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ARTICLE IN PRESS Vaccine xxx (2014) xxx–xxx
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Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice Rhonda KuoLee a , Greg Harris a , Hongbin Yan b , H. Howard Xu c , Wayne J. Conlan a , Girishchandra B. Patel a , Wangxue Chen a,d,∗ a
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Department of Chemistry, Brock University, St. Catharines, ON L2S 3A1, Canada c Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA 90032, USA d Department of Biology, Brock University, St. Catharines, Ontario L2S 3A1, Canada b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 24 January 2014 Accepted 25 February 2014 Available online xxx Keywords: Acinetobacter baumannii Pneumonia Mucosal immunization B cells Neutrophils Mice
a b s t r a c t Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24 h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcR␥, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines. © 2014 Crown. Published by Elsevier Ltd. All rights reserved.
1. Introduction Acinetobacter baumannii is a significant cause of healthcareacquired infections worldwide with the respiratory tract being an important entry portal of infection [1,2]. Mortality rates of 35–70% have been reported for A. baumannii pneumonia [3]. In addition, the rapid emergence of multi-drug resistant strains [4–8] has made A. baumannii infections increasingly difficult to treat. Therefore,
∗ Corresponding author at: Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Room 3100, Ottawa, Ontario K1A 0R6, Canada. Tel.: +1 613 991 0924; fax: +1 613 952 9092. E-mail address: [email protected] (W. Chen).
there is an urgent need for the development of nonantibiotic-based intervention strategies to combat this pathogen [9]. Vaccination is one of the most effective measures for infection control, and is likely to be unaffected by the drug resistance of the pathogen. Indeed, several laboratories have begun to develop A. baumannii vaccines for active and passive immunization of targeted populations [10–12]. Vaccines based on inactivated whole cells [12] as well as several potential protective antigens [10,11,13–17] are being evaluated. However, essentially all experimental vaccine studies to date have been limited to evaluating protection against systemic infections and utilized either immunocompromised (such as neutropenic or diabetic) mice [8,11] or virulence-enhancing substance (such as porcine mucin) [12,16]. For more predictive determination of the efficacy of vaccines against
http://dx.doi.org/10.1016/j.vaccine.2014.02.083 0264-410X/© 2014 Crown. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: KuoLee R, et al. Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.083
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A. baumannii pneumonia, a pulmonary challenge model would be useful. We recently demonstrated that LAC-4, a clinical isolate of A. baumannii, caused 100% mortality in conventional BALB/c and C57BL/6 mice after an intranasal (i.n.) administration [18]. The infection caused acute pneumonia and bacteremia that closely resemble the course of human infection [18]. Here we used this model together with an inactivated whole cell vaccine to evaluate the feasibility of developing mucosal vaccines against respiratory A. baumannii infection and to investigate the potential protective mechanisms of such vaccines.
Table 1 Serum preparation information. Serum
Donor mouse strain
Immunization/infection
Collection time
Hyperimmune Convalescent Naïve
BALB/c BALB/c BALB/c
107 ffLAC-4 (i.n., day 0, 14, 21) 5 × 106 CFU LAC-4 (i.n.) –
Day 48 Day 7a Agematched
a
All convalescent mice had shown clinical signs before recovering from infection.
neutral buffered formalin, processed by standard paraffin embedding methods, and stained with hematoxylin–eosin [19].
2. Materials and methods 2.5. Cytokine and chemokine assays 2.1. Mice Eight- to twelve-week-old, specific-pathogen-free BALB/c and C57BL/6 mice were purchased from Charles River Laboratories (St. Constant, Quebec). Igh-Jtm1Dhu [B cell KO (knock-out)] and Fcer1gtm1Rav (FcR␥ KO) mice on the BALB/c background, and their age- and sex-matched control (wild-type, WT) mice were purchased from Taconic Farms, Inc. (Hudson, New York). The animals were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals, and all experimental procedures were approved by the institutional animal care committee. 2.2. Intranasal immunization and challenge Formalin-killed A. baumannii LAC-4 cells (ffLAC-4) were prepared as described previously [18] and intranasally administered in 50 l saline on day 0, 14 and 21 to mice under isoflurane anesthesia. Fecal pellets and sera were collected from individual mice at day 28. Naïve and immunized mice were challenged with LAC4 intranasally (i.n.) (108 CFU) or intraperitoneally (106 CFU) at day 42. Actual inoculum was verified by quantitative bacteriology. Body weight, clinical scores, and survival of the mice were monitored [19]. In some experiments, groups of five mice were sacrificed before challenge (0 h), or at 4 and 24 h post-challenge (hpc), and various tissues were aseptically removed and used for quantitative bacteriology or histopathology. In addition, blood samples were collected for bacteriology or serum separation. Lungs were lavaged to harvest bronchoalveolar lavage (BAL) fluid for total and differential cell counts [20] and the supernatant was collected and stored at −80 ◦ C. 2.3. ELISA for A. baumannii whole cell-specific IgA and IgG antibodies Levels of A. baumannii whole cell-specific antibodies in sera and mucosal samples were determined by ELISA [21]. Briefly, 96-well microplates were pre-coated with 106 ffLAC-4 cells/well in 100 l of sodium bicarbonate buffer (pH 9.6). Samples were either prediluted (1:2 for fecal, 1:100 for serum IgG and 1:20 for serum IgA) or undiluted (BAL fluid) for the assays. 2.4. Quantitative bacteriology and histopathology The lungs and spleens were homogenized in sterile saline. The number of viable A. baumannii in the respective organs were determined by plating 100 l aliquots of 10-fold serial dilutions of the homogenates on brain–heart infusion agar plates [19]. Blood samples were similarly cultured to assess bacteremia. For histopathology, the tissues were immediately immersed in 10%
Cytokine and chemokine levels in sera and BAL fluid were measured with 21-plex Milliplex MAP® mouse cytokine/chemokine kits (Millipore, Ltd. Billerica, MA) using a Luminex® 100IS system (Luminex, Austin, TX). Samples were assayed in duplicate, and the cytokine/chemokine concentrations were calculated against the standards, using Beadview® software (ver 1.03, Upstate) [19]. 2.6. Neutrophil depletion Groups of five immunized BALB/c mice were treated intraperitoneally with either 25 g rat anti-mouse monoclonal antibody RB6-8C5 (RB6) in 0.25 ml saline for in vivo depletion of neutrophils or equivalent amount of purified rat IgG (Sigma–Aldrich, St. Louis, MO) as a control [19,22]. The treatment was given 18 h before and at the time (0 h) of i.n. LAC-4 challenge. The specificity and efficacy of the RB6 antibody have been previously established by us and others [19,22,23]. 2.7. Passive serum transfer protection To assess the potential role of antibodies in the protection against i.n. challenge with LAC-4, various mouse sera preparations were transferred by i.p. injection to groups of naïve BALB/c mice (Table 1). Serum transfer was administered 24 h before (500 l per mouse) and at the time (0 h, 250 l per mouse) of i.n. challenge with 1.6 × 108 CFU of LAC-4. In one experiment, hyperimmune or naïve sera (50 l per mouse) was administered by i.n. instillation to mice 16 h before the i.n. challenge with 1.1 × 108 CFU of LAC-4. 2.8. Statistical analysis Data are presented as means ± standard deviation (SD) for each group. Differences were assessed by Student’s t test, one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc multiple comparison tests, when appropriate. Survival rates were analyzed by log-rank test followed by Bonferroni’s method for multiple comparison of survival curve. Differences were considered significant when p < 0.05. 3. Results and discussion 3.1. Intranasal immunization of mice with inactivated LAC-4 cells induces antigen-specific mucosal and systemic antibody responses and protects against lethal respiratory or systemic LAC-4 challenge Although recent studies have convincingly demonstrated the efficacy of experimental vaccination against a systemic A. baumannii challenge [12,16], the potential of these vaccines for protection against respiratory challenge has not been reported. To
Please cite this article in press as: KuoLee R, et al. Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.083
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OD405nm
4.0
3.0
*** ***
Sera IgG1
***
Sera IgG2a
OD405nm
A
3
3.0 2.0
2.0
***
1.0
1.0 0.0
0.0
5 x 103
Naïve
5 x 105
5 x 107
5 x 105
5 x 107
***
0.8
***
Sera IgA
*
0.6
Feces IgA
0.5
***
OD405nm
OD405nm
5 x 103
Naïve
0.4 0.2
***
0.4
***
0.3 0.2 0.1
0.0
5 x 103
Naïve
5 x 105
0.0
5 x 107
B
4
OD405
5 x 105
5 x 107
BALB/C
Naive Immunized
BAL
***
3
5 x 103
Naïve
C57BL/6
2
* **
1 0
IgA
IgG1
IP challenge 8.4 x 105 CFU
IN challenge 7 x 107 CFU
* *
Percent Survival
100 80 60 40 20 0 0
1
2
3
4
5
6
7
8
9
*
100
Percent Survival
C
C57BL/6
IgG2a
80 60 40 20 0 0
10
1
BALB/C
Percent Survival
80 60 40 20 0 1
2
3
4
5
6
7
Day post-challenge
3
4
5
6
7
8
9
10
8
9
10
* *
100
Percent Survival
* *
100
0
2
Day post-challenge
Day post-challenge
80
5 x 107 CFU 5 x 105 CFU 5 x 103 CFU Naïve
60 40 20 0 0
1
2
3
4
5
6
7
8
9
10
Day post-challenge
Fig. 1. Intranasal immunization with inactivated A. baumannii LAC-4 cells induces robust protection against a lethal i.n. or i.p. challenge with LAC-4. Groups (n = 5) of female BALB/c and C57BL/6 mice were intranasally immunized with 5 × 103 , 5 × 105 or 5 × 107 CFU equivalent of ffLAC-4 at day 0, 14 and 21. At day 28, blood and fecal pellets (A) were collected from all mice and BAL fluid (B) from mice immunized with 5 × 107 CFU for the measurement of specific IgA and IgG (IgG1 and IgG2a) antibodies by ELISA assays. The mice were subsequently challenged at day 42 with LAC-4 via the i.n. (7 × 107 CFU) or the i.p. (8.4 × 105 CFU) route and their survival rates were monitored daily for up to 10 days (C). *p < 0.05, **p < 0.01 and ***p < 0.005 vs. naïve mice, respectively.
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10
Log10 CFU/organ
examine this possibility, groups of BALB/c and C57BL/6 mice were intranasally immunized with varying numbers of ffLAC-4 cells and then challenged with lethal doses of LAC-4 either i.n. or i.p. As seen in Fig. 1A, both BALB/c and C57BL/c mice immunized with 5 × 107 ffLAC-4 developed high levels of ffLAC-4-specific serum IgG1 and IgG2a responses. This vaccination dose also induced moderate serum and fecal (an indication of mucosal response) ffLAC-4-specific IgA responses (Fig. 1A). Mucosal and systemic antibody responses in mice vaccinated with 5 × 105 ffLAC-4 cells were also observed in BALB/c mice, although at a lower magnitude, but not at all in the C57BL/6 mice. Mice vaccinated with 5 × 103 ffLAC-4 cells failed to develop detectable serum or fecal antibody responses (Fig. 1A). In addition, immunization of BALB/c mice with 5 × 107 ffLAC-4 induced significant amount of specific IgA, IgG1 and IgG2a in the BAL fluid (Fig. 1B). More significantly, all mice immunized with 5 × 107 ffLAC-4 cells survived the lethal i.n. (7 × 107 CFU) and the i.p. (8.4 × 105 CFU) challenges of LAC-4 (Fig. 1C). At the 5 × 105 ffLAC-4 immunization dose, only the BALB/c mice survived both the i.n. and the i.p. challenge. At this dose, 100% of the C57BL/6 mice survived i.n. challenge while only 20% survived i.p. challenge (Fig. 1C). Immunization with 5 × 103 ffLAC-4 cells provided no survival advantage for either mouse strain against i.n. or i.p. challenge (Fig. 1C). These results suggest that intranasal immunization might be effective in protecting against both systemic and respiratory infections with A. baumannii.
Lung
***
8 6 4 2 0 4
24
Hours post-challenge 7
Log10 CFU/organ
4
Spleen
6 5 4
***
3 2 1 0 4
24
Hours post-challenge 3.2. Quantitative bacteriology and histopathology
Log10 CFU/ml
To determine if intranasal ffLAC-4 immunization promotes pulmonary clearance of LAC-4 and limits its systemic dissemination, immunized or naive mice were challenged intranasally with 1.6 × 108 CFU of LAC-4 at day 42. Mice were euthanized at 4 and 24 hpc for quantitative bacteriology and histopathology. At 4 hpc, tissue and blood bacterial burdens of naïve and ffLAC-4-immunized mice were similar, with the burdens in the spleen being much lower than in the lung and only negligible bacteria detected in the blood (Fig. 2). At 24 hpc, LAC-4 burdens in the lungs of the immunized mice were significantly lower (>10-fold) than those in the lungs of the naïve mice (Fig. 2). In addition, while significant numbers of LAC-4 were recovered from the spleens and blood of the naïve mice, indicating extrapulmonary dissemination, only small numbers of LAC-4 were recovered from the spleens of the immunized mice, and the bacterial numbers in the blood were barely above the detectable limit (Fig. 2). Histopathologically, the lungs from naïve mice at 24 hpc showed moderate infiltration of mixed inflammatory cells in the perivascular and peribronchial space and lymphatic dilation (Fig. 3A) with the occasional presence of moderate numbers of mixed neutrophils and mononuclear cells in the airway lumen (Fig. 3A). The histopathological changes in the lungs of immunized mice killed at this time point were much milder and limited largely to the alveolar space where small to moderate numbers of neutrophils and macrophages were seen (Fig. 3B). The histological changes in the spleens and livers between the naïve and immunized mice killed at 24 hpc were relatively mild and comparable and there were no abnormal changes in the kidneys of any infected mice (data not shown). These results suggest that the protection induced by i.n. ffLAC-4 immunization is likely due to the actions of both pulmonary and systemic protective immunity, which controls the otherwise rapid local bacterial replication in the lungs and substantially restricts the extrapulmonary dissemination of this hypervirulent strain, rather than preventing the attachment and initial colonization of the pathogen at the site of entry since the bacterial burdens in the lungs of immunized and naïve mice at 4 hpc were similar.
6
Blood
5 4 3 ***
2 1 0 4
24
Hours post-challenge IN immunized
Naïve
Fig. 2. Intranasal immunization with inactivated LAC-4 cells reduces lung bacterial burdens and limits the extrapulmonary dissemination and bacteremia in vaccinated mice. Groups of female BALB/c mice (n = 5) were intranasally immunized with 107 ffLAC-4 cells at day 0, 14 and 21, and intranasally challenged with 1.6 × 108 CFU of LAC-4 at day 42. A. baumannii burdens in the lungs, spleen and blood in the naïve and immunized mice were determined by quantitative bacteriology at 4 and 24 h post-challenge. The data are presented as mean log10 CFU ± SD, and represent one of at least two experiments with similar results. The detection limits (dotted lines) for bacterial burdens were 1.3 log10 CFU/organ for lungs and spleen, and 1.0 log10 CFU/ml blood, respectively. ***p < 0.001.
3.3. Cytokine and chemokine responses To understand the protective mechanism induced by intranasal ffLAC-4 immunization, we compared the serum and BAL proinflammatory cytokine and chemokine levels between the immunized and naïve mice following i.n. LAC-4 challenge (Fig. 4). At 4 hpc, comparable amounts of interleukin (IL)-6, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-␣ were detected in the BAL fluid from the immunized and naïve mice (Fig. 4A). The levels of IL-1, monocyte chemotactic protein (MCP)-1 or regulated on activation, normal T cell expressed and secreted (RANTES) were generally
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Fig. 3. Photomicrographs of lung histopathology following i.n. challenge with LAC-4 in naïve (A) and ffLAC-4-immunized (B) mice. Groups of female BALB/c mice (n = 5) were intranasally immunized with 107 ffLAC-4 cells at day 0, 14 and 21, and intranasally challenged with 1.6 × 108 CFU of LAC-4 at day 42. The mice were sacrificed at 24 h post-challenge and tissues collected for histopathology. (A) The lung from a naïve, challenged mouse showing moderate inflammatory cell infiltration in the perivascular and peribronchial areas (arrows), and within the airway lumen (arrowhead). (B) The lung from an immunized, challenged mouse showing the mild inflammatory cell infiltration in the perivascular and peribronchial areas (arrows). H&E, Bar = 100 m.
24
0
40000
KC
30000
*** 20000 10000 0
4
24
5000 4000
60000 40000 20000 0
4
24
Concentration (pg/ml)
Hours post-challenge 25000 20000
α TNF-α
MCP-1
2000
***
1000 0
4
2500
RANTES
***
2000 1500 1000 500 0
4
0
24
24
8000
KC
20000
***
10000
0
4
24
40000
MIP-2
30000 20000 10000 0
*** 4
24
Hours post-challenge
***
6000 4000 2000 0
4
24
Hours post-challenge
40000
MCP-1
30000 20000 10000 0
Hours post-challenge
50000
IL-6
*** 4
24
Hours post-challenge
2000
RANTES
1500 1000 500 0
*** 4
24
Hours post-challenge
Hours post-challenge
IN immunized Naïve 4
30000
24
15000
5000
4
10000
Hours post-challenge
24
*
10000
*** 0
Hours post-challenge Concentration (pg/ml)
Concentration (pg/ml)
80000
MIP-2
100
24
3000
Hours post-challenge
100000
200
Hours post-challenge Concentration (pg/ml)
Concentration (pg/ml)
Hours post-challenge
4
300
Concentration (pg/ml)
20000
IL-1β β
Concentration (pg/ml)
4
***
400
Concentration (pg/ml)
0
40000
Concentration (pg/ml)
500
IL-6
Concentration (pg/ml)
1000
B
60000
Concentration (pg/ml)
IL-1β β
Similar to BAL fluid, the majority of cytokines/chemokines were low or below the detection limit in the sera at 4 hpc, except for moderate and comparable amounts of IL-6 and KC detected in both groups of mice (Fig. 4B). Compared to 4 hpc, the serum levels of all the cytokines/chemokines assayed were generally substantially higher at 24 hpc and levels in the naïve mice were significantly higher (p < 0.001) than those in the immunized mice (Fig. 4B).
Concentration (pg/ml)
1500
Concentration (pg/ml)
A
Concentration (pg/ml)
below the detection limit (
ARTICLE IN PRESS Vaccine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice Rhonda KuoLee a , Greg Harris a , Hongbin Yan b , H. Howard Xu c , Wayne J. Conlan a , Girishchandra B. Patel a , Wangxue Chen a,d,∗ a
Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Department of Chemistry, Brock University, St. Catharines, ON L2S 3A1, Canada c Department of Biological Sciences, California State University, Los Angeles, Los Angeles, CA 90032, USA d Department of Biology, Brock University, St. Catharines, Ontario L2S 3A1, Canada b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 24 January 2014 Accepted 25 February 2014 Available online xxx Keywords: Acinetobacter baumannii Pneumonia Mucosal immunization B cells Neutrophils Mice
a b s t r a c t Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24 h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcR␥, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines. © 2014 Crown. Published by Elsevier Ltd. All rights reserved.
1. Introduction Acinetobacter baumannii is a significant cause of healthcareacquired infections worldwide with the respiratory tract being an important entry portal of infection [1,2]. Mortality rates of 35–70% have been reported for A. baumannii pneumonia [3]. In addition, the rapid emergence of multi-drug resistant strains [4–8] has made A. baumannii infections increasingly difficult to treat. Therefore,
∗ Corresponding author at: Human Health Therapeutics, National Research Council Canada, 100 Sussex Drive, Room 3100, Ottawa, Ontario K1A 0R6, Canada. Tel.: +1 613 991 0924; fax: +1 613 952 9092. E-mail address: [email protected] (W. Chen).
there is an urgent need for the development of nonantibiotic-based intervention strategies to combat this pathogen [9]. Vaccination is one of the most effective measures for infection control, and is likely to be unaffected by the drug resistance of the pathogen. Indeed, several laboratories have begun to develop A. baumannii vaccines for active and passive immunization of targeted populations [10–12]. Vaccines based on inactivated whole cells [12] as well as several potential protective antigens [10,11,13–17] are being evaluated. However, essentially all experimental vaccine studies to date have been limited to evaluating protection against systemic infections and utilized either immunocompromised (such as neutropenic or diabetic) mice [8,11] or virulence-enhancing substance (such as porcine mucin) [12,16]. For more predictive determination of the efficacy of vaccines against
http://dx.doi.org/10.1016/j.vaccine.2014.02.083 0264-410X/© 2014 Crown. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: KuoLee R, et al. Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.083
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2
A. baumannii pneumonia, a pulmonary challenge model would be useful. We recently demonstrated that LAC-4, a clinical isolate of A. baumannii, caused 100% mortality in conventional BALB/c and C57BL/6 mice after an intranasal (i.n.) administration [18]. The infection caused acute pneumonia and bacteremia that closely resemble the course of human infection [18]. Here we used this model together with an inactivated whole cell vaccine to evaluate the feasibility of developing mucosal vaccines against respiratory A. baumannii infection and to investigate the potential protective mechanisms of such vaccines.
Table 1 Serum preparation information. Serum
Donor mouse strain
Immunization/infection
Collection time
Hyperimmune Convalescent Naïve
BALB/c BALB/c BALB/c
107 ffLAC-4 (i.n., day 0, 14, 21) 5 × 106 CFU LAC-4 (i.n.) –
Day 48 Day 7a Agematched
a
All convalescent mice had shown clinical signs before recovering from infection.
neutral buffered formalin, processed by standard paraffin embedding methods, and stained with hematoxylin–eosin [19].
2. Materials and methods 2.5. Cytokine and chemokine assays 2.1. Mice Eight- to twelve-week-old, specific-pathogen-free BALB/c and C57BL/6 mice were purchased from Charles River Laboratories (St. Constant, Quebec). Igh-Jtm1Dhu [B cell KO (knock-out)] and Fcer1gtm1Rav (FcR␥ KO) mice on the BALB/c background, and their age- and sex-matched control (wild-type, WT) mice were purchased from Taconic Farms, Inc. (Hudson, New York). The animals were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals, and all experimental procedures were approved by the institutional animal care committee. 2.2. Intranasal immunization and challenge Formalin-killed A. baumannii LAC-4 cells (ffLAC-4) were prepared as described previously [18] and intranasally administered in 50 l saline on day 0, 14 and 21 to mice under isoflurane anesthesia. Fecal pellets and sera were collected from individual mice at day 28. Naïve and immunized mice were challenged with LAC4 intranasally (i.n.) (108 CFU) or intraperitoneally (106 CFU) at day 42. Actual inoculum was verified by quantitative bacteriology. Body weight, clinical scores, and survival of the mice were monitored [19]. In some experiments, groups of five mice were sacrificed before challenge (0 h), or at 4 and 24 h post-challenge (hpc), and various tissues were aseptically removed and used for quantitative bacteriology or histopathology. In addition, blood samples were collected for bacteriology or serum separation. Lungs were lavaged to harvest bronchoalveolar lavage (BAL) fluid for total and differential cell counts [20] and the supernatant was collected and stored at −80 ◦ C. 2.3. ELISA for A. baumannii whole cell-specific IgA and IgG antibodies Levels of A. baumannii whole cell-specific antibodies in sera and mucosal samples were determined by ELISA [21]. Briefly, 96-well microplates were pre-coated with 106 ffLAC-4 cells/well in 100 l of sodium bicarbonate buffer (pH 9.6). Samples were either prediluted (1:2 for fecal, 1:100 for serum IgG and 1:20 for serum IgA) or undiluted (BAL fluid) for the assays. 2.4. Quantitative bacteriology and histopathology The lungs and spleens were homogenized in sterile saline. The number of viable A. baumannii in the respective organs were determined by plating 100 l aliquots of 10-fold serial dilutions of the homogenates on brain–heart infusion agar plates [19]. Blood samples were similarly cultured to assess bacteremia. For histopathology, the tissues were immediately immersed in 10%
Cytokine and chemokine levels in sera and BAL fluid were measured with 21-plex Milliplex MAP® mouse cytokine/chemokine kits (Millipore, Ltd. Billerica, MA) using a Luminex® 100IS system (Luminex, Austin, TX). Samples were assayed in duplicate, and the cytokine/chemokine concentrations were calculated against the standards, using Beadview® software (ver 1.03, Upstate) [19]. 2.6. Neutrophil depletion Groups of five immunized BALB/c mice were treated intraperitoneally with either 25 g rat anti-mouse monoclonal antibody RB6-8C5 (RB6) in 0.25 ml saline for in vivo depletion of neutrophils or equivalent amount of purified rat IgG (Sigma–Aldrich, St. Louis, MO) as a control [19,22]. The treatment was given 18 h before and at the time (0 h) of i.n. LAC-4 challenge. The specificity and efficacy of the RB6 antibody have been previously established by us and others [19,22,23]. 2.7. Passive serum transfer protection To assess the potential role of antibodies in the protection against i.n. challenge with LAC-4, various mouse sera preparations were transferred by i.p. injection to groups of naïve BALB/c mice (Table 1). Serum transfer was administered 24 h before (500 l per mouse) and at the time (0 h, 250 l per mouse) of i.n. challenge with 1.6 × 108 CFU of LAC-4. In one experiment, hyperimmune or naïve sera (50 l per mouse) was administered by i.n. instillation to mice 16 h before the i.n. challenge with 1.1 × 108 CFU of LAC-4. 2.8. Statistical analysis Data are presented as means ± standard deviation (SD) for each group. Differences were assessed by Student’s t test, one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc multiple comparison tests, when appropriate. Survival rates were analyzed by log-rank test followed by Bonferroni’s method for multiple comparison of survival curve. Differences were considered significant when p < 0.05. 3. Results and discussion 3.1. Intranasal immunization of mice with inactivated LAC-4 cells induces antigen-specific mucosal and systemic antibody responses and protects against lethal respiratory or systemic LAC-4 challenge Although recent studies have convincingly demonstrated the efficacy of experimental vaccination against a systemic A. baumannii challenge [12,16], the potential of these vaccines for protection against respiratory challenge has not been reported. To
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OD405nm
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Fig. 1. Intranasal immunization with inactivated A. baumannii LAC-4 cells induces robust protection against a lethal i.n. or i.p. challenge with LAC-4. Groups (n = 5) of female BALB/c and C57BL/6 mice were intranasally immunized with 5 × 103 , 5 × 105 or 5 × 107 CFU equivalent of ffLAC-4 at day 0, 14 and 21. At day 28, blood and fecal pellets (A) were collected from all mice and BAL fluid (B) from mice immunized with 5 × 107 CFU for the measurement of specific IgA and IgG (IgG1 and IgG2a) antibodies by ELISA assays. The mice were subsequently challenged at day 42 with LAC-4 via the i.n. (7 × 107 CFU) or the i.p. (8.4 × 105 CFU) route and their survival rates were monitored daily for up to 10 days (C). *p < 0.05, **p < 0.01 and ***p < 0.005 vs. naïve mice, respectively.
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10
Log10 CFU/organ
examine this possibility, groups of BALB/c and C57BL/6 mice were intranasally immunized with varying numbers of ffLAC-4 cells and then challenged with lethal doses of LAC-4 either i.n. or i.p. As seen in Fig. 1A, both BALB/c and C57BL/c mice immunized with 5 × 107 ffLAC-4 developed high levels of ffLAC-4-specific serum IgG1 and IgG2a responses. This vaccination dose also induced moderate serum and fecal (an indication of mucosal response) ffLAC-4-specific IgA responses (Fig. 1A). Mucosal and systemic antibody responses in mice vaccinated with 5 × 105 ffLAC-4 cells were also observed in BALB/c mice, although at a lower magnitude, but not at all in the C57BL/6 mice. Mice vaccinated with 5 × 103 ffLAC-4 cells failed to develop detectable serum or fecal antibody responses (Fig. 1A). In addition, immunization of BALB/c mice with 5 × 107 ffLAC-4 induced significant amount of specific IgA, IgG1 and IgG2a in the BAL fluid (Fig. 1B). More significantly, all mice immunized with 5 × 107 ffLAC-4 cells survived the lethal i.n. (7 × 107 CFU) and the i.p. (8.4 × 105 CFU) challenges of LAC-4 (Fig. 1C). At the 5 × 105 ffLAC-4 immunization dose, only the BALB/c mice survived both the i.n. and the i.p. challenge. At this dose, 100% of the C57BL/6 mice survived i.n. challenge while only 20% survived i.p. challenge (Fig. 1C). Immunization with 5 × 103 ffLAC-4 cells provided no survival advantage for either mouse strain against i.n. or i.p. challenge (Fig. 1C). These results suggest that intranasal immunization might be effective in protecting against both systemic and respiratory infections with A. baumannii.
Lung
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Log10 CFU/ml
To determine if intranasal ffLAC-4 immunization promotes pulmonary clearance of LAC-4 and limits its systemic dissemination, immunized or naive mice were challenged intranasally with 1.6 × 108 CFU of LAC-4 at day 42. Mice were euthanized at 4 and 24 hpc for quantitative bacteriology and histopathology. At 4 hpc, tissue and blood bacterial burdens of naïve and ffLAC-4-immunized mice were similar, with the burdens in the spleen being much lower than in the lung and only negligible bacteria detected in the blood (Fig. 2). At 24 hpc, LAC-4 burdens in the lungs of the immunized mice were significantly lower (>10-fold) than those in the lungs of the naïve mice (Fig. 2). In addition, while significant numbers of LAC-4 were recovered from the spleens and blood of the naïve mice, indicating extrapulmonary dissemination, only small numbers of LAC-4 were recovered from the spleens of the immunized mice, and the bacterial numbers in the blood were barely above the detectable limit (Fig. 2). Histopathologically, the lungs from naïve mice at 24 hpc showed moderate infiltration of mixed inflammatory cells in the perivascular and peribronchial space and lymphatic dilation (Fig. 3A) with the occasional presence of moderate numbers of mixed neutrophils and mononuclear cells in the airway lumen (Fig. 3A). The histopathological changes in the lungs of immunized mice killed at this time point were much milder and limited largely to the alveolar space where small to moderate numbers of neutrophils and macrophages were seen (Fig. 3B). The histological changes in the spleens and livers between the naïve and immunized mice killed at 24 hpc were relatively mild and comparable and there were no abnormal changes in the kidneys of any infected mice (data not shown). These results suggest that the protection induced by i.n. ffLAC-4 immunization is likely due to the actions of both pulmonary and systemic protective immunity, which controls the otherwise rapid local bacterial replication in the lungs and substantially restricts the extrapulmonary dissemination of this hypervirulent strain, rather than preventing the attachment and initial colonization of the pathogen at the site of entry since the bacterial burdens in the lungs of immunized and naïve mice at 4 hpc were similar.
6
Blood
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Hours post-challenge IN immunized
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Fig. 2. Intranasal immunization with inactivated LAC-4 cells reduces lung bacterial burdens and limits the extrapulmonary dissemination and bacteremia in vaccinated mice. Groups of female BALB/c mice (n = 5) were intranasally immunized with 107 ffLAC-4 cells at day 0, 14 and 21, and intranasally challenged with 1.6 × 108 CFU of LAC-4 at day 42. A. baumannii burdens in the lungs, spleen and blood in the naïve and immunized mice were determined by quantitative bacteriology at 4 and 24 h post-challenge. The data are presented as mean log10 CFU ± SD, and represent one of at least two experiments with similar results. The detection limits (dotted lines) for bacterial burdens were 1.3 log10 CFU/organ for lungs and spleen, and 1.0 log10 CFU/ml blood, respectively. ***p < 0.001.
3.3. Cytokine and chemokine responses To understand the protective mechanism induced by intranasal ffLAC-4 immunization, we compared the serum and BAL proinflammatory cytokine and chemokine levels between the immunized and naïve mice following i.n. LAC-4 challenge (Fig. 4). At 4 hpc, comparable amounts of interleukin (IL)-6, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-␣ were detected in the BAL fluid from the immunized and naïve mice (Fig. 4A). The levels of IL-1, monocyte chemotactic protein (MCP)-1 or regulated on activation, normal T cell expressed and secreted (RANTES) were generally
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Fig. 3. Photomicrographs of lung histopathology following i.n. challenge with LAC-4 in naïve (A) and ffLAC-4-immunized (B) mice. Groups of female BALB/c mice (n = 5) were intranasally immunized with 107 ffLAC-4 cells at day 0, 14 and 21, and intranasally challenged with 1.6 × 108 CFU of LAC-4 at day 42. The mice were sacrificed at 24 h post-challenge and tissues collected for histopathology. (A) The lung from a naïve, challenged mouse showing moderate inflammatory cell infiltration in the perivascular and peribronchial areas (arrows), and within the airway lumen (arrowhead). (B) The lung from an immunized, challenged mouse showing the mild inflammatory cell infiltration in the perivascular and peribronchial areas (arrows). H&E, Bar = 100 m.
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Similar to BAL fluid, the majority of cytokines/chemokines were low or below the detection limit in the sera at 4 hpc, except for moderate and comparable amounts of IL-6 and KC detected in both groups of mice (Fig. 4B). Compared to 4 hpc, the serum levels of all the cytokines/chemokines assayed were generally substantially higher at 24 hpc and levels in the naïve mice were significantly higher (p < 0.001) than those in the immunized mice (Fig. 4B).
Concentration (pg/ml)
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Concentration (pg/ml)
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Concentration (pg/ml)
below the detection limit (
