IgG (Sigma) diluted 1:5000 in PBS-T and DAB staining.
Fig. 1. Serum and salivary antibody responses to mucosal immunization with OmpA. (A) IgG antibody titers in sera from intranasal-immunized mice. (B) IgA antibody titers in saliva from intranasal-immunized mice. Antibody titers were determined as the reciprocal of the dilution of sera or saliva yielding 50% of the maximum A450 above the background. The control is too low, not to be seen.
2.11. Human sera samples and anti-OmpA immunoglobulin G (IgG) characterization Sera from 13 healthy individuals not suffering from any infectious diseases at the time of sampling and sera from seven patients with positive blood culture for A. baumannii were obtained at The First Affiliated Hospital of Chongqing Medical University in a study
2.10. Challenge studies In the sepsis model, intraperitoneal-challenge experiments were performed. BALB/c mice were challenged 1 week after the last intranasal immunization with A. baumannii. A. baumannii strains were grown for 18 h at 37 C in MuellereHinton broth and then adjusted to the appropriate concentration in physiologic saline. Bacterial concentrations were determined by plating on blood agar. Mice were infected by intraperitoneal injection with 0.2 ml of the bacterial suspension and were carefully monitored for survival for 15 days. The lethal dose (LD) values for the strains used in challenge studies were determined by infecting groups of eight mice with 5fold dilutions of bacteria and analyzing the survival data.
Fig. 2. Expression of OmpA in clinical strains of multidrug-resistant Acinetobacter baumannii. (A) PCR analysis of multidrug-resistant A. baumannii. Molecular mass markers are indicated at the right. M, DNA markers. (B) Western blot analysis of A. baumannii strains. N, lysate of untransformed E. coli as a negative control. Recombinant OmpA served as a positive control. Lanes A-E, five different multidrug-resistant A. baumannii strains.
X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
23
Fig. 3. Protection by OmpA in a model of sepsis caused by multidrug-resistant A. baumannii. BALB/c mice were intranasally immunized with recombinant OmpA and then intraperitoneally challenged with five different multidrug-resistant A. baumannii strains (A strain, 5.0 108 cfu; B strain, 2 108 cfu; C strain, 5 107 cfu; D strain, 3 108; E strain, 3 107 cfu). Control mice (n ¼ 12) were sham immunized with CT only. Mucosal immunization with OmpA significantly protected the BALB/c mice (P < 0.05) from an intraperitoneal lethal challenge with A. baumannii.
approved by the Ethical Committees of Chongqing Medical University and The First Affiliated Hospital of Chongqing Medical University. These sera samples were analyzed for anti-OmpA IgG levels by ELISA using recombinant OmpA protein as coating antigens as described previously. 2.12. Statistical analyses The differences between the overall survival rates for the groups of mice were analyzed by Fisher's exact test. A nonparametric test (Mann-Whitney) was used to compare the concentrations of antiOmpA antibodies. The limit for statistical significance was a P value of 0.05. 3. Results 3.1. Immune responses elicited by mucosal immunization with recombinant OmpA The recombinant OmpA protein was firstly expressed and
purified (data not shown). ELISA assays were performed in order to evaluate the ability of intranasal immunization with OmpA to induce antibody production in BALB/c mice. The results are shown in Fig. 1. The results showed that the nasal route, with CT as a mucosal adjuvant, was a successful route that could be used to induce sera IgG antibodies against purified OmpA. nasal immunization with OmpA in CT was clearly evident for induction of IgA antibodies to OmpA in saliva. However, intranasal immunization with CT alone as a control did not lead to a systemic and mucosal anti-OmpA antibody response. 3.2. Characterization of OmpA expression in clinical multidrugresistant A. baumannii strains Different multidrug-resistant A. baumannii clinical isolates have been characterized in our previous study by pulsed-field gel electrophoresis [20]. We therefore characterized OmpA expression in these clinical strains. The results revealed that bands corresponding to OmpA were detected in the 20 clinical multidrug-resistant strains of A. baumannii tested (Fig. 2A). Using specific primers,
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X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
4. Discussion
Fig. 4. OmpA protein antigen was immunologic in healthy persons and patients with invasive A. baumannii infections. Sera samples were analyzed for anti-OmpA IgG levels by ELISA using recombinant proteins as coating antigens. Each data point represents an individual's data. A horizontal line denotes the median anti-OmpA IgG titer for the group. Antibody titers are expressed as ELISA units (OD, 450 nm) at 1:1000 serum dilutions.
PCR amplification exhibited single bands of identical size (1071 bp for OmpA) in all strains. These PCR products were then cloned into PMD-18T vectors, and subsequent DNA sequencing showed that OmpA in multidrug-resistant A. baumannii strains were highly conserved (data not shown). Furthermore, five multidrug-resistant strains of A. baumannii were randomly selected to analyze OmpA protein expression. Western blot analysis demonstrated that hyperimmune anti-sera specific for OmpA reacted with a single band of molecular mass 38 kDa in whole-cell lysates isolated from each of the strains tested, and anti-OmpA did not react with a lysate of the untransformed E. coli expression strain from which the recombinant protein was purified. 3.3. Protection against A. baumannii invasive infection by mucosal immunization with OmpA Since mucosal immunization with OmpA induced specific antiOmpA antibodies, we next determined if the immune response produced by mucosal vaccination with OmpA could protect mice from a challenge with multidrug-resistant A. baumannii strains. After analysis of anti-OmpA antibodies, BALB/c mice underwent an intraperitoneal challenge with five different clinical multidrugresistant A. baumannii strains characterized by pulsed-field gel electrophoresis [20]. In the sepsis model, the LD80 values for the five strains were 2.0 108 cfu (A strain), 1 108 cfu (B strain), 2.5 107 cfu (C strain), 1 108 (D strain)and 1 107 cfu (E strain). As shown in Fig. 3, the survival rates for mice that received intranasal OmpA in CT were significantly higher than those for mice that received CT alone as a control upon lethal challenge with multidrug-resistant A. baumannii strains (P < 0.05 in all cases). 3.4. Characterization of specific anti-OmpA antibodies in humans Next, 11 sera obtained from healthy subjects and seven sera from patients with positive blood culture for A. baumannii were analyzed for anti-OmpA IgG levels. In the group of healthy individuals, the median value for anti-OmpA IgG ELISA units was about 12,000 (Fig. 4). In the patient group, the median value for anti-OmpA IgG ELISA units was about 15,000. However, there was no significant difference in anti-OmpA IgG between the healthy and patient groups.
A. baumannii has emerged as a highly troublesome global pathogen. Treatment is complicated by high levels of antibiotic resistance, necessitating alternative means to prevent or treat A. baumannii infections. Due to the limitations of antibiotic treatment, there is a need to actively develop new drugs to control infections of multiple drug-resistant A. baumannii. Vaccination is the most promising and effective approach. Surface proteins play a fundamental role in the interaction between the bacterial cell and its environment [22e27]. OmpA of A. baumannii is a major porin protein that plays an important role in pathogenesis [28]. Studies have indicated that OmpA activates dendritic cells, which are important for determining the nature of the immune response against A. baumannii. The N-terminal region of OmpA has been reported to be responsible for host cell death [29]. In another study, OMVs from A. baumannii induced apoptosis of the host cells, whereas this effect was not detected in the OMVs from an OmpA mutant, thereby reflecting OmpAdependent host cell death. Taken together, the OmpA protein of A. baumannii is a potential antigen target for the development of a vaccine. In the current study, we found that mucosal immunization with recombinant OmpA induced both systemic and mucosal antiOmpA antibodies. In fact, intranasal vaccination may offer an alternative approach to current strategies since it induces a mucosal as well as a systemic immune response, while systemic immunization does not induce a mucosal immune response. Additionally, intranasal immunization with OmpA protected BALB/ c mice against death caused by five different clinical multidrugresistant A. baumannii strains characterized by pulsed-field gel electrophoresis as described in our previous study [20]. The lethal doses of these five strains were each unique. However, in this intraperitoneal-sepsis model, mucosal immunization with OmpA was effective in protecting against all the different strains tested, which was consistent with the results that different clinical multidrug-resistant expressed OmpA at both the gene and protein level without heterogeneity. Indicating that this protection was antibody mediated. The clinical targets of a vaccine are individuals with a high risk of infection such as patients with mechanical ventilation problems, patients previously treated with antibiotics or who have previously stayed in the ICU, as well as wounded military personal and patients with long-term hormone use, all of whom are susceptible to A. baumannii infections [30e32]. A recombinant protein vaccine antigen should be safe, effective and inexpensive. Although vaccines based on inactivated whole cells and attenuated strains are able to elicit antibodies against multiple surface proteins, the administration of whole organisms raises potential safety concerns [33]. An additional potential benefit of using recombinant protein subunit vaccines is that outer membrane vesicles are relatively complicated and expensive to manufacture [34]. In conclusion, our present results indicate that mucosal immunization with OmpA appears to be a novel, noninvasive vaccine approach that could protect mice against multidrug-resistant A. baumannii infections. Considering that OmpA is immunogenic in humans, the findings of the present study should stimulate attempts to extend the development of OmpA as a promising A. baumannii protein vaccine component. However, it is also apparent that protection against infection by mucosal immunization with OmpA is dependent on CT, which is not an acceptable adjuvant for humans due to its toxicity. Future studies will hopefully improve OmpA protein vaccine efficacy by the use of more efficient adjuvants.
X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
Acknowledgments This project was supported by National Natural Science Foundation grants of China (Grant No. 81272545 and No. 81471992).
[17]
[18]
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Active and passive immunization protects against lethal, extreme drug resistant-Acinetobacter baumannii infection, PLoS One 7 (2012) e29446. D.G. Moriel, S.A. Beatson, D.J. Wurpel, J. Lipman, G.R. Nimmo, D.L. Paterson, et al., Identification of novel vaccine candidates against multidrug-resistant Acinetobacter baumannii, PLoS One 8 (2013) e77631. L. Lin, B. Tan, P. Pantapalangkoor, T. Ho, A.M. Hujer, M.A. Taracila, et al., Acinetobacter baumannii rOmpA vaccine dose alters immune polarization and immunodominant epitopes, Vaccine 31 (2013) 313e318. n, Outer membrane vesicles as an M.J. McConnell, C. Rumbo, G. Bou, J. Pacho acellular vaccine against Acinetobacter baumannii, Vaccine 29 (2011) 5705e5710. W. Dai, S. Huang, S. Sun, J. Cao, L. Zhang, Nosocomial spread of carbapenemresistant A. baumannii (types ST75 and ST137) carrying blaOXA-23-like gene with an upstream ISAba1 in a Chinese hospital, Infect. Genet. Evol. 14 (2013) 98e101. M. Vaneechoutte, L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. de Vos, G. Claeys, Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis, J. Clin. Microbiol. 33 (1995) 11e15. G. Lindahl, M. Stalhammar-Carlemalm, T. Areschoug, Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens, Clin. Microbiol. Rev. 18 (2005) 102e127. H.S. Courtney, J.B. Dale, D.L. Hasty, in: Y.N. An, R.J. Friedman (Eds.), Handbook of Bacterial Adhesion: Principles,Methods and Applications, Human Press, Totowa, NJ, 2002, pp. 553e579. J. Lin, S. Huang, Q. Zhang, Outer membrane proteins: key players for bacterial adaptation in host niches, Microbes Infect. 4 (2002) 325e331. H.H. Niemann, W.D. Schubert, D.W. Heinz, Adhesins and invasins of pathogenic bacteria: a structural view, Microbes Infect. 6 (2004) 101e112. H. Ton-That, L.A. Marraffini, O. Schneewind, Protein sorting to the cell wall envelope of Gram-positive bacteria, Biochim. Biophys. Acta 1694 (2004) 269e278. R. Janulczyk, M. Rasmussen, Improved pattern for genome-based screening identifies novel cell walleattached proteins in gram-positive bacteria, Infect. Immun. 69 (2001) 4019e4026. J.S. Lee, J.C. Lee, C.M. Lee, I.D. Jung, Y.I. Jeong, E.Y. Seong, et al., Outer membrane protein A of A. baumannii induces differentiation of CD4þ T cells toward a Th1 polarizing phenotype through the activation of dendritic cells, Biochem. Pharmacol. 74 (2007) 86e97. J.S. Jin, S.O. Kwon, D.C. Moon, M. Gurung, J.H. Lee, S.I. Kim, et al., A. baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles, PLoS One 6 (2011) e17027. L.S. Munoz-Price, R.A. Weinstein, Acinetobacter infection, N. Engl. J. Med. 358 (2008) 1271e1281. A. Jones, D. Morgan, A. Walsh, J. Turton, D. Livermore, T. Pitt, et al., Importation of multidrug-resistant Acinetobacter spp infections with casualties from Iraq, Lancet Infect. Dis. 6 (2006) 317e318. D.M. Sengstock, R. Thyagarajan, J. Apalara, A. Mira, T. Chopra, K.S. Kaye, Multidrug-Resistant A. baumannii: an emerging pathogen among older adults in community hospitals and nursing homes, Clin. Infect. Dis. 50 (2010) 1617e1618. nimo Pacho n, Active and passive immunization Michael J. McConnell, Jero against A. baumannii using an inactivated whole cell vaccine, Vaccine 29 (2011) 1e5. pez-Rojas, F. DocoboM.J. McConnell, J. Domínguez-Herrera, Y. Smani, R. Lo rez, J. Pacho n, Vaccination with outer membrane complexes elicits rapid Pe protective immunity to multidrug-resistant A. baumannii, Infect. Immun. 79 (2011) 518e526.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii Xiaojiao Zhang 1, Tianxiang Yang 1, Ju Cao, Jide Sun, Wei Dai, Liping Zhang* Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 10 April 2016 Accepted 28 April 2016 Available online 29 April 2016
Multidrug-resistant Acinetobacter baumannii (A. baumannii) is a rapidly emerging pathogen causing infections with high mortality rates due to inadequate medical treatment. New ways to prevent and treat such infections are of a critical medical need. In this study, intranasal vaccination with A. baumannii outer membrane protein A (OmpA) induced both systemic and mucosal antibodies. After challenge intraperitoneally by clinical strains of multidrug-resistant A. baumannii, mice immunized with OmpA had a significantly higher survival rate than control mice. The OmpA protein level tested positive by western blot in clinical strains of A. baumannii. Furthermore, characterization of human sera for anti-OmpA immunoglobulin G (IgG) antibody levels demonstrated that OmpA protein was immunogenic in healthy individuals and patients with A. baumannii invasive infections. In conclusion, to the best of our knowledge, this is the first study protective efficacy of mucosal immunization with OmpA as a protein antigen against multidrug-resistant A. Baumannii. © 2016 Elsevier Ltd. All rights reserved.
Keywords: A. Baumannii Protein Vaccine
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.1. Bacterial strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.3. Collecting saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4. Collecting blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.5. Cloning, expression, and purification of recombinant OmpA in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.6. Production of hyperimmune mouse sera against OmpA in BALB/c mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.7. Active immunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.8. Enzyme-linked immunosorbent assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.9. Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.10. Challenge studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11. Human sera samples and anti-OmpA immunoglobulin G (IgG) characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.12. Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.1. Immune responses elicited by mucosal immunization with recombinant OmpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2. Characterization of OmpA expression in clinical multidrug-resistant A. baumannii strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3. Protection against A. baumannii invasive infection by mucosal immunization with OmpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4. Characterization of specific anti-OmpA antibodies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
* Corresponding author. Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing, China. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2016.04.019 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
4.
21
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1. Introduction Acinetobacter baumannii has become an increasingly important human pathogen that causes various infections, including bacteremia, meningitis, pneumonia, endocarditis, skin and soft tissue infections and urinary tract infections [1,2]. In the last decade, A. baumannii has emerged as one of the most multidrug-resistant pathogens throughout the world [3e5]. In most cases, it is thought that infections are acquired after exposure to A. baumannii that persists on contaminated hospital equipment or by contact with healthcare personnel that have been exposed to the organism through contact with an infected patient. Infections caused by multidrug-resistant A. baumannii are associated with prolonged hospitalization, tremendous health care costs and high rates of death despite treatment [6e10]. Even more concerning is the increasing resistance of A. baumannii to both colistin and tigecycline [11e13]. However, both colistin and tigecycline which are metabolized through the kidney, are toxic especially in children. Innovative therapeutic approaches such as the development of new drugs and vaccines are necessary to treat infectious diseases caused by multidrug-resistant A. baumannii. It is likely that the most effective and rapid intervention for multidrug-resistant resistant A. baumannii will be a therapeutic vaccine. However, a lack of understanding of the protective immunity against A. baumannii has caused little progress in the development of an efficacious vaccine. Vaccines based on surface-exposed and secreted proteins are already commercially available and others are in development [14,15]. Outer membrane protein A (OmpA) has been found to be a predominant target of humoral immunity during bacterial infections [14e20]. In this study, we aimed to evaluate the protective effects of mucosal immunization with OmpA against A. baumannii infections. 2. Materials and methods 2.1. Bacterial strains The A. baumannii strain ATCC 19606 is an antibiotic susceptible reference strain, and clinic strains included in this study are multidrug-resistant clinical isolates previously characterized by pulsed-field gel electrophoresis [20,21]. The clinical isolates were confirmed as A. baumannii by a fully-automatic germ analysis system and amplified intrinsic blaOXA-51, and detailed information on bacterial strains has bee described in our previous study [20]. Escherichia coli (E. coli)DH5a competent Escherichia coli (Invitrogen) was used as the host for performing plasmid cloning. Recombinant proteins were expressed in BL21 competent E. coli (Novagen). 2.2. Animals 150 BABL/c mice, SPF grade, female, 6e8weeks, 18e22 g, bought from the Center of Laboratory of Chongqing Medical University. All animal experiments were approved by the respective ethics committees of ChongQing Medical University. 2.3. Collecting saliva Each
mouse
was
primed
with
2%
Grose
pilocarpine
hydrochloride (0.75 mg/100 g), Saliva was collected from each mouse by the insertion of absorbent sticks (Polyfiltronics) to the mouth. After 5 min, the sticks were transferred to phosphatebuffered saline, pH 7.4 (PBS), containing 10.0 mg of protease inhibitor (Sigma) per ml to prevent the proteolysis of antibodies. The dissolved saliva was pooled for each group of CBA/N mice and was stored at 70 C. 2.4. Collecting blood Pricking Orbital intravenous to collect blood, blood was stored at 70 C. 2.5. Cloning, expression, and purification of recombinant OmpA in E. coli The full-length AbOmpA gene (1317 bp, GenBank accession number AY485227) was amplified by PCR. A chromosomal preparation of A. baumannii (ATCC 19606) was used as the PCR substrate. The upstream primer 50 -ACAGGATCCATGAAATTGAGTCGTATT30 was designed to carry a BamHI restriction site and the downstream primer 50 -ACAAGCTTTTATTGAGCTGCTGCA-30 carried a HindIII restriction site. The PCR products digested with BamHI and HindIII were ligated into the pET28a expression vector. BL21/ pET28a cells harboring the OmpA gene were grown in Luria-Bertani (LB) medium at 37 C, and recombinant proteins were overexpressed with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 25 C, for 4 h. After sonication of bacterial cells, the pellet containing the inclusion bodies was discarded and the supernatant containing the soluble form of AbOmpA was collected by centrifugation. The 6X-His tagged protein was purified over a Ni-agarose affinity column according to the manufacturer's instructions (Qiagen). The column was equilibrated with binding buffer (20 mM TriseHCl, 500 mM NaCl and 5 mM imidazole) and was then combined with sample proteins. His-tagged OmpA was eluted by an elution buffer (20 mM TriseHCl, 500 mM NaCl and 300 mM imidazole). Imidazole and NaCl washed away or diluted through a Centrifugal Filter Devices(Amicon) and the samples were dissolved in phosphate-buffered saline (PBS, pH7.4) to reduce the salt concentrationrAbOmpA was analyzed for purity by SDS-PAGE (10% separation gel, Bio-Rad) after denaturation with the sample buffer at 100 C for 5min.After electrophoresis, the gels were stained with 0.1% (w/v) Coomassie blue R-250 and analyzed using the Gel Imaging System(Bio-Rad). 2.6. Production of hyperimmune mouse sera against OmpA in BALB/ c mice Hyperimmune mouse sera specific for OmpA (anti-OmpA) were generated by intraperitoneal immunization of BALB/c mice with recombinant OmpA protein. Every mouse was primed with 100 ml 1.5%sodium pentobarbital solution to anesthetize. Each mouse was primed with 10 mg of OmpA, emulsified in complete Freund's adjuvant (CFA, 1:1 ratio, v/v) on day zero, and boosted with the same concentration of recombinant protein emulsified in incomplete Freund's adjuvant (IFA, 1:1 ratio, v/v) on day 14. On day 28, each BALB/c mouse received the last dose of 10 mg of antigen in sterile PBS. At the sametime, the control mice were used PBS to
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replace OmpA, operation was same. Pooled sera from blood collected 14 days after the final immunization were stored at 20 C for future assays. 2.7. Active immunization In mucosal immunization studies, Every mouse was primed with 100 ml 1.5% sodium pentobarbital solution to anesthetize. groups of BALB/c mice (10 per group) were immunized three times intranasal vaccination with 10 mg of recombinant antigens in cholera toxin (CT, Sigma). The dose of the antigen solution corresponded to a mixture of 25 ml of antigen and 25 ml (0.4 mg/ml) of CT. The intranasal immunization was carried out with the BALB/c mouse held in a supine position with the head down while 10 ml of the antigen solution was delivered slowly with a micropipette onto the nares. At the sametime, the control mice were used PBS to replace OmpA, operation was same. Mice were immunized and boosted at 3 weeks, and then serum and saliva were harvested 1 week after boosting. 2.8. Enzyme-linked immunosorbent assay (ELISA) Ninety-six-well ELISA plates were coated with recombinant OmpA (10 mg/ml, 100 ml per well in PBS) overnight at 4 C and blocked with 200 ml of 10% fetal calf serum (Sigma) in PBS for 3 h at room temperature. Individual sera or saliva from immunized mice were tested in duplicate. Sera and saliva samples (100 ml) were added and serially diluted in PBS. After 1 h of incubation at 37 C, the plates were washed three times with 250 ml of PBS containing 0.05% Tween 20 (PBS-T) and titers of IgG and IgA were determined by the addition of 100 ml of peroxidase conjugated goat anti-mouse IgG (Sigma) and peroxidase-conjugated goat anti-mouse IgA (Sigma), respectively. Following incubation for an additional hour at 37 C, the plates were washed six times and the color reaction for the ELISA assay was developed by adding 100 ml of tetramethylbenzidine (Sigma). The assay was allowed to react for 15 min at 37 C and stopped with 50 ml 2% H2SO4. The plates were read spectrophotometrically at 450 nm and the endpoint titers were calculated for all samples. 2.9. Western blot analysis Whole-cell lysates were prepared from A. baumannii strains by boiling cell suspensions containing SDS-PAGE sample buffer (Ambresco) for 5 min. After electrophoresis in 10% polyacrylamide gels, the proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes for immunoblotting with BALB/c hyperimmune mouse sera diluted 1:1000. The detection of antigens was performed by an indirect antibody immunoassay using HRP-labeled anti-mouse IgG (Sigma) diluted 1:5000 in PBS-T and DAB staining.
Fig. 1. Serum and salivary antibody responses to mucosal immunization with OmpA. (A) IgG antibody titers in sera from intranasal-immunized mice. (B) IgA antibody titers in saliva from intranasal-immunized mice. Antibody titers were determined as the reciprocal of the dilution of sera or saliva yielding 50% of the maximum A450 above the background. The control is too low, not to be seen.
2.11. Human sera samples and anti-OmpA immunoglobulin G (IgG) characterization Sera from 13 healthy individuals not suffering from any infectious diseases at the time of sampling and sera from seven patients with positive blood culture for A. baumannii were obtained at The First Affiliated Hospital of Chongqing Medical University in a study
2.10. Challenge studies In the sepsis model, intraperitoneal-challenge experiments were performed. BALB/c mice were challenged 1 week after the last intranasal immunization with A. baumannii. A. baumannii strains were grown for 18 h at 37 C in MuellereHinton broth and then adjusted to the appropriate concentration in physiologic saline. Bacterial concentrations were determined by plating on blood agar. Mice were infected by intraperitoneal injection with 0.2 ml of the bacterial suspension and were carefully monitored for survival for 15 days. The lethal dose (LD) values for the strains used in challenge studies were determined by infecting groups of eight mice with 5fold dilutions of bacteria and analyzing the survival data.
Fig. 2. Expression of OmpA in clinical strains of multidrug-resistant Acinetobacter baumannii. (A) PCR analysis of multidrug-resistant A. baumannii. Molecular mass markers are indicated at the right. M, DNA markers. (B) Western blot analysis of A. baumannii strains. N, lysate of untransformed E. coli as a negative control. Recombinant OmpA served as a positive control. Lanes A-E, five different multidrug-resistant A. baumannii strains.
X. Zhang et al. / Microbial Pathogenesis 96 (2016) 20e25
23
Fig. 3. Protection by OmpA in a model of sepsis caused by multidrug-resistant A. baumannii. BALB/c mice were intranasally immunized with recombinant OmpA and then intraperitoneally challenged with five different multidrug-resistant A. baumannii strains (A strain, 5.0 108 cfu; B strain, 2 108 cfu; C strain, 5 107 cfu; D strain, 3 108; E strain, 3 107 cfu). Control mice (n ¼ 12) were sham immunized with CT only. Mucosal immunization with OmpA significantly protected the BALB/c mice (P < 0.05) from an intraperitoneal lethal challenge with A. baumannii.
approved by the Ethical Committees of Chongqing Medical University and The First Affiliated Hospital of Chongqing Medical University. These sera samples were analyzed for anti-OmpA IgG levels by ELISA using recombinant OmpA protein as coating antigens as described previously. 2.12. Statistical analyses The differences between the overall survival rates for the groups of mice were analyzed by Fisher's exact test. A nonparametric test (Mann-Whitney) was used to compare the concentrations of antiOmpA antibodies. The limit for statistical significance was a P value of 0.05. 3. Results 3.1. Immune responses elicited by mucosal immunization with recombinant OmpA The recombinant OmpA protein was firstly expressed and
purified (data not shown). ELISA assays were performed in order to evaluate the ability of intranasal immunization with OmpA to induce antibody production in BALB/c mice. The results are shown in Fig. 1. The results showed that the nasal route, with CT as a mucosal adjuvant, was a successful route that could be used to induce sera IgG antibodies against purified OmpA. nasal immunization with OmpA in CT was clearly evident for induction of IgA antibodies to OmpA in saliva. However, intranasal immunization with CT alone as a control did not lead to a systemic and mucosal anti-OmpA antibody response. 3.2. Characterization of OmpA expression in clinical multidrugresistant A. baumannii strains Different multidrug-resistant A. baumannii clinical isolates have been characterized in our previous study by pulsed-field gel electrophoresis [20]. We therefore characterized OmpA expression in these clinical strains. The results revealed that bands corresponding to OmpA were detected in the 20 clinical multidrug-resistant strains of A. baumannii tested (Fig. 2A). Using specific primers,
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4. Discussion
Fig. 4. OmpA protein antigen was immunologic in healthy persons and patients with invasive A. baumannii infections. Sera samples were analyzed for anti-OmpA IgG levels by ELISA using recombinant proteins as coating antigens. Each data point represents an individual's data. A horizontal line denotes the median anti-OmpA IgG titer for the group. Antibody titers are expressed as ELISA units (OD, 450 nm) at 1:1000 serum dilutions.
PCR amplification exhibited single bands of identical size (1071 bp for OmpA) in all strains. These PCR products were then cloned into PMD-18T vectors, and subsequent DNA sequencing showed that OmpA in multidrug-resistant A. baumannii strains were highly conserved (data not shown). Furthermore, five multidrug-resistant strains of A. baumannii were randomly selected to analyze OmpA protein expression. Western blot analysis demonstrated that hyperimmune anti-sera specific for OmpA reacted with a single band of molecular mass 38 kDa in whole-cell lysates isolated from each of the strains tested, and anti-OmpA did not react with a lysate of the untransformed E. coli expression strain from which the recombinant protein was purified. 3.3. Protection against A. baumannii invasive infection by mucosal immunization with OmpA Since mucosal immunization with OmpA induced specific antiOmpA antibodies, we next determined if the immune response produced by mucosal vaccination with OmpA could protect mice from a challenge with multidrug-resistant A. baumannii strains. After analysis of anti-OmpA antibodies, BALB/c mice underwent an intraperitoneal challenge with five different clinical multidrugresistant A. baumannii strains characterized by pulsed-field gel electrophoresis [20]. In the sepsis model, the LD80 values for the five strains were 2.0 108 cfu (A strain), 1 108 cfu (B strain), 2.5 107 cfu (C strain), 1 108 (D strain)and 1 107 cfu (E strain). As shown in Fig. 3, the survival rates for mice that received intranasal OmpA in CT were significantly higher than those for mice that received CT alone as a control upon lethal challenge with multidrug-resistant A. baumannii strains (P < 0.05 in all cases). 3.4. Characterization of specific anti-OmpA antibodies in humans Next, 11 sera obtained from healthy subjects and seven sera from patients with positive blood culture for A. baumannii were analyzed for anti-OmpA IgG levels. In the group of healthy individuals, the median value for anti-OmpA IgG ELISA units was about 12,000 (Fig. 4). In the patient group, the median value for anti-OmpA IgG ELISA units was about 15,000. However, there was no significant difference in anti-OmpA IgG between the healthy and patient groups.
A. baumannii has emerged as a highly troublesome global pathogen. Treatment is complicated by high levels of antibiotic resistance, necessitating alternative means to prevent or treat A. baumannii infections. Due to the limitations of antibiotic treatment, there is a need to actively develop new drugs to control infections of multiple drug-resistant A. baumannii. Vaccination is the most promising and effective approach. Surface proteins play a fundamental role in the interaction between the bacterial cell and its environment [22e27]. OmpA of A. baumannii is a major porin protein that plays an important role in pathogenesis [28]. Studies have indicated that OmpA activates dendritic cells, which are important for determining the nature of the immune response against A. baumannii. The N-terminal region of OmpA has been reported to be responsible for host cell death [29]. In another study, OMVs from A. baumannii induced apoptosis of the host cells, whereas this effect was not detected in the OMVs from an OmpA mutant, thereby reflecting OmpAdependent host cell death. Taken together, the OmpA protein of A. baumannii is a potential antigen target for the development of a vaccine. In the current study, we found that mucosal immunization with recombinant OmpA induced both systemic and mucosal antiOmpA antibodies. In fact, intranasal vaccination may offer an alternative approach to current strategies since it induces a mucosal as well as a systemic immune response, while systemic immunization does not induce a mucosal immune response. Additionally, intranasal immunization with OmpA protected BALB/ c mice against death caused by five different clinical multidrugresistant A. baumannii strains characterized by pulsed-field gel electrophoresis as described in our previous study [20]. The lethal doses of these five strains were each unique. However, in this intraperitoneal-sepsis model, mucosal immunization with OmpA was effective in protecting against all the different strains tested, which was consistent with the results that different clinical multidrug-resistant expressed OmpA at both the gene and protein level without heterogeneity. Indicating that this protection was antibody mediated. The clinical targets of a vaccine are individuals with a high risk of infection such as patients with mechanical ventilation problems, patients previously treated with antibiotics or who have previously stayed in the ICU, as well as wounded military personal and patients with long-term hormone use, all of whom are susceptible to A. baumannii infections [30e32]. A recombinant protein vaccine antigen should be safe, effective and inexpensive. Although vaccines based on inactivated whole cells and attenuated strains are able to elicit antibodies against multiple surface proteins, the administration of whole organisms raises potential safety concerns [33]. An additional potential benefit of using recombinant protein subunit vaccines is that outer membrane vesicles are relatively complicated and expensive to manufacture [34]. In conclusion, our present results indicate that mucosal immunization with OmpA appears to be a novel, noninvasive vaccine approach that could protect mice against multidrug-resistant A. baumannii infections. Considering that OmpA is immunogenic in humans, the findings of the present study should stimulate attempts to extend the development of OmpA as a promising A. baumannii protein vaccine component. However, it is also apparent that protection against infection by mucosal immunization with OmpA is dependent on CT, which is not an acceptable adjuvant for humans due to its toxicity. Future studies will hopefully improve OmpA protein vaccine efficacy by the use of more efficient adjuvants.
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Acknowledgments This project was supported by National Natural Science Foundation grants of China (Grant No. 81272545 and No. 81471992).
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