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Previous Article | Next Article 
Infection and Immunity, November 2003, p. 6329-6337, Vol. 71, No. 11
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.11.6329-6337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Debra C. Alperin,1 Linda K. Norton,1 and Stephen A. Hines1*
Department of Veterinary Microbiology and Pathology,1 Department of Veterinary Clinical Sciences, Washington State University, Pullman, Washington 99164-7040,2 Department of Veterinary Pathobiology, Purdue University, West Lafayette, Indiana 47907-20273
Received 24 March 2003/ Returned for modification 27 May 2003/ Accepted 5 August 2003
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ABSTRACT |
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INTRODUCTION |
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The exact mechanism of the protective immune response is unknown; however, there is substantial evidence that immune clearance of R. equi in horses is the result of a type 1-like pulmonary recall response. Adult horses challenged with virulent strains of R. equi effectively clear organisms from the lungs in association with an increase in pulmonary CD4+ and CD8+ T-lymphocytes (18). As determined by a flow cytometric method for detection of intracytoplasmic cytokines in bronchoalveolar lavage fluid (BALF) cells, clearance is associated with an increased number of T-lymphocytes that produce gamma interferon (IFN-
) (20). Pulmonary T-lymphocytes collected following challenge proliferate in response to stimulation with R. equi antigen and produce increased levels of IFN- but not interleukin-4 (26). Importantly, the adult horse model demonstrates the protective phenotype and likely reflects the type of response that an effective vaccine has to induce in foals. Thus, immune adult horses provide a model for identifying protective responses against R. equi. A vaccine that effectively induced these protective responses early in life would be used widely in foals and would have a significant impact on morbidity and mortality. However, the antigens that stimulate this protective immune response are unknown. In order to further investigate immunogenic antigens of R. equi, we modeled our approach on previous research performed with the closely related organism Mycobacterium tuberculosis.
R. equi and M. tuberculosis are both nocardioform actinomycetes that have genomes with high G+C contents and cell walls containing mycolic acid (16). These organisms are also similar in that they both infect and survive within alveolar macrophages, where they replicate within the phagosome and prevent phagolysosomal fusion (7, 17, 36, 45). Studies to identify targets of protective immune responses against M. tuberculosis have recently focused on secreted proteins present in culture filtrate supernatants (CFS). Secreted proteins of M. tuberculosis are released into the medium during growth and accumulate over time (2). Several studies have demonstrated that actively secreted proteins of M. tuberculosis induce cell-mediated responses in animals and humans (4, 6, 9, 21, 33, 44). The evidence that secreted proteins may be important in immunity to M. tuberculosis includes observations that CD4+ T-lymphocytes from immune mice proliferate and produce IFN- in response to these antigens (5) and that CD4+ T-lymphocytes from humans with active minimal tuberculosis secrete IFN- when they are stimulated with M. tuberculosis CFS proteins (9, 44). Furthermore, peripheral blood mononuclear cells from tuberculin-positive humans proliferate when they are stimulated with the 10- and 38-kDa M. tuberculosis secreted antigens (28, 29). A number of studies have demonstrated that immunization with secretory proteins of M. tuberculosis or with DNA encoding secreted antigens in CFS provides protective immunity in guinea pigs and mice when the animals are subsequently challenged with M. tuberculosis (10, 12, 21, 27, 31, 32).
Due to the similarity between R. equi and M. tuberculosis and the finding that M. tuberculosis CFS proteins are immunoprotective, we hypothesized that proteins in R. equi CFS would be immunogenic in infected foals and stimulate a type 1-like recall response in immune adult horses. To test this hypothesis, R. equi CFS proteins were identified by using immunoblots probed with sera from R. equi-infected foals and immune horses. In addition, the CFS was evaluated for the presence of previously described proteins encoded by the R. equi virulence plasmid. The CFS was then examined for the ability to induce proliferation of and IFN- expression by pulmonary T-lymphocytes isolated from the lungs of horses immune to R. equi.
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MATERIALS AND METHODS |
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Preparation of CFS of R. equi ATCC 33701, R. equi ATCC 33701 PC, and C. psuedotuberculosis. Cultures were centrifuged at 5,200 x g for 15 min at 4°C with a Beckman-Coulter Avanti J-25, and the supernatants were sterile filtered by using a 0.22-µm-pore-size membrane (Millipore, Bedford, Mass.). The supernatants were concentrated 100-fold by using a 10,000-molecular-weight concentrator (Centricon Plus-80; Amicon, Danvers, Mass.) according to the manufacturer's instructions and were dialyzed by extensive washing with sterilized, distilled water while they were in the ultrafiltration device. DMEM containing 10% BHI was sterile filtered and concentrated exactly like the bacterial CFS preparations for use as a medium-alone negative control in immunoblotting, lymphoproliferation, and real-time reverse transcription (RT)-PCR analyses. The protein contents of all preparations were determined by performing a Micro-BCA protein assay with an albumin standard according to the manufacturer's instructions (Pierce, Rockford, Ill.).
Preparation of soluble R. equi antigen. R. equi ATCC 33701 was grown in BHI medium for 72 h at 37°C and pH 7.0 with shaking, harvested by centrifugation at 5,000 x g for 15 min, and washed with sterile phosphate-buffered saline. Two milliliters of the bacterial pellet was resuspended in 10 ml of phosphate-buffered saline and frozen at -20°C for 2 h. The pellet was thawed, sonicated for 3 min, centrifuged at 12,000 x g for 15 min, and then disrupted by three freeze-thaw cycles at -80°C. After the third cycle, the bacterial homogenate was centrifuged again at 25,000 x g for 1 h to separate the soluble components from the intact bacteria and debris. The supernatant was collected to obtain the soluble R. equi antigen used in proliferation assays and real-time RT-PCR. The protein content was determined by a Micro-BCA protein assay.
SDS-PAGE and immunoblotting. DMEM-BHI, CFS, and bacterial pellets were boiled with a solution containing 350 mM Tris-Cl, 33% glycerol, 3.3% sodium dodecyl sulfate (SDS), 3.3% ß-mercaptoethanol, and 0.004% bromphenol blue; separated under nonreducing conditions by SDS-polyacrylamide gel electrophoresis (PAGE) by using a 12% acrylamide separation gel; and transferred electrophoretically to a nitrocellulose membrane (Hybond; Amersham Pharmacia Biotech Inc., Piscataway, N.J.). For the experiment whose results are shown in Fig. 5B, a 12% 2D-Prep gel (Bio-Rad Laboratories, Hercules, Calif.) was used, and 1.74 mg of 48-h CFS protein was loaded in a lane covering the entire gel in order to accurately interpret protein band comigration. For all other immunoblots, CFS and/or pellet antigen was loaded into individual lanes, as indicated below. Nitrocellulose membranes were incubated with sera from foals infected with R. equi (verified by isolation from transtracheal wash fluid), sera from immune horses 2 weeks postchallenge with R. equi ATCC 33701 (horses were shown to be immune by effective clearance of R. equi infection) (26), an anti-VapA monoclonal antibody (monoclonal antibody 10G5, provided by Shinji Takai) (38), or a monospecific rabbit antiserum to detect the presence of VapC, VapD, or VapE. VapA is a surface protein, and VapC, VapD, and VapE have been characterized previously as secreted proteins encoded by the R. equi ATCC 33701 virulence plasmid (11). Horseradish peroxidase-conjugated goat anti-horse, goat anti-rabbit, or goat anti-mouse immunoglobulin G (Kirkegaard & Perry, Gaithersburg, Md.) was used as a secondary antibody at a 1:20,000 dilution. The primary antibody dilutions used are given in the figure legends, and sera were obtained from the following foals and horses: foal 07 (see Fig. 2 and 5B); foals 136, CA11, and 151 (see Fig. 3A); and horses N124, 20, and 02 (see Fig. 3B and 4). Bound antibodies were detected by using a chemiluminescent substrate kit (ECL; Amersham Pharmacia Biotech Inc.).
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BAL and pulmonary challenge. All animal experiments were conducted in compliance with relevant federal guidelines and the Animal Care and Use Program of Washington State University. Previous studies have shown that peripheral blood mononuclear cells are not a sensitive indicator of the local pulmonary responses involved in clearance of R. equi (18); therefore, T-lymphocytes were isolated from the lungs of horses. Three clinically normal thoroughbred horses (horses 44, 71, and 02, which were 5, 17, and 28 years old, respectively) were studied. Two weekly bronchoalveolar lavages (BALs) were performed on each horse as previously described (18). Briefly, horses were mildly sedated with butorphanol and xylazine. An endoscope was passed nasally and directed into the right and left cranial lobar bronchi. A total of 540 ml of 0.9% sodium chloride-0.06% sodium bicarbonate (pH 6.5) was instilled into the right and left lungs in three 180-ml doses. After each instillation of 180 ml of saline, the BALF was aspirated for analysis. During the first BAL procedure (day 0) after both lungs had been completely aspirated, each lung was inoculated with 2 x 107 R. equi cells suspended in 5 ml of saline, and the endoscope was flushed with 60 ml of air just prior to removal. The second BAL (day 7) was performed by using the same procedure, except that no R. equi was instilled. On the day of each BAL procedure, blood was obtained via jugular venipuncture and submitted to the Washington State University Clinical Pathology Laboratory for determination of fibrinogen levels and complete blood counts. Following each BAL, the horses were placed in a stall for 1 week and monitored for fluctuations in rectal temperature and pulse and respiration rates. Previous work in our lab has shown that when infected in one lung, adult horses effectively clear the infection without major clinical signs in association with local, antigen-specific recall responses. Here we chose to infect both lungs in order to recover the maximum number of cells associated with a local recall response. Each horse infected in this study developed a transient fever of 39.4 to 40°C; however, all three horses effectively cleared the infection and had fibrinogen levels within the normal range 1 week postinfection. One horse developed a mild upper respiratory infection, and thus a more in-depth assessment was conducted, which included auscultation of the lungs with a rebreathing bag. This horse effectively cleared the respiratory infection 1 week after challenge, as determined by a lack of abnormal lung sounds and a negative BALF culture; thus, no antibiotics were administered.
BALF cell preparation.
The BALF was centrifuged at 400 x g for 8 min. The pelleted cells were resuspended and washed three times with RPMI (Gibco) containing 0.05 mg of gentamicin per ml, 25 mM HEPES, 3.5 x 10-6 M ß-mercaptoethanol, 100 mM L-glutamine (RPMI), and 2.5 µg of Fungizone per ml. If red blood cells were grossly apparent, cell pellets were incubated with 0.87% ammonium chloride for 5 min after the first wash. Cells were resuspended in RPMI containing 10% horse serum at a concentration of 2 x 106 cells/ml for a 1-h macrophage adherence analysis in 175-cm2 tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, N.Y.). Nonadherent cells were washed and resuspended at a concentration of 2 x 106 cells/ml for proliferation assays and quantification of IFN- mRNA.
Proliferation assay. BALF cells were assayed in quadruplicate wells in round-bottom 96-well microplates (Sarstadt Inc., Newton, N.C.) as previously described (18). Briefly, 4 x 105 cells were incubated with R. equi CFS, C. pseudotuberculosis CFS, or concentrated DMEM-BHI (all at four concentrations, 1, 2.5, 10, and 25 µg/ml) or with soluble R. equi antigen (2.5 and 10 µg/ml) for 96 h at 37°C in 5% CO2. Optimal stimulation was achieved with 10 µg of protein/ml (data not shown); therefore, 10 µg of DMEM-BHI per ml and each antigen were used in proliferation assays and in cytokine analyses. As a positive control, cells were also stimulated with pokeweed mitogen at a concentration of 5 µg/ml for 36 h at 37°C in 5% CO2. The cells were incubated for an additional 21 h after addition of 0.25 µCi of [3H]thymidine and were harvested with a Tomtec cell harvester. The uptake of [3H]thymidine was measured by liquid scintillation counting with a ß-plate reader (Wallac, Gaithersburg, Md.).
Quantification of IFN- mRNA.
For antigen-specific IFN- expression, cells were incubated as described above for the proliferation assay. After incubation for 24 h in 25- and 75-cm2 tissue culture flasks (Corning Inc., Corning, N.Y.) before and after intrabronchial challenge, respectively, the cultures were harvested as previously described (26). A culture period of 24 h was chosen to measure IFN- mRNA expression based on previous time course studies (15, 26; David Horohov, personal communication). RNA was immediately extracted with an RNeasy kit (Qiagen Inc., Valencia, Calif.) used according to the manufacturer's specifications. To eliminate contaminating DNA, RNA was subjected to a DNase treatment for 30 min at 37°C. The concentrations of RNA in samples were estimated by determining the optical densities at 260 and 280 nm, and cDNA was generated by using 1 µg of RNA with murine leukemia virus reverse transcriptase and random hexamers in a reaction for 20 min at 42°C. To confirm that no genomic DNA was present, portions of the DNase-treated RNA samples were used in cDNA reaction mixtures without reverse transcriptase (no-reverse transcriptase controls). The cDNA was then used to detect expression of IFN- by real-time PCR. BAL cells from a horse that was not challenged with R. equi were stimulated with pokeweed mitogen for 24 h under similar conditions and used to generate a linear standard curve for IFN- transcription in real-time PCR.
Expression of equine IFN- and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in BAL cells was determined by performing real-time PCR with previously described primers (26). Briefly, the fluorescent dye SYBR Green was used to measure amplified products in a thermocycler (iCycler; Bio-Rad Laboratories). Reactions (volume of reaction mixtures, 25 µl) were performed in 96-well plates containing 50 ng of cDNA, 1x SYBR Green buffer, 1.5 mM MgCl2, each deoxynucleoside triphosphate at a concentration of 1 mM, 0.04 U of uracil N-glycosylase (Amperase), and 0.2 U of AmpliTaq Gold polymerase. For each 96-well plate, a standard curve for IFN- and GAPDH was generated by using a dilution series of cDNA from a horse that was not challenged with R. equi. The resulting curves were used as standards to control for variation between plates. All samples were analyzed in triplicate, and amplification was performed by using the following procedure: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s. Transcript levels were determined for each sample by comparing the threshold cycle values of IFN- and GAPDH to the corresponding standard curves. The transcript levels of IFN- were normalized by using the transcript level value for GAPDH from the same sample (IFN-/GAPDH). In each real-time PCR, reaction mixtures containing no cDNA were included to control for extraneous DNA within the reagents.
Statistical analyses.
Statistical analyses were performed by using the SAS statistical package, version 8 (SAS Institute Inc., Cary, N.C.). Significant differences in proliferative responses and IFN- expression on days 0 and 7 were determined by using a randomized complete block design, followed by the Bonferroni multiple-comparison test. Significant differences in proliferative responses and IFN- expression between samples obtained before and after challenge were detected by using a repeated-measure randomized complete block design, followed by the Bonferroni multiple-comparison test. For all statistical analyses, the horse-antigen interaction was used as the error term for horse (block effect) and antigen. The blocking effect was significant in the analyses; thus, the randomized complete block design was used in this study. Data that were not normally distributed were transformed by using the square root transformation; transformation of data did not change the significance in any analysis. P values less than 0.05 were considered significant.
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RESULTS |
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The protein bands identified in CFS by this immunoblot analysis were divided into two subgroups based on relative migration in SDS-PAGE gels. A group of five distinct proteins with calculated molecular masses of 12 to 24 kDa were present in CFS after 4 h of culture and at all subsequent times. The locations of these bands appeared to be slightly different after 48 and 144 h of culture than after 4 h, and the bands were slightly more intense at 48 h and slightly less intense at 144 h. A second group of CFS proteins had calculated molecular masses between 56 and 99 kDa. The migration patterns of the proteins in this molecular mass range were different after 4 and 48 h of culture, and the intensities of the bands in this part of the blot were significantly diminished after 144 h of culture. An additional broad band at 50 kDa was detected in the 144-h CFS (Fig. 2, lane 8) and was also present in the 4- and 48-h pellets. In accordance with the observed increase in the growth of bacteria between 48 and 144 h (Fig. 1), the amount of antigen in the pellet increased during this time (Fig. 2, lanes 7, 9, and 10). Media from cultures in the late logarithmic growth phase (e.g., 144 h) contained increasing amounts of nonsecreted, cytoplasmic proteins due to passive release from dead and dying bacteria (2). Since our goal was to identify proteins that are actively secreted by live bacteria, 48-h CFS was used for further characterization.
In order to determine whether the proteins identified in CFS were uniquely recognized by antibodies from the one foal or were more broadly recognized by serum antibodies from all infected horses, CFS and pellet antigen were probed by using sera from 12 additional foals with active rhodococcal pneumonia and by using sera from five adult horses. The results for three representative foal sera and three representative horse sera are shown in Fig. 3A and B, respectively. The adult horses had been challenged with virulent R. equi 2 weeks prior to serum collection and were shown to be immune (see Materials and Methods) (26). Similar to the initial serum tested, sera from all six animals reacted with five distinct protein bands in the12- to 24-kDa range of the 48-h CFS blot (Fig. 3, lanes CFS). When reacted with the 48-h pellet antigen, all sera recognized the characteristic 15- to 17-kDa doublet attributed to VapA (Fig. 3, lanes P). Again, the migration pattern of proteins identified in CFS was different from the pattern of proteins identified in the bacterial pellet. Sera from the foals, but not sera from the immune adults, also recognized a small number of CFS proteins that migrated at higher molecular masses, between 55 and 99 kDa (Fig. 3A, lanes CFS). However, the number and migration pattern of these higher-molecular-mass proteins were not consistent for all foals.
Presence of plasmid-associated proteins in R. equi CFS. The R. equi virulence plasmid is required for the bacterium to replicate and survive within the phagosome and to cause clinical disease in foals (14). Therefore, the 48-h CFS from both the plasmid-bearing strain R. equi ATCC 33701 and the plasmid-cured strain R. equi ATCC 33701 PC were probed with sera from immune adult horses to determine whether the low-molecular-mass immunogenic protein bands were associated with the virulence plasmid. The distinct 12- to 24-kDa protein bands that were present in CFS from the plasmid-bearing strain were not detected in the CFS from the plasmid-cured strain (Fig. 4). These results support the hypothesis that the 12- to 24-kDa immunogenic proteins in CFS are encoded by genes on the virulence plasmid or the hypothesis that the expression of these proteins is regulated by genes on the virulence plasmid. The CFS from both the plasmid-cured strain and the virulent strain produced two to four protein bands at calculated molecular masses between 45 and 98 kDa that reacted with the sera from adult horses, although the reactivity was inconsistent (Fig. 4).
Identification of secreted proteins VapC, VapD, and VapE in R. equi CFS. To demonstrate that the VapA surface protein was not present in the 48-h CFS and to confirm that the 15- to 17-kDa protein in the 48-h pellet antigen recognized by the equine sera was in fact VapA, an immunoblot was probed with a monoclonal antibody to VapA (Fig. 5A). The characteristic 15- to 17-kDa VapA doublet was present in the lane containing the bacterial pellet (Fig. 5A, lane 5) but did not appear in the lane containing the CFS preparation (Fig. 5A, lane 4). Interestingly, however, the anti-VapA monoclonal antibody also reacted with a group of protein bands in the low-molecular-mass region of the CFS. The 48-h CFS contained six protein bands at 11, 12, 13, 17, 21, and 24 kDa, which are the predicted molecular masses of molecules that were reactive with the monoclonal antibody (Fig. 5A, lane 4). The monoclonal antibody did not react with any protein migrating in the higher-molecular-mass region of the immunoblot. As expected, no protein bands were identified when the anti-VapA antibody was used to probe CFS or pellet antigen from a plasmid-cured derivative of R. equi virulent strain ATCC 33701 (data not shown).
A previous study showed that VapA is not excreted into the medium and is therefore present only in bacterial pellet preparations (11). However, one explanation for the presence in R. equi CFS of proteins that are reactive with the anti-VapA antibody is shedding of VapA from the bacterial surface, possibly due to cleavage. An alternative explanation is active secretion of additional virulence-associated proteins related to VapA. The Vap proteins are encoded by a family of genes clustered in the pathogenicity island of the R. equi virulence plasmid. Recent work has shown that the 10G5 monoclonal antibody binds an epitope within a conserved region shared by multiple Vap proteins (42).
To further characterize R. equi CFS for the presence of secreted Vap proteins, the 48-h CFS was probed with monospecific rabbit antisera to VapC, VapD, and VapE (11) (Fig. 5B). These previously identified proteins are coordinately regulated with VapA (11). All three monospecific antisera recognized protein bands present in the CFS (Fig. 5B) and not in the pellet (data not shown). The anti-VapC and anti-VapD antisera reacted with bands at 12 and 15 kDa, respectively. Additionally, the anti-VapE antiserum identified a triplet, the dominant band of which migrated at 17 kDa. The protein bands identified by anti-VapC, anti-VapE, and possibly anti-VapD comigrated with protein bands recognized by equine serum (Fig. 5B).
R. equi CFS-specific lymphoproliferative responses. Immune adult horses effectively clear a challenge dose of virulent R. equi in association with a pulmonary recall response (18). The R. equi CFS specificity of lymphocytes recovered from BALF of three immune adult horses was evaluated in proliferation assays prior to challenge (day 0) and 1 week after challenge (day 7). Cells were stimulated with pokeweed mitogen, R. equi CFS, and (as a positive control) SRA, which contains VapA. The negative controls included DMEM-BHI (growth medium alone) and C. pseudotuberculosis CFS. Proliferation was evaluated by measuring incorporation of [3H]thymidine and was expressed in counts per minute. Prior to challenge, there was no significant difference in proliferation between antigen-stimulated BALF cells and nonstimulated BALF cells (Fig. 6A). This result also indicates that none of the antigens used had a mitogenic effect. In contrast, BALF cells obtained at day 7 postchallenge specifically proliferated in response to R. equi CFS and SRA, and the responses were significantly greater than the R. equi-specific responses before challenge (P < 0.01) (Fig. 6B). Additionally, the R. equi CFS-specific and SRA-specific responses after challenge were significantly greater (P < 0.05 and P < 0.01, respectively) than the proliferative responses of BALF cells stimulated with DMEM-BHI and C. pseudotuberculosis CFS, as well as the proliferative responses of nonstimulated BALF cells at day 7 (Fig. 6B). BALF cells from all the horses proliferated when they were stimulated with pokeweed mitogen (data not shown).
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expression by BALF cells.
Prior to challenge and on day 7 after challenge with R. equi, BALF cells were analyzed by real-time RT-PCR to detect expression of IFN- transcripts. Cells were stimulated for 24 h with R. equi CFS, C. pseudotuberculosis CFS, R. equi SRA, and DMEM-BHI as the growth medium control. The efficiency of the real-time PCR was >0.90 (data not shown). The cytokine transcript levels were normalized by determining the level of GAPDH transcripts for each sample. The data were expressed as mean normalized levels of IFN- expression for each of three horses to more clearly demonstrate individual variation. On day 0, the mean normalized IFN- transcript levels for all three horses were low for all antigen-stimulated cells (Fig. 7). However, in one horse the IFN- mRNA levels for R. equi SRA-stimulated cells were significantly greater than the levels for cells stimulated with DMEM-BHI and C. pseudotuberculosis CFS, as well as the levels for nonstimulated cells (horse 44; P < 0.01) (Fig. 7B). Importantly, on day 7 postchallenge the mean normalized IFN- transcript levels for cells stimulated with R. equi CFS and R. equi SRA for all three horses were significantly greater than the levels on day 0 (P < 0.001). Furthermore, after challenge the R. equi CFS- and SRA-specific IFN- transcript levels were significantly greater than the levels for cells stimulated with DMEM-BHI, C. pseudotuberculosis CFS, and medium alone (nonstimulated) for each horse (P < 0.01) (Fig. 7).
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DISCUSSION |
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Like M. tuberculosis CFS, R. equi CFS antigen is a complex mixture of bacterial proteins, which have been partially defined at this point. Equine antibodies were used to identify CFS proteins that were immunogenic in horses. Although different culture conditions may have influenced the composition of CFS, the CFS produced for use in our experiments did contain immunogenic proteins, as shown by equine antibody recognition. Importantly, the induction of antibodies in infected foals and horses also demonstrated that these proteins are expressed in vivo. Therefore, the use of antibodies allows initial identification of potentially important immunogens.
Ten to twelve CFS proteins were identified based on their migration in SDS-PAGE gels and their reactivities with equine sera in immunoblots. The most interesting of these CFS antigens was a group of five proteins that migrated between 12 and 24 kDa and were recognized by all equine sera. These proteins also appeared to be reactive with a monoclonal antibody that binds an epitope shared by multiple Vap proteins. Our failure to detect any of the 12- to 24-kDa bands in CFS from a plasmid-cured strain suggested that these low-molecular-mass CFS proteins are either encoded by or regulated by the R. equi virulence plasmid. Significantly, R. equi CFS contained VapC, VapD, and VapE, which are low-molecular-mass proteins that are encoded by genes in the pathogenicity island of the virulence plasmid and have previously been shown to be secreted. Furthermore, equine sera recognized proteins comigrating with VapC, VapE, and possibly VapD in immunoblots. Together, these data suggest that the immunogenic proteins identified in R. equi CFS are produced during natural infection, are recognized by immune adult horses, and are associated with the R. equi virulence plasmid.
Importantly, T-lymphocytes obtained from the lungs of adult horses following immune clearance of virulent R. equi proliferated in response to CFS antigen and produced IFN-. Although the correlation is imperfect, the induction of IFN- remains the best predictor of antigens that are capable of inducing significant protection in animal models of tuberculosis (4, 33, 44). There is substantial evidence that clearance of R. equi infection, like clearance of M. tuberculosis infection, requires a type 1-like T-lymphocyte response and that immune clearance is mediated by IFN- secretion. For example, a neutralizing antibody to IFN- blocked clearance of R. equi in immunocompetent mice that normally clear a pulmonary challenge (22). Furthermore, adoptive transfer of an R. equi-specific Th1 cell line producing IFN- into susceptible mice resulted in clearance of R. equi infection from the lungs. In contrast, in susceptible mice infused with an R. equi-specific Th2 cell line producing interleukin-4 and not IFN- there was no clearance and pulmonary lesions developed (23).
The CFS-specific induction of IFN- in the lungs of immune adult horses postchallenge also matches what is known about the requirements for immunity to R. equi. Previous work has demonstrated that when horses are challenged with plasmid-cured R. equi, there is a lack of IFN- production by pulmonary T-lymphocytes when these horses are compared to horses challenged with plasmid-bearing R. equi (20). Additionally, live virulent R. equi, rather than killed or avirulent R. equi, elicits protective immunity to R. equi infection (40). Thus, the IFN- response described in this study may have been stimulated by R. equi CFS proteins expressed in association with the virulent genotype, including proteins encoded by the virulence plasmid. Furthermore, M. tuberculosis research has indicated that low-molecular-mass CFS proteins with molecular masses between 6 and 31 kDa initially identified by antibodies proved to be important T-lymphocyte antigens in pulmonary tuberculosis (2-4, 6, 33, 35). Therefore, the pulmonary T-lymphocyte response identified in immune horses may also have been stimulated by the low-molecular-mass, plasmid-associated CFS proteins identified by equine antibodies.
The data reported here support our hypothesis that secreted antigens found in R. equi CFS are targets of the protective immune response in horses. The next steps in characterizing the targets of this protective immune response are to identify the specific proteins in CFS that stimulate IFN- production and to determine the contribution of proteins encoded by the virulence plasmid. Once specific CFS antigens capable of inducing an immunodominant response in immune horses are identified, their abilities to effectively stimulate protective immune responses when they are used to immunize naïve foals can be investigated. Although an efficacious vaccine may ultimately include both nonsecreted and secreted proteins, the observations made in this study provide a strong rationale for further study of R. equi CFS antigens as vaccine candidates.
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ACKNOWLEDGMENTS |
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We thank Sinji Takai for providing monoclonal antibody 10G5. We also thank Molly Loaiza for excellent technical assistance and are grateful for the help of Charles Gaskins with the statistical analyses.
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FOOTNOTES |
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Present address: Department of Pathology, Microbiology, and Immunology, University of California, Davis, Davis, CA 95616
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| 1. | Allen, G., M. Yeargan, L. R. R. Costa, and R. Cross. 1995. Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1. J. Virol. 69:606-612.[Abstract] |
| 2. | Andersen, P., D. Askgaard, L. Ljungqvist, J. Bennedsen, and I. Heron. 1991. Proteins released from Mycobacterium tuberculosis during growth. Infect. Immun. 59:1905-1910. |
| 3. | Andersen, P., and I. Heron. 1993. Specificity of a protective immune response against Mycobacterium tuberculosis. Infect. Immun. 61:844-851. |
| 4. | Andersen, P. 1994. The T cell response to secreted antigens of Mycobacterium tuberculosis. Immunobiology 191:537-547.[Medline] |
| 5. | Andersen, P., A. B. Andersen, A. L. Sorensen, and S. Nagai. 1995. Recall of long-lived immunity to Mycobacterium tuberculosis infection mice. J. Immunol. 154:3359-3372.[Abstract] |
| 6. | Arend, S. M., P. Andersen, K. E. van Meijgaarden, R. L. V. Skjot, Y. W. Subronto, J. T. van Dissel, and T. H. M. Ottenhoff. 2000. Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10. J. Infect. Dis. 181:1850-1854.[CrossRef][Medline] |
| 7. | Armstrong, J. A., and P. D. Hart. 1971. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134:713-740.[Abstract] |
| 8. | Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted  ß+ T-cells. Nature 372:691-694.[CrossRef][Medline] |
| 9. | Boesen, H., B. N. Jensen, T. Wilcke, and P. Andersen. 1995. Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis. Infect. Immun. 63:1491-1497.[Abstract] |
| 10. | Brandt, L., M. Elhay, I. Rosenkrands, E. B. Lindblad, and P. Andersen. 2000. ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect. Immun. 68:791-795. |
| 11. | Byrne, B. A., J. F. Prescott, G. H. Palmer, S. Takai, V. M. Nicholson, D. C. Alperin, and S. A. Hines. 2001. Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins. Infect. Immun. 69:650-656. |
| 12. | Coler, N. R., A. Campos-Neto, P. Ovendale, F. H. Day, S. P. Fling, L. Zhu, N. Serbina, J. L. Flynn, S. G. Reed, and M. R. Alderson. 2001. Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis. J. Immun. 166:6227-6235. |
| 13. | Emmons, W., B. Reichwein, and D. L. Winslow. 1991. Rhodoccocus equi infection in the patient with AIDS: literature review and report of an unusual case. Rev. Infect. Dis. 13:91-96.[Medline] |
| 14. | Giguère, S., M. K. Hondalus, J. A. Yager, P. Darrah, D. M. Mosser, and J. F. Prescott. 1999. Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infect. Immun. 67:3548-3557. |
| 15. | Giguère, S., and J. F. Prescott. 1999. Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction. Vet. Immunol. Immunopathol. 67:1-15.[CrossRef][Medline] |
| 16. | Goodfellow, M. 1987. Taxonomic status of Rhodococcus equi. Vet. Microbiol. 14:205-209.[CrossRef][Medline] |
| 17. | Heitala, S. K., and A. A. Ardands. 1987. Interaction of Rhodococcus equi with phagocytic cells from Rhodococcus equi-exposed and non-exposed foals. Vet. Microbiol. 14:307-320.[CrossRef][Medline] |
| 18. | Hines, M. T., K. M. Paasch, D. C. Alperin, G. H. Palmer, N. C. Westhoff, and S. A. Hines. 2001. Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses. Vet. Immunol. Immunopathol. 79:101-113.[CrossRef][Medline] |
| 19. | Hines, S. A., S. T. Kanaly, B. A. Byrne, and G. H. Palmer. 1997. Immunity to Rhodococcus equi. Vet. Microbiol. 56:177-186.[CrossRef][Medline] |
| 20. | Hines, S. A., D. M. Stone, M. T. Hines, A. D. C. Alperin, L. Norton, and T. C. McGuire. 2003. Clearance of virulent but not avirulent Rhodococcus equi from the lungs of adult horses is associated with intracytoplasmic gamma interferon production by CD4+ and CD8+ T lymphocytes. Clin. Diagn. Lab. Immunol. 10:208-215. |
| 21. | Horowitz, M. A., B. E. Lee, B. J. Dillon, and G. Harth. 1995. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92:1530-1534. |
| 22. | Kanaly, S. T., S. A. Hines, and G. H. Palmer. 1995. Cytokine modulation alters pulmonary clearance of Rhodococcus equi and development of granulomatous pneumonia. Infect. Immun. 63:3037-3041.[Abstract] |
| 23. | Kanaly, S. T., S. A. Hines, and G. H. Palmer. 1996. Transfer of a CD4+ Th1 cell line to nude mice effects clearance of Rhodococcus equi from the lung. Infect. Immun. 64:1126-1132.[Abstract] |
| 24. | Kauffman, S. H. E., and J. Hess. 1999. Impact of intracellular location of and antigen display by intracellular bacteria: implications for vaccine development. Immun. Lett. 65:81-84.[CrossRef][Medline] |
| 25. | Kedlaya, I., M. B. Ing, and S. S. Wong. 2001. Rhodococcus equi infections in immunocompetent hosts: case report and review. Clin. Infect. Dis. 32:e39-e47. |
| 26. | Lopez, A. M., M. T. Hines, G. H. Palmer, D. C. Alperin, and S. A. Hines. 2002. Identification of pulmonary T-lymphocytes and serum antibody isotype responses associated with protection against Rhodococcus equi. Clin. Diagn. Lab. Immunol. 9:1270-1276. |
| 27. | Lozes, E., K. Huygen, J. Content, O. Denis, D. L. Montgomery, A. M. Yawman, P. Vandenbussche, J. Van Vooren, A. Drowart, J. B. Ulmer, and M. A. Liu. 1997. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 15:830-833.[CrossRef][Medline] |
| 28. | Madi, N. M., R. Al-Attiya, F. A. Shaban, A. S. Mustafa, A. T. Abal, F. Ofung, H. G. Wiker, P. Ravn, and P. Andersen. 1998. Effective chemotherapy restores T-cell responses to M. tuberculosis antigens in tuberculosis patients, p. 439-442. In G. P. Talwar, I. Nath, N. K. Ganguly, and K. V. S. Rao (ed.), Proceedings of the 10th International Immunology Congress, 1998. Monduzzi Editore, S.p.A., Bologna, Italy. |
| 29. | Mendez-Samperio, P., L. Gonzalez-Garcia, P. R. Pineda-Fragoso, and E. Ramos-Sanchez. 1995. Specificity of T cells in human resistance to Mycobacterium tuberculosis infection. Cell. Immunol. 162:194-201.[CrossRef][Medline] |
| 30. | Nagai, S., H. G. Wiker, M. Harboe, and M. Kinomoto. 1991. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect. Immun. 59:372-382. |
| 31. | Olsen, A. W., L. A. H. Van Pinxteren, L. M. Okkels, P. B. Rasmussen, and P. Andersen. 2001. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Infect. Immun. 69:2773-2778. |
| 32. | Sinha, R. K., I. Verma, and G. K. Khuller. 1997. Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra. Vaccine 15:689-699.[CrossRef][Medline] |
| 33. | Smith, S. A., R. Brookes, M. R. Klein, A. S. Malin, P. T. Lukey, A. S. King, G. S. Ogg, A. V. S. Hill, and H. M. Dockrell. 2000. Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A. J. Immunol. 165:7088-7095. |
| 34. | Sonnenberg, M. G., and J. T. Belisle. 1997. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect. Immun. 65:4515-4524.[Abstract] |
| 35. | Sorrensen, A. L., S. Nagai, G. Houen, P. Andersen, and A. B. Andersen. 1995. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 63:1710-1717.[Abstract] |
| 36. | Takai, S., T. Michizoe, K. Matsumura, M. Nagai, H. Sato, and S. Tsubaki. 1985. Correlation of in vitro properties of Rhodococcus (Corynebacterium) equi with virulence for mice. Microbiol. Immunol. 29:1175-1184.[Medline] |
| 37. | Takai, S., M. Ile, Y. Watanabe, S. Tsubaki, and T. Sekizaki. 1992. Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus equi: temperature-dependent expression and location of the antigens. Infect. Immun. 60:2995-2997. |
| 38. | Takai, S., M. Iie, C. Kobayashi, T. Morishita, T. Nishio, T. Ishida, T. Fujimura, Y. Sasaki, and S. Tsubaki. 1993. Monoclonal antibody specific to virulence-associated 15- to17-kilodalton antigens of Rhodococcus equi. J. Clin. Microbiol. 31:2780-2782. |
| 39. | Takai, S. 1997. Epidemiology of Rhodococcus equi infections: a review. Vet. Microbiol. 56:167-176.[CrossRef][Medline] |
| 40. | Takai, S., C. Kobayashi, K. Murakami, Y. Sasaki, and S. Tsubaki. 1999. Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice. FEMS Immunol. Med. Microbiol. 24:1-9.[CrossRef][Medline] |
| 41. | Tan, C., J. F. Prescott, M. C. Patterson, and V. M. Nicholson. 1995. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity. Can. J. Vet. Res. 59:51-59.[Medline] |
| 42. | Taouji, S., E. Bréard, A. Peyret-Lacombe, S. Pronost, G. Fortier, and C. Collobert-Laugier. 2002. Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA of Rhodococcus equi. FEMS Immunol. Med. Microbiol. 34:299-306.[CrossRef][Medline] |
| 43. | Verville, T. D., M. M. Huycke, R. A. Greenfield, D. P. Fine, T. L. Kuhls, and L. N. Slater. 1994. Rhodococcus equi infection of humans. 12 cases and a review of the literature. Medicine 73:119-132.[Medline] |
| 44. | Weldingh, K., and P. Andersen. 1999. Immunological evaluation of novel Mycobacterium tuberculosis culture filtrate proteins. FEMS Immunol. Med. Microbiol. 23:159-164.[Medline] |
| 45. | Zink, M. C., J. A. Yager, J. F. Prescott, and M. A. Fernando. 1987. Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages. Vet. Microbiol. 14:295-305.[CrossRef][Medline] |
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