Evaluation of Protection Afforded by Brucella abortus and Brucella melitensis Unmarked Deletion Mutants Exhibiting Different Rates of Cleara
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《感染与免疫杂志》
Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4467
ABSTRACT
Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The asp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The asp24 mutants also display superior protection compared to manBA and virB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.
INTRODUCTION
Brucella species are small, facultative, gram-negative, intracellular coccobacilli that are classified phylogenetically within the -2 subdivision of Proteobacteria. Several species of Brucella are the etiologic agents of brucellosis, a disease affecting numerous animal species that is also a zoonotic disease for humans (4, 15, 17, 29, 39). Animal exposure most commonly occurs through inhalation, in utero exposure from infected mothers to calves, ingestion of infected fetal tissues, or in some cases may be sexually transmitted (29). Acute infections in animal hosts are observed after Brucella organisms invade the chorionic trophoblast cells of the placenta, resulting in abortion of the fetus. Persistence is then classified by the organism's ability to reside in reproductive tissues and the mammary gland and lymph nodes, chronically shedding into the milk (18). Animal brucellosis can be a serious cause of economic loss due to abortion and infertility and also represents a public health threat (22, 40).
In the United States, Brucella abortus infection in cattle has been controlled by the combined use of vaccination, testing for exposure, and slaughter of seropositive animals, but the disease has not been successfully eradicated in wildlife species. Commonly, S19 and RB51 have been used for vaccination, but these vaccines are not ideal for all wildlife species at risk. S19 may cause abortion when administered to pregnant animals, and RB51 fails to protect most wildlife species from challenge infection (12, 14). As a result, improved vaccines need to be designed that will combine safety and efficacy to all species at risk, including domestic herds and wildlife. The scope of Brucella research has also expanded to include the demand for better protection and reaction of the United States against potential bioterrorist threats. Superior vaccine strains are therefore in demand for improved protection of agricultural and wildlife reservoirs as well as direct intervention in human infection.
Numerous attenuated mutants have been identified in Brucella species via signature-tagged mini-Tn5 mutagenesis to identify Brucella genes encoding factors or products vital for survival and virulence within the host (2, 17, 25, 32). Mutants defective for genes required early in infection are rapidly cleared from both the mouse model and from macrophages in culture. Mutants defective for genes required later in infection are retained longer in the mouse model and, depending on the defect, may or may not show any difference in the macrophage assay (24). Since mice infected via intraperitoneal injection with wild-type Brucella abortus retain bacteria within the spleen for up to 24 weeks postinfection, mutant persistence in the spleen can therefore be compared to wild-type persistence in the spleen to determine the degree of attenuation of the mutant (25). Mutants may be classified according to their rate of clearance from the mouse.
In order to evaluate protection as a function of vaccine persistence, mutants exhibiting different rates of clearance were selected for further study. Rough mutants were selected due to overall interest in their use, such as the approved rough vaccine strain RB51, due to a lack of interference with diagnostic tests (34, 43, 45, 46, 49). Reliance on O-antigen for serological diagnosis of brucellosis is based on potent antibody responses directed against this cell surface antigen, and interest in the use of rough organisms as vaccines stems from the ease of detection of anti-O-antigen antibodies (34, 46). The rapid clearance of rough organisms, however, is often considered a weakness of rough vaccine strains, preventing a long-term protective immune response (34). Furthermore, the immunity induced by RB51 appears to vary from host to host and is ineffective in several wildlife species (12, 14).
Because of the controversy surrounding the use of rough vaccines, we chose to evaluate another highly attenuated but smooth organism, a virB mutant. The virB type IV secretion system forms a complex serving to secrete or export macromolecules (5, 7). Mutations in this operon attenuate Brucella survival in macrophages and in the mouse model, suggesting that a completely functional VirB system is necessary for the establishment and maintenance of infection (5-8, 13, 30). It is a logical step to construct a knockout of this gene, since it is part of a macrophage/host cell-specific virulence mechanism identified in Brucella species.
Mutants that are not rapidly cleared in the mouse model have been shown to exhibit superior protective immunity; thus, it was desirable to correlate vaccine efficacy with survival of the vaccine strain (24). Based on preliminary evidence demonstrating persistence in macrophages and delayed attenuation in the mouse model, the asp24 gene was selected as a deletion candidate for this study (GenBank accession number AE009508). The gene encoding Asp24 had never been identified in mutant screens, consistent with extremely late or reduced attenuation.
MATERIALS AND METHODS
Bacteria and bacterial cultures. Escherichia coli cultures were routinely grown on Luria-Bertani (Difco Laboratories) plates overnight at 37°C with or without supplemental kanamycin (100 mg/liter), carbenicillin (100 mg/liter), or chloramphenicol (50 mg/liter). All wild-type, vaccine, and unmarked deletion strains were routinely grown on tryptic soy agar (TSA; Difco Laboratories) at 37°C in an atmosphere containing 5% (vol/vol) CO2. Virulent B. abortus strain S2308 was obtained from Billy Deyoe at the National Animal Disease Center in Ames, Iowa. Brucella melitensis bv. 1 (16M) was obtained from ATCC and reisolated by this lab from an aborted goat fetus. These strains were used as virulent challenge organisms and to generate vaccine candidates via deletion mutagenesis. Strain 19 was obtained from the National Veterinary Services Laboratory in Ames, Iowa. Rev 1 INRA was obtained from Menachem Banai. All bacterial strains were stored frozen at –80°C in medium supplemented with 50% (vol/vol) glycerol (Table 1).
Transfection of Brucella was performed via electroporation, using cultures prepared from the frozen stocks and grown to confluence on TSA at 37°C for 3 days. Bacteria were harvested into phosphate-buffered saline (PBS, pH 7.4; Gibco) to yield a final suspension containing approximately 4 x 1011 CFU/ml, as estimated turbidometrically using a Klett meter.
Inoculation doses used to infect mice were prepared the day of challenge following growth on solid medium, and the bacterial density was estimated using a Klett meter as described above. Inoculum doses were prepared, and serial dilution was performed retrospectively to accurately determine the number of organisms inoculated.
Recombinant plasmid construction. In order to construct vectors to eliminate genes of interest, primers were designed to amplify sequences flanking the genes. These flanking regions are referred to as the 5' and the 3' fragments and were joined to one another using specially designed PCR primers (Table 2). Importantly, the reverse primer of the 5' fragment and the forward primer of the 3' fragment include approximately 5 to 10 nucleotides of sequence complementary to the opposite fragment and a terminal restriction site. The 5' and 3' fragments were amplified in separate reactions, gel purified, and used as templates for a second round of PCR (38). The forward primer of the 5' fragment and the reverse primer of the 3' fragment were utilized in a second round of PCR to engineer a product that represented the ligation of the 5' and 3' fragments. The ends of this joined product were removed by restriction digestion at sites engineered into the primers. The final fragment was gel purified and ligated to pBluescript II KS(+) (Stratagene). A kanamycin cassette was inserted between the 5' and 3' fragments following amplification via PCR from the plasmid pKD4 using primers containing the compatible restriction site located within the overlap between the fragments. pKD4 contains nptII from Tn5 flanked by FLP recombination target sites (11). These constructs are referred to as the marked plasmids (Table 1).
The construction of the plasmid for creation of unmarked deletion mutants entails cloning of the original overlapping PCR product (lacking the kanamycin cassette) into plasmid pEX18Ap, which contains sacB, using the appropriate restriction enzymes (23). This construct is referred to here as the unmarked plasmid (Table 1).
Selection of marked deletion mutants. Marked deletion mutants were created in B. melitensis and B. abortus via allelic exchange following electroporation of the marked plasmid into either 16M or S2308, respectively. Bacteria were grown as described above and pelleted via centrifugation at 1,700 x g for 15 min at 4°C. All subsequent steps were performed on ice or at 4°C. The cell pellet was washed three times with ice-cold sterile water under the same conditions. After the final wash, the cells were resuspended in 1 ml sterile water. The bacterial cell suspension was used in each electroporation with approximately 1 μg DNA in a prechilled 1-mm gap cuvette (Bio-Rad, California) and shocked in a BTX electroporation apparatus set at 2.2 to 2.5 kV and 246 . SOC-B (6% [wt/vol] tryptic soy broth, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) medium was immediately added to the cuvette, transferred to microcentrifuge tubes, and incubated overnight at 37°C with agitation (31). Following incubation, the entire culture was plated onto TSA containing kanamycin. Colonies were replica plated onto TSA containing kanamycin and onto plates containing carbenicillin. Marked deletion mutants from allelic exchange should be kanamycin resistant (Kmr) and carbenicillin sensitive (Carbs). Verification of mutant genotypes was obtained via PCR and Southern blot analysis to ensure that the gene of interest was deleted and the kanamycin cassette was retained.
Selection of unmarked deletion mutants. The unmarked plasmid, containing the sacB gene, ligated 5' and 3' fragments, and bla gene, was used for electroporation into marked deletion strains. Electroporation conditions were identical to those described for the construction of marked mutants. Following electroporation, cells were plated onto TSA containing carbenicillin to select for the first homologous recombination, i.e., a cointegration. Colonies were replica plated onto sucrose plates (TSA without salt, containing 6% [wt/vol] sucrose, without antibiotic) and to TSA containing carbenicillin. Colonies that grew on carbenicillin (Carbr) but not sucrose (Sucs) were cointegrates with a functional sacB gene. Resolution of cointegration occurs spontaneously and was selected for by inoculating 5 ml of sucrose broth (TSB, without salt or antibiotics, and supplemented with 6% [wt/vol] sucrose) and incubating for 24 h with agitation at 37°C, with subsequent plating onto sucrose-containing medium. All knockout candidates were verified via PCR and Southern blot analysis to demonstrate gene deletion as well as loss of the kanamycin cassette.
Clearance of mutants from mice. Survival or persistence of mutants was evaluated following intraperitoneal inoculation of groups of 4- to 6-week-old female BALB/c mice with 1 x 105 to 1 x 106 CFU/ml unmarked deletion mutant or wild-type organisms. Mice were euthanized via carbon dioxide asphyxiation at various times postinfection, depending upon the anticipated clearance rate of the mutant. At each time point, spleens were collected and weighed, homogenized in 1 ml PBS, and serially diluted, and 200-μl aliquots of all dilutions were plated onto TSA. Recovered bacteria were enumerated to evaluate the persistence of each individual organism.
Efficacy studies. The mouse model was used to evaluate efficacies of various unmarked deletion mutants against subsequent virulent infection. Groups of 6 to 10 female 4- to 6-week-old BALB/c mice were vaccinated via intraperitoneal injection of 1 x 106 CFU/ml of unmarked deletion mutant or PBS in nave controls. Mice were subsequently challenged with 1 x 104 CFU homologous wild-type strain at 12, 16, or 20 weeks postvaccination. One week after the virulent challenge, the mice were euthanized, and spleens were extracted and weighed, homogenized in 1 ml PBS, serially diluted, and plated onto TSA to enumerate recovery of the challenge organism.
To evaluate cross-Brucella species protection, groups of five female 4- to 6-week-old BALB/c mice were vaccinated as described above and subsequently challenged with 1 x 104 CFU heterologous wild-type strain 16 weeks postvaccination. One week after the virulent challenge, the mice were euthanized and challenge organisms recovered were enumerated as described above. For both experiments, vaccine efficacy is presented in units of protection (U), representing the bacterial (challenge organism) burden in the spleen as a measure of protective immunity (log10 wild type recovered from unvaccinated mice minus log10 wild type recovered from vaccinates).
Immunological analysis. Correlates of protective immunity were evaluated via splenocyte cytokine memory responses. Splenocytes were pressed through fine mesh into complete RPMI (RPMI 1640 [GIBCO Laboratories, Grand Island, N.Y.] containing L-glutamine, 10% [vol/vol] fetal bovine serum [HyClone Laboratories, Logan, Utah], 1% [vol/vol] sodium pyruvate, 0.1% [vol/vol] -mercaptoethanol, 1% [vol/vol] penicillin-streptomycin). Cells were pelleted at 1,700 x g for 10 min; 10 ml ACK red blood cell lysis buffer (0.15 M NH4Cl, 10.0 mM KHCO3, 0.1 mM Na2EDTA) was added to the pellet for 5 min. Splenocytes were pelleted and washed three times with PBS, enumerated using a hemacytometer, and then resuspended in complete RPMI to be seeded in triplicate at a density of 2 x 105 cells/well in 96-well tissue culture plates (Costar, Massachusetts). Samples were stimulated for 3 days with either 1 x 108 CFU heat-killed homologous Brucella strain or controls, including unstimulated cells for a baseline level and cells stimulated with 2 μg/ml (wt/vol) concanavalin A (ConA), a positive control mitogen. Supernatants were collected at day 3 and frozen at –80°C. Enzyme-linked immunosorbent assays (ELISAs) were performed on supernatants for gamma interferon (IFN-) and interleukin-10 (IL-10) using monoclonal antibody pairs according to the manufacturer's protocol (PeproTech, New Jersey).
Statistical analysis. Data from survival of mutants as well as in efficacy studies were expressed as mean CFU ± the standard error and were presented graphically as the log10 CFU of Brucella recovered per spleen. Culture-negative spleens were assigned a value of 4 CFU, which is below the limit of detection of 5 CFU/spleen.
For survival of unmarked strains in mice, analyses of variance (ANOVAs) were performed to compare individual vaccine groups to the wild-type control group of the same species. Efficacy studies compared vaccinated and subsequently challenged mice to mice receiving PBS as a vaccine control which were challenged with wild-type organisms. ANOVAs were performed on these samples as well, comparing the vaccinated groups to nonvaccinated controls. For all ANOVAs, P values less than 0.05 were considered statistically significant.
RESULTS
Safety of attenuated unmarked deletion mutants in the mouse model. Infection of mice with unmarked deletion mutants was used to evaluate clearance of the vaccine strain, modeling field vaccination conditions. Compared to 2308, the unmarked rough B. abortus manBA deletion mutant (BAmanBA) was significantly attenuated by 3.4 logs at 1 week postinfection (P < 0.0001), 2.4 logs at 2 weeks postinfection (P < 0.0001), and undetected by 5 weeks postinfection (P < 0.0001). Between 1 and 2 weeks postinfection, the levels of detectable vaccine strain in the spleens remained unchanged (Fig. 1A). Unmarked rough B. melitensis deletion mutants of manBA (BMmanBA) demonstrated a similar clearing trend as BAmanBA. Compared to wild-type 16M, the mutant was attenuated 2.5 logs at 1 week (P = 0.01), 1.3 logs at 2 weeks (P = 0.001), and undetected by 5 weeks postinfection (P = 0.001) (Fig. 1B). Numbers of bacteria in the spleen remained unchanged between 1 and 2 weeks postinfection, as was observed in the B. abortus mutant. From these data we consider the manBA unmarked deletion to be highly attenuated.
FIG. 1. Kinetics of clearance of unmarked deletion mutants from mice. Four to five female BALB/c mice were infected with 2308, BAasp24, BAvirB2, or BAmanBA (A) or 16 M, BMasp24, BMvirB2, or BMmanBA (B) at a dose of 1 x 106 CFU/mouse. Mice were sacrificed at various time points, and remaining Brucella organisms persisting in the spleens were enumerated and plotted as the mean CFU/spleen ± the standard error. Statistical significance is based upon an ANOVA comparing the deletion mutant to the wild-type strain and is listed in the text. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU.
Mice infected with unmarked B. abortus virB2 (BAvirB2) deletion mutants were attenuated 1.6 logs at 2 weeks postinfection (P = 0.003), though the attenuation compared to 2308 was not as severe as what was demonstrated with the BAmanBA mutant at the same time point. BAvirB2 was attenuated 4.7 logs at 6 weeks postinfection (P < 0.0001), which was just above the limit of detection, and was undetected at 10 weeks postinfection (P < 0.0001) (Fig. 1A). Unmarked B. melitensis virB2 (BMvirB2) demonstrated similar survival characteristics to the B. abortus mutant. In this case, BMvirB2 was reduced 2.2 logs compared to 16M (P = 0.001). By 6 weeks postinfection, the mutant strain was undetected from the mouse spleens (P < 0.0001), which held constant at 10 weeks as well (P = 0.002) (Fig. 1B). From these data, we consider both BAvirB2 and BMvirB2 to be attenuated at a moderate rate compared to wild-type strains, since they are not cleared as rapidly as manBA deletion mutants.
The two asp24 deletion mutants were the first set of gene deletions that portrayed varied survival phenotypes in the mouse model, depending upon which species of Brucella the deletion was created. For the B. abortus deletion (BAasp24), the mutant was highly attenuated (4.6 log reduction) by 6 weeks postinfection (P < 0.0001), barely detectable and 5.0 logs reduced at 8 weeks (P < 0.0001), and below the limit of detection at 10 weeks (P < 0.0001) (Fig. 1A). In B. melitensis, however, BMasp24 was not reduced in the spleen at a significant level until 16 weeks postinfection, where it persisted at 1.2 logs lower than 16M (P = 0.04) (Fig. 1B). One difficulty in assessing the difference between 16M and the knockout arises from the fact that 16M itself clears at a rapid rate compared to 2308 in mice. As such, differences between 16M and knockouts are not as pronounced, especially for mutants that exhibit a delay in attenuation, such as asp24.
Efficacy of B. abortus mutants against 2308 challenge infection. To evaluate the vaccine potential of the selected unmarked mutants, the level of protection provided against virulent challenge infection was assessed. Degree of efficacy was determined by subtracting the mean CFU/spleen recovered from mice after vaccination for either 12, 16, or 20 weeks plus 1 week challenge with 2308 (corresponding to 13, 17, and 21 weeks postvaccination, respectively) from the mean CFU/spleen recovered from age-matched nonvaccinated but challenged controls. The U value obtained is relative only to the nonvaccinated controls challenged with the wild type at each particular time point.
At 13 weeks postvaccination, mice were protected by BAasp24 at 4.7 U and S19-vaccinated mice at 4.3 U, both significantly greater than nave controls (P < 0.0001 for both). Mutants that clear more quickly from the host, BAvirB2 and BAmanBA, protected mice to a lesser degree at 1.5 and 0.9 U, respectively, but significantly better than nave mice (P = 0.004 and P = 0.001) (Fig. 2A.). At this time point, BAasp24 protected mice significantly better than S19, BAvirB2, and BAmanBA (P = 0.01, P < 0.004, and P < 0.001, respectively). There was no significant difference between BAvirB2 and BAmanBA vaccinates (P = 0.22), and both strains protected mice significantly less than S19 (P = 0.004 and P = 0.0002, respectively).
FIG. 2. Homologous efficacy of B. abortus and B. melitensis vaccine strains. Groups of three to seven female BALB/c mice were vaccinated with 1 x 106 CFU unmarked B. abortus vaccine strain, S19, or PBS. Groups were allowed to rest for either 12 weeks (A and D), 16 weeks (B and E), or 20 weeks (C and F) postvaccination. At these time points, mice were subsequently challenged with 1 x 104 CFU 2308 (A to C) or 16M (D to F) and allowed to rest 1 week. After 1 week, mice were euthanized via CO2 asphyxiation and spleens were collected. Data are reported as the log10 recovery of Brucella from spleens, with individual mice in each treatment group represented. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU. , P < 0.05 compared to nave animals by ANOVA; #, P < 0.05 compared to accepted vaccine strain (S19 or Rev 1) by ANOVA.
At 17 weeks postvaccination, recovery of the challenge organism 2308 from a single mouse vaccinated with BAasp24 was just above the limit of detection. The challenge organism was not recovered from the three remaining mice, which appeared to exhibit sterile immunity. Overall protection for this group was 4.7 U relative to nave controls (P < 0.0001). S19 protected mice to a similarly high degree at this time point as well, at 4.4 U (P < 0.0001). Mice exhibited 0.9 U protection when vaccinated with BAvirB2 (P = 0.002) or BAmanBA (P = 0.03), suggesting that persistent yet attenuated mutant strains provide better protection than rapidly cleared mutants (Fig. 2B). At this time point, the protection provided by BAasp24 against infection was superior to BAvirB2 and BAmanBA (P < 0.0001 and P = 0.0004, respectively), though there was no significant difference compared to S19 at this time point (P = 0.22). There was no significant difference between BAvirB2 and BAmanBA groups (P = 0.84), and both deletion mutants protected mice significantly less than S19 (P < 0.0001 and P = 0.0003, respectively).
At 21 weeks postvaccination, BAasp24 again provided superior protection at 4.5 U (P < 0.0001), followed by S19 at 4.3 U (P = 0.0001), BAvirB2 at 1.8 U (P = 0.001), and BAmanBA at 0.07 U (P = 0.86) relative to nave controls (Fig. 2C). Similar to earlier time points, BAasp24 provided significantly higher levels of protection than BAvirB2 and BAmanBA (P < 0.0001 for both strains) but no significant difference relative to S19 (P = 0.59). Vaccination with BAvirB2 elicited significantly better protection in mice than BAmanBA at this time point (P = 0.002), although neither protected as well as S19 (P = 0.0004 and P < 0.0001, respectively).
Efficacy of B. melitensis mutants against 16M challenge infection. At 13 weeks postvaccination, BMasp24-vaccinated mice exhibited the highest level of protection against virulent B. melitensis challenge relative to nave controls, at 3.7 U (P < 0.0001). Rev 1-vaccinated mice were protected at 2.4 U (P = 0.002), BMvirB2-vaccinated mice were protected at 1.9 U, and BMmanBA-vaccinated mice were protected at 1.5 U (P = 0.04 for both strains) compared to nave controls (Fig. 2D). At this time point, BMasp24 protected mice significantly better than Rev 1, BMvirB2, and BMmanBA (P = 0.02, P = 0.003, and P = 0.02, respectively). There was no significant difference between BMvirB2 or BMmanBA vaccinates (P = 0.67), and there was no significant difference in protection for these strains compared to Rev 1 (P = 0.16 and P = 0.45, respectively).
At 17 weeks postvaccination, BMasp24-vaccinated mice exhibited the highest level of protection at 5.2 U (P < 0.0001), Rev 1 at 3.7 U (P < 0.0001), BMvirB2 at 3.4 U (P < 0.0001), and BMmanBA at 3.4 U (P = 0.0002) against virulent B. melitensis compared to nave controls (Fig. 2E). At this time point, BMasp24 protected mice significantly greater than Rev 1 and BMvirB2 (P = 0.006 and P = 0.003, respectively), although there was no significant difference between BMasp24 and BMmanBA due to variation between mice (P = 0.07). There was no significant difference in protection between BMvirB2 or BMmanBA vaccinates (P = 0.92), and there was no difference for either of these strains compared to Rev 1(P = 0.35 and P = 0.72, respectively).
At 21 weeks postvaccination, BMasp24 provided the highest level of protection relative to nave mice at 4.7 U (P < 0.0001), followed by Rev 1 at 4.4 U (P = 0.0002) (Fig. 2F). The BMasp24 mutant also protected mice significantly better than mice vaccinated with BMvirB2 (P = 0.003). The BMmanBA mutant protected mice at 4.1 U and BMvirB2 protected at 2.8 U (P = 0.0001 and P < 0.0001). The protection provided by BMasp24 vaccination was equivalent to Rev 1 (P = 0.31) but was statistically indistinguishable from BMmanBA (P = 0.07) due to variations between mice. There was no significant difference in protection between BMvirB2 and BMmanBA (P = 0.14) or between Rev 1 and BMmanBA (P = 0.42), although Rev 1 vaccination elicited better protection than BMvirB2 (P = 0.04).
Efficacy of unmarked mutants against heterologous Brucella species. Mice were challenged with a heterologous wild-type strain to determine if the vaccine would be protective against another species of Brucella. When challenged with 2308, the BMasp24 mutant provided the greatest degree of protection compared to nave control groups, at 4.2 U (P < 0.0001) (Fig. 3A). BMvirB2 provided 3.3 U (P < 0.0001), and BMmanBA provided the least protection at 1.6 U (P = 0.02). These data indicate that, similar to the homologous efficacy data, an attenuated mutant that persists for a longer period in the host protects better than mutants that clear rapidly. Mice that were vaccinated with BMasp24 were protected better than BMvirB2, though this difference was not significant (P = 0.12). Both BMasp24 and BMvirB2 strains elicited significantly better protection than BMmanBA (P = 0.003 and P = 0.045, respectively).
FIG. 3. Heterologous efficacy of B. abortus and B. melitensis vaccine strains. Groups of 5 to 10 mice were vaccinated with 1 x 106 CFU of unmarked B. melitensis vaccine strain or PBS (A) or 1 x 106 CFU of unmarked B. abortus vaccine strains or the PBS control (B). Groups were allowed to rest for 16 weeks postvaccination, at which time they were subsequently challenged with 1 x 104 CFU 2308 (A) or 16M (B) and allowed to rest for 1 week. After 1 week, mice were euthanized via CO2 asphyxiation and spleens were collected. Data are reported as the log10 recovery of Brucella from spleens, with individual mice in each treatment group represented. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU. , P < 0.05 compared to nave animals by ANOVA.
Vaccination with BAasp24 provided the highest level of protection against challenge with 16M at 5.1 U relative to nave mice (P < 0.0001) (Fig. 3B). Mice vaccinated with the BAvirB2 mutant were significantly protected at 2.1 U (P = 0.0001), and mice vaccinated with BAmanBA were protected at 1.4 U (P = 0.002) relative to nave mice. BAasp24-vaccinated mice were protected better relative to mice vaccinated with either BAvirB2 (P = 0.0002) or BAmanBA (P = 0.002). Although BAvirB2 elicited better protection than BAmanBA, the difference was not significant between these two groups (P = 0.95).
Production of cytokines from splenocytes of vaccinated mice. Mouse cytokines were evaluated at 21 weeks in an attempt to identify correlates of protective immunity associated with the different vaccine strains. Splenocytes were isolated from mice and stimulated as described above. Splenocytes from mice vaccinated with BMasp24 produced the highest amount of IFN- after stimulation with heat-killed B. melitensis (P < 0.0001) compared to nave mice stimulated with the same antigen (Fig. 4A). Splenocytes from the BMvirB2 vaccination group also produced a significant amount of IFN- compared to nave mice (P = 0.02). There was no significant difference in IFN- production between BMasp24 and BMvirB2 vaccine groups (P = 0.12). Splenocytes from mice vaccinated with BMmanBA did not produce significant amounts of IFN- relative to nave mice (data not shown).
FIG. 4. IFN- production from mouse splenocytes. Mice were vaccinated with 1 x 106 CFU of B. melitensis deletion mutants (A) or B. abortus deletion mutants (B) for 21 weeks, with nave mice kept as controls. Mice were then euthanized and splenocytes were isolated to measure in vitro IFN- responses. Splenocytes were stimulated with either ConA, 1 x 108 CFU heat-killed 16M (A) or 2308 (B) organisms, or unstimulated for a baseline cytokine concentration. IFN- production (in pg/ml) was detected after 3 days of growth in the presence of the stimulant and assayed as described in the text, via ELISA.
Splenocytes from BAasp24-vaccinated mice produced a significant amount of IFN- compared to nave mice when stimulated in the presence of heat-killed B. abortus (P = 0.002) (Fig. 4B). Production of this cytokine was also greater, though not significantly, than in BAvirB2 vaccinates (P = 0.37). Levels of IFN- from the BAvirB2 splenocytes, though elevated, were not significant compared to nave mice (P = 0.14).
Relative to nave controls, IL-10 production from splenocytes of BMasp24 and BMvirB2 vaccinates was significantly increased (P = 0.02 and P = 0.03, respectively), and these levels were not significantly different from one another (P = 0.09) (Fig. 5A). Levels of IL-10 from the BMmanBA group were not significantly different from nave mice (data not shown).
FIG. 5. IL-10 production from mouse splenocytes. Mice were vaccinated with 1 x 106 CFU of B. melitensis deletion mutants (A) or B. abortus deletion mutants (B) for 21 weeks, with nave mice kept as controls. Mice were then euthanized, and splenocytes were isolated to measure in vitro IL-10 responses. Splenocytes were stimulated with either ConA, 1 x 108 CFU heat-killed 16M (A) or 2308 (B), or unstimulated for a baseline cytokine concentration. IL-10 production (in pg/ml) was detected after 3 days of growth in the presence of the stimulant and assayed as described in the text, via ELISA.
IL-10 production was significantly increased compared to nave controls for BAasp24-vaccinated mice only (P = 0.05) (Fig. 5B). There was no significant increase in levels between any of the other vaccine groups stimulated with heat-killed Brucella.
DISCUSSION
Since it is not desirable to release an antibiotic resistance cassette into the field, particularly a transposon, removal of this resistance while retaining attenuation of the mutant is ideal. By utilizing homologous recombination between the bacterial chromosome and a plasmid carrying cloned chromosomal sequences and a selectable marker, unmarked, in-frame deletions can be created via allelic exchange. Mutants generated in this fashion were used in the mouse model to demonstrate a correlation between persistence of the vaccine candidate efficacy.
Genes of interest were chosen based upon differences in survival characteristics of the resulting mutants. Brucella abortus mutants deficient in O-antigen exhibit reduced virulence in the host and thus have been considered safe candidate live vaccines (1, 26, 27, 35, 44, 48). Rough mutants are taken up in greater numbers than smooth strains by macrophages and human monocytes, exhibit altered trafficking and host defense responses, and may cause necrotic cell death of the macrophages (27, 28, 42, 44). Due to their defect in O-antigen expression, rough mutants do not interfere with current diagnostic tests but have often proved mildly successful for protection of target livestock and wildlife species (1, 10, 12, 14, 26, 35, 48). A virB deletion was chosen because of its inability to persist within macrophages due to altered trafficking, despite the presence of O-antigen (7, 9, 13). The contribution of Asp24 to intracellular survival is unknown, but the upregulated expression of this gene in macrophages and in response to low pH has suggested a role in the establishment of an intracellular niche leading to chronic infection (33). Since deletions in asp24 possess both intact O-antigen and the type IV secretion system, it is an ideal candidate to evaluate the importance of both virulence criteria in vaccine efficacy.
The safety of selected vaccine candidates was evaluated based on clearance of unmarked deletion mutants using the mouse model. These experiments mimic utilization of the mutant as a vaccine in the field and may be used to estimate the duration of persistence of the vaccine strain under field conditions. Evaluation of colonization noncompetitively is important, since the presence of the wild-type organism can influence clearance of attenuated mutants from the host and may suggest enhanced rates of clearance (unpublished results). In these experiments, we constructed deletion mutants exhibiting a range of survival characteristics in the mouse model. The rough mutants, BAmanBA and BMmanBA, were significantly reduced in number by 1 week postvaccination but required 5 weeks postvaccination to be cleared completely. The virB2 mutants, BAvirB2 and BMvirB2, cleared more gradually than the manBA deletions, suggesting that their defect affects survival at a later stage of infection. Lastly, BAasp24 and BMasp24 cleared from mice at a much slower rate, with BAasp24 significantly reduced between 8 and 10 weeks postinfection and BMasp24 significantly reduced after 16 weeks, suggesting that Asp24 may only be required for persistence of the organism. The differences in survival exhibited by these mutants provided an opportunity to evaluate the relationship between protection and persistence of the vaccine strain.
Protection against homologous challenge (i.e., identical vaccine and challenge species) was most successful with the persistent BAasp24 and BMasp24 mutants. These mutants protected mice against infection at or near sterile levels at all time points examined. We consider these mutants to be the best candidates for novel attenuated vaccine strains, since they protect mice against infection better than current vaccine strains. In contrast, the rapid clearance of BAvirB2, BMvirB2, BAmanBA, and BMmanBA may enhance their safety as vaccine strains but provides significantly reduced levels of protection compared to asp24 mutants or currently available vaccine strains. Due to their enhanced safety, however, their use may be supported if delivered using controlled release, where the duration and presentation to the host can be extended to improve efficacy.
In protection studies against heterologous challenge, both BAasp24 and BMasp24 again protected mice significantly against challenge, with very few challenge organisms isolated. These results suggest that survival beyond 8 to 10 weeks does not enhance protection, and since prolonged survival may have side effects, the use of BAasp24 as a vaccine strain against other species of Brucella may be recommended. BAasp24 may be a protective vaccine strain in the event of a bioterrorist attack or from natural exposure to nonvaccinated, infected animals, providing cross-species protection.
The degree of protection provided by different vaccine candidates was compared with the cytokine profiles elicited from splenocytes in order to identify any correlates of protective immunity that may be used to predict potency or efficacy. The experiments described were designed to evaluate long-term immune response, mimicking field conditions, in which vaccination is expected to protect against challenge exposure occurring at undefined later dates.
IFN-, a Th1 cytokine involved in pathogen clearance from activated macrophages, has been well defined as a critical factor in the control of Brucella abortus infections in mice via enhanced activation of macrophages and intracellular killing (3, 21, 40). Infection in mice is more severe when the animals are treated with antibody to IFN-, and brucellosis becomes a fatal infection in IFN- knockout mice, confirming the critical role for IFN- in the early stages of infection (3, 36, 37, 40).
Vaccine candidates asp24 and virB2 produced significant levels of IFN- in response to stimulation with heat-killed Brucella relative to nave mice. However, significant differences in efficacy determined by reduced recovery of the challenge strain were not matched by significant differences in IFN- production. As such, there did not seem to be an absolute correlation of protection with IFN- in this study, since mice vaccinated with asp24 mutants demonstrated significantly higher protection than mice vaccinated with virB2 mutants at all time points tested. Although increased levels of this cytokine may reflect an improved memory response with subsequent protection against challenge, it appears that IFN-, although necessary, is not sufficient to provide protection against infection. IFN- was not induced significantly in mice vaccinated with a rough mutant, manBA. In other studies evaluating cytokine responses from splenocytes from mice vaccinated with rough mutants, a similar result was published (1).
IL-10 is generally considered an antiinflammatory molecule, thought to reduce negative effects caused by proinflammatory cytokines such as IFN- (47). Evidence suggests that inhibition of IL-10 activity results in improved clearance of B. abortus from the spleens of infected mice (16, 20). Since BALB/c mice are known to mount a very strong inflammatory response early in Brucella infection, Th2 cytokines such as IL-10 may be necessary to prevent overwhelming the host and to limit the influx of phagocytic cells and potential spread of the disease (3, 19, 41). Recently, it has been shown that IL-10 develops proinflammatory qualities during active inflammatory responses (47). The fact that IL-10 was generally increased in these experiments compared to nave controls for BMasp24, BAasp24, and BMvirB2 may be related to the observed levels of protection, suggesting a proinflammatory role in the mouse model.
In this study, mice were vaccinated for 20 weeks, and since previous research has never considered such late time points in the mouse, direct comparisons cannot be made to published work. Splenocytes may not be the ideal tissue to evaluate long-term cytokine responses in animals vaccinated for extended periods, and another tissue such as lymph nodes may need to be considered to identify correlates of immunity associated with protection.
Overall, the mouse is a sensitive and popular model for evaluating brucellosis. This model has been invaluable for characterization of genetic components necessary for virulence and is an affordable and consistent method for evaluation of potential vaccine candidates (15, 18, 25). Mutants considered safe and protective in the mouse model are often used as candidates for vaccines in target animal species. The mouse model cannot be used, however, to gauge the effect of persistence on the colonization of reproductive tissues, thus, the need for other animal models after the initial murine screen. Since the mouse is not an exact model of disease in target livestock and wildlife species, vaccine candidates from the mouse will need to be reevaluated in a suitable animal model. The asp24 deletion mutants were the most protective in the mouse screen against both homologous and heterologous challenge infections, and perhaps their protective ability in target species may be superior to the other candidates in that model as well.
ACKNOWLEDGMENTS
Melissa Kahl-McDonagh was supported by USDA Food and Agricultural Sciences National Needs Graduate Fellowship Grant 98-38420-5806 (CSREES) and an NIH Training Grant Predoctoral Fellowship Award. Research was funded by USDA/CSREES-NRICGP 99-35204-7550 and NIH 1RO1 AI48496-01.
pEX18Ap was kindly given to the Ficht lab by Herbert Schweizer.
FOOTNOTES
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ABSTRACT
Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The asp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The asp24 mutants also display superior protection compared to manBA and virB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.
INTRODUCTION
Brucella species are small, facultative, gram-negative, intracellular coccobacilli that are classified phylogenetically within the -2 subdivision of Proteobacteria. Several species of Brucella are the etiologic agents of brucellosis, a disease affecting numerous animal species that is also a zoonotic disease for humans (4, 15, 17, 29, 39). Animal exposure most commonly occurs through inhalation, in utero exposure from infected mothers to calves, ingestion of infected fetal tissues, or in some cases may be sexually transmitted (29). Acute infections in animal hosts are observed after Brucella organisms invade the chorionic trophoblast cells of the placenta, resulting in abortion of the fetus. Persistence is then classified by the organism's ability to reside in reproductive tissues and the mammary gland and lymph nodes, chronically shedding into the milk (18). Animal brucellosis can be a serious cause of economic loss due to abortion and infertility and also represents a public health threat (22, 40).
In the United States, Brucella abortus infection in cattle has been controlled by the combined use of vaccination, testing for exposure, and slaughter of seropositive animals, but the disease has not been successfully eradicated in wildlife species. Commonly, S19 and RB51 have been used for vaccination, but these vaccines are not ideal for all wildlife species at risk. S19 may cause abortion when administered to pregnant animals, and RB51 fails to protect most wildlife species from challenge infection (12, 14). As a result, improved vaccines need to be designed that will combine safety and efficacy to all species at risk, including domestic herds and wildlife. The scope of Brucella research has also expanded to include the demand for better protection and reaction of the United States against potential bioterrorist threats. Superior vaccine strains are therefore in demand for improved protection of agricultural and wildlife reservoirs as well as direct intervention in human infection.
Numerous attenuated mutants have been identified in Brucella species via signature-tagged mini-Tn5 mutagenesis to identify Brucella genes encoding factors or products vital for survival and virulence within the host (2, 17, 25, 32). Mutants defective for genes required early in infection are rapidly cleared from both the mouse model and from macrophages in culture. Mutants defective for genes required later in infection are retained longer in the mouse model and, depending on the defect, may or may not show any difference in the macrophage assay (24). Since mice infected via intraperitoneal injection with wild-type Brucella abortus retain bacteria within the spleen for up to 24 weeks postinfection, mutant persistence in the spleen can therefore be compared to wild-type persistence in the spleen to determine the degree of attenuation of the mutant (25). Mutants may be classified according to their rate of clearance from the mouse.
In order to evaluate protection as a function of vaccine persistence, mutants exhibiting different rates of clearance were selected for further study. Rough mutants were selected due to overall interest in their use, such as the approved rough vaccine strain RB51, due to a lack of interference with diagnostic tests (34, 43, 45, 46, 49). Reliance on O-antigen for serological diagnosis of brucellosis is based on potent antibody responses directed against this cell surface antigen, and interest in the use of rough organisms as vaccines stems from the ease of detection of anti-O-antigen antibodies (34, 46). The rapid clearance of rough organisms, however, is often considered a weakness of rough vaccine strains, preventing a long-term protective immune response (34). Furthermore, the immunity induced by RB51 appears to vary from host to host and is ineffective in several wildlife species (12, 14).
Because of the controversy surrounding the use of rough vaccines, we chose to evaluate another highly attenuated but smooth organism, a virB mutant. The virB type IV secretion system forms a complex serving to secrete or export macromolecules (5, 7). Mutations in this operon attenuate Brucella survival in macrophages and in the mouse model, suggesting that a completely functional VirB system is necessary for the establishment and maintenance of infection (5-8, 13, 30). It is a logical step to construct a knockout of this gene, since it is part of a macrophage/host cell-specific virulence mechanism identified in Brucella species.
Mutants that are not rapidly cleared in the mouse model have been shown to exhibit superior protective immunity; thus, it was desirable to correlate vaccine efficacy with survival of the vaccine strain (24). Based on preliminary evidence demonstrating persistence in macrophages and delayed attenuation in the mouse model, the asp24 gene was selected as a deletion candidate for this study (GenBank accession number AE009508). The gene encoding Asp24 had never been identified in mutant screens, consistent with extremely late or reduced attenuation.
MATERIALS AND METHODS
Bacteria and bacterial cultures. Escherichia coli cultures were routinely grown on Luria-Bertani (Difco Laboratories) plates overnight at 37°C with or without supplemental kanamycin (100 mg/liter), carbenicillin (100 mg/liter), or chloramphenicol (50 mg/liter). All wild-type, vaccine, and unmarked deletion strains were routinely grown on tryptic soy agar (TSA; Difco Laboratories) at 37°C in an atmosphere containing 5% (vol/vol) CO2. Virulent B. abortus strain S2308 was obtained from Billy Deyoe at the National Animal Disease Center in Ames, Iowa. Brucella melitensis bv. 1 (16M) was obtained from ATCC and reisolated by this lab from an aborted goat fetus. These strains were used as virulent challenge organisms and to generate vaccine candidates via deletion mutagenesis. Strain 19 was obtained from the National Veterinary Services Laboratory in Ames, Iowa. Rev 1 INRA was obtained from Menachem Banai. All bacterial strains were stored frozen at –80°C in medium supplemented with 50% (vol/vol) glycerol (Table 1).
Transfection of Brucella was performed via electroporation, using cultures prepared from the frozen stocks and grown to confluence on TSA at 37°C for 3 days. Bacteria were harvested into phosphate-buffered saline (PBS, pH 7.4; Gibco) to yield a final suspension containing approximately 4 x 1011 CFU/ml, as estimated turbidometrically using a Klett meter.
Inoculation doses used to infect mice were prepared the day of challenge following growth on solid medium, and the bacterial density was estimated using a Klett meter as described above. Inoculum doses were prepared, and serial dilution was performed retrospectively to accurately determine the number of organisms inoculated.
Recombinant plasmid construction. In order to construct vectors to eliminate genes of interest, primers were designed to amplify sequences flanking the genes. These flanking regions are referred to as the 5' and the 3' fragments and were joined to one another using specially designed PCR primers (Table 2). Importantly, the reverse primer of the 5' fragment and the forward primer of the 3' fragment include approximately 5 to 10 nucleotides of sequence complementary to the opposite fragment and a terminal restriction site. The 5' and 3' fragments were amplified in separate reactions, gel purified, and used as templates for a second round of PCR (38). The forward primer of the 5' fragment and the reverse primer of the 3' fragment were utilized in a second round of PCR to engineer a product that represented the ligation of the 5' and 3' fragments. The ends of this joined product were removed by restriction digestion at sites engineered into the primers. The final fragment was gel purified and ligated to pBluescript II KS(+) (Stratagene). A kanamycin cassette was inserted between the 5' and 3' fragments following amplification via PCR from the plasmid pKD4 using primers containing the compatible restriction site located within the overlap between the fragments. pKD4 contains nptII from Tn5 flanked by FLP recombination target sites (11). These constructs are referred to as the marked plasmids (Table 1).
The construction of the plasmid for creation of unmarked deletion mutants entails cloning of the original overlapping PCR product (lacking the kanamycin cassette) into plasmid pEX18Ap, which contains sacB, using the appropriate restriction enzymes (23). This construct is referred to here as the unmarked plasmid (Table 1).
Selection of marked deletion mutants. Marked deletion mutants were created in B. melitensis and B. abortus via allelic exchange following electroporation of the marked plasmid into either 16M or S2308, respectively. Bacteria were grown as described above and pelleted via centrifugation at 1,700 x g for 15 min at 4°C. All subsequent steps were performed on ice or at 4°C. The cell pellet was washed three times with ice-cold sterile water under the same conditions. After the final wash, the cells were resuspended in 1 ml sterile water. The bacterial cell suspension was used in each electroporation with approximately 1 μg DNA in a prechilled 1-mm gap cuvette (Bio-Rad, California) and shocked in a BTX electroporation apparatus set at 2.2 to 2.5 kV and 246 . SOC-B (6% [wt/vol] tryptic soy broth, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) medium was immediately added to the cuvette, transferred to microcentrifuge tubes, and incubated overnight at 37°C with agitation (31). Following incubation, the entire culture was plated onto TSA containing kanamycin. Colonies were replica plated onto TSA containing kanamycin and onto plates containing carbenicillin. Marked deletion mutants from allelic exchange should be kanamycin resistant (Kmr) and carbenicillin sensitive (Carbs). Verification of mutant genotypes was obtained via PCR and Southern blot analysis to ensure that the gene of interest was deleted and the kanamycin cassette was retained.
Selection of unmarked deletion mutants. The unmarked plasmid, containing the sacB gene, ligated 5' and 3' fragments, and bla gene, was used for electroporation into marked deletion strains. Electroporation conditions were identical to those described for the construction of marked mutants. Following electroporation, cells were plated onto TSA containing carbenicillin to select for the first homologous recombination, i.e., a cointegration. Colonies were replica plated onto sucrose plates (TSA without salt, containing 6% [wt/vol] sucrose, without antibiotic) and to TSA containing carbenicillin. Colonies that grew on carbenicillin (Carbr) but not sucrose (Sucs) were cointegrates with a functional sacB gene. Resolution of cointegration occurs spontaneously and was selected for by inoculating 5 ml of sucrose broth (TSB, without salt or antibiotics, and supplemented with 6% [wt/vol] sucrose) and incubating for 24 h with agitation at 37°C, with subsequent plating onto sucrose-containing medium. All knockout candidates were verified via PCR and Southern blot analysis to demonstrate gene deletion as well as loss of the kanamycin cassette.
Clearance of mutants from mice. Survival or persistence of mutants was evaluated following intraperitoneal inoculation of groups of 4- to 6-week-old female BALB/c mice with 1 x 105 to 1 x 106 CFU/ml unmarked deletion mutant or wild-type organisms. Mice were euthanized via carbon dioxide asphyxiation at various times postinfection, depending upon the anticipated clearance rate of the mutant. At each time point, spleens were collected and weighed, homogenized in 1 ml PBS, and serially diluted, and 200-μl aliquots of all dilutions were plated onto TSA. Recovered bacteria were enumerated to evaluate the persistence of each individual organism.
Efficacy studies. The mouse model was used to evaluate efficacies of various unmarked deletion mutants against subsequent virulent infection. Groups of 6 to 10 female 4- to 6-week-old BALB/c mice were vaccinated via intraperitoneal injection of 1 x 106 CFU/ml of unmarked deletion mutant or PBS in nave controls. Mice were subsequently challenged with 1 x 104 CFU homologous wild-type strain at 12, 16, or 20 weeks postvaccination. One week after the virulent challenge, the mice were euthanized, and spleens were extracted and weighed, homogenized in 1 ml PBS, serially diluted, and plated onto TSA to enumerate recovery of the challenge organism.
To evaluate cross-Brucella species protection, groups of five female 4- to 6-week-old BALB/c mice were vaccinated as described above and subsequently challenged with 1 x 104 CFU heterologous wild-type strain 16 weeks postvaccination. One week after the virulent challenge, the mice were euthanized and challenge organisms recovered were enumerated as described above. For both experiments, vaccine efficacy is presented in units of protection (U), representing the bacterial (challenge organism) burden in the spleen as a measure of protective immunity (log10 wild type recovered from unvaccinated mice minus log10 wild type recovered from vaccinates).
Immunological analysis. Correlates of protective immunity were evaluated via splenocyte cytokine memory responses. Splenocytes were pressed through fine mesh into complete RPMI (RPMI 1640 [GIBCO Laboratories, Grand Island, N.Y.] containing L-glutamine, 10% [vol/vol] fetal bovine serum [HyClone Laboratories, Logan, Utah], 1% [vol/vol] sodium pyruvate, 0.1% [vol/vol] -mercaptoethanol, 1% [vol/vol] penicillin-streptomycin). Cells were pelleted at 1,700 x g for 10 min; 10 ml ACK red blood cell lysis buffer (0.15 M NH4Cl, 10.0 mM KHCO3, 0.1 mM Na2EDTA) was added to the pellet for 5 min. Splenocytes were pelleted and washed three times with PBS, enumerated using a hemacytometer, and then resuspended in complete RPMI to be seeded in triplicate at a density of 2 x 105 cells/well in 96-well tissue culture plates (Costar, Massachusetts). Samples were stimulated for 3 days with either 1 x 108 CFU heat-killed homologous Brucella strain or controls, including unstimulated cells for a baseline level and cells stimulated with 2 μg/ml (wt/vol) concanavalin A (ConA), a positive control mitogen. Supernatants were collected at day 3 and frozen at –80°C. Enzyme-linked immunosorbent assays (ELISAs) were performed on supernatants for gamma interferon (IFN-) and interleukin-10 (IL-10) using monoclonal antibody pairs according to the manufacturer's protocol (PeproTech, New Jersey).
Statistical analysis. Data from survival of mutants as well as in efficacy studies were expressed as mean CFU ± the standard error and were presented graphically as the log10 CFU of Brucella recovered per spleen. Culture-negative spleens were assigned a value of 4 CFU, which is below the limit of detection of 5 CFU/spleen.
For survival of unmarked strains in mice, analyses of variance (ANOVAs) were performed to compare individual vaccine groups to the wild-type control group of the same species. Efficacy studies compared vaccinated and subsequently challenged mice to mice receiving PBS as a vaccine control which were challenged with wild-type organisms. ANOVAs were performed on these samples as well, comparing the vaccinated groups to nonvaccinated controls. For all ANOVAs, P values less than 0.05 were considered statistically significant.
RESULTS
Safety of attenuated unmarked deletion mutants in the mouse model. Infection of mice with unmarked deletion mutants was used to evaluate clearance of the vaccine strain, modeling field vaccination conditions. Compared to 2308, the unmarked rough B. abortus manBA deletion mutant (BAmanBA) was significantly attenuated by 3.4 logs at 1 week postinfection (P < 0.0001), 2.4 logs at 2 weeks postinfection (P < 0.0001), and undetected by 5 weeks postinfection (P < 0.0001). Between 1 and 2 weeks postinfection, the levels of detectable vaccine strain in the spleens remained unchanged (Fig. 1A). Unmarked rough B. melitensis deletion mutants of manBA (BMmanBA) demonstrated a similar clearing trend as BAmanBA. Compared to wild-type 16M, the mutant was attenuated 2.5 logs at 1 week (P = 0.01), 1.3 logs at 2 weeks (P = 0.001), and undetected by 5 weeks postinfection (P = 0.001) (Fig. 1B). Numbers of bacteria in the spleen remained unchanged between 1 and 2 weeks postinfection, as was observed in the B. abortus mutant. From these data we consider the manBA unmarked deletion to be highly attenuated.
FIG. 1. Kinetics of clearance of unmarked deletion mutants from mice. Four to five female BALB/c mice were infected with 2308, BAasp24, BAvirB2, or BAmanBA (A) or 16 M, BMasp24, BMvirB2, or BMmanBA (B) at a dose of 1 x 106 CFU/mouse. Mice were sacrificed at various time points, and remaining Brucella organisms persisting in the spleens were enumerated and plotted as the mean CFU/spleen ± the standard error. Statistical significance is based upon an ANOVA comparing the deletion mutant to the wild-type strain and is listed in the text. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU.
Mice infected with unmarked B. abortus virB2 (BAvirB2) deletion mutants were attenuated 1.6 logs at 2 weeks postinfection (P = 0.003), though the attenuation compared to 2308 was not as severe as what was demonstrated with the BAmanBA mutant at the same time point. BAvirB2 was attenuated 4.7 logs at 6 weeks postinfection (P < 0.0001), which was just above the limit of detection, and was undetected at 10 weeks postinfection (P < 0.0001) (Fig. 1A). Unmarked B. melitensis virB2 (BMvirB2) demonstrated similar survival characteristics to the B. abortus mutant. In this case, BMvirB2 was reduced 2.2 logs compared to 16M (P = 0.001). By 6 weeks postinfection, the mutant strain was undetected from the mouse spleens (P < 0.0001), which held constant at 10 weeks as well (P = 0.002) (Fig. 1B). From these data, we consider both BAvirB2 and BMvirB2 to be attenuated at a moderate rate compared to wild-type strains, since they are not cleared as rapidly as manBA deletion mutants.
The two asp24 deletion mutants were the first set of gene deletions that portrayed varied survival phenotypes in the mouse model, depending upon which species of Brucella the deletion was created. For the B. abortus deletion (BAasp24), the mutant was highly attenuated (4.6 log reduction) by 6 weeks postinfection (P < 0.0001), barely detectable and 5.0 logs reduced at 8 weeks (P < 0.0001), and below the limit of detection at 10 weeks (P < 0.0001) (Fig. 1A). In B. melitensis, however, BMasp24 was not reduced in the spleen at a significant level until 16 weeks postinfection, where it persisted at 1.2 logs lower than 16M (P = 0.04) (Fig. 1B). One difficulty in assessing the difference between 16M and the knockout arises from the fact that 16M itself clears at a rapid rate compared to 2308 in mice. As such, differences between 16M and knockouts are not as pronounced, especially for mutants that exhibit a delay in attenuation, such as asp24.
Efficacy of B. abortus mutants against 2308 challenge infection. To evaluate the vaccine potential of the selected unmarked mutants, the level of protection provided against virulent challenge infection was assessed. Degree of efficacy was determined by subtracting the mean CFU/spleen recovered from mice after vaccination for either 12, 16, or 20 weeks plus 1 week challenge with 2308 (corresponding to 13, 17, and 21 weeks postvaccination, respectively) from the mean CFU/spleen recovered from age-matched nonvaccinated but challenged controls. The U value obtained is relative only to the nonvaccinated controls challenged with the wild type at each particular time point.
At 13 weeks postvaccination, mice were protected by BAasp24 at 4.7 U and S19-vaccinated mice at 4.3 U, both significantly greater than nave controls (P < 0.0001 for both). Mutants that clear more quickly from the host, BAvirB2 and BAmanBA, protected mice to a lesser degree at 1.5 and 0.9 U, respectively, but significantly better than nave mice (P = 0.004 and P = 0.001) (Fig. 2A.). At this time point, BAasp24 protected mice significantly better than S19, BAvirB2, and BAmanBA (P = 0.01, P < 0.004, and P < 0.001, respectively). There was no significant difference between BAvirB2 and BAmanBA vaccinates (P = 0.22), and both strains protected mice significantly less than S19 (P = 0.004 and P = 0.0002, respectively).
FIG. 2. Homologous efficacy of B. abortus and B. melitensis vaccine strains. Groups of three to seven female BALB/c mice were vaccinated with 1 x 106 CFU unmarked B. abortus vaccine strain, S19, or PBS. Groups were allowed to rest for either 12 weeks (A and D), 16 weeks (B and E), or 20 weeks (C and F) postvaccination. At these time points, mice were subsequently challenged with 1 x 104 CFU 2308 (A to C) or 16M (D to F) and allowed to rest 1 week. After 1 week, mice were euthanized via CO2 asphyxiation and spleens were collected. Data are reported as the log10 recovery of Brucella from spleens, with individual mice in each treatment group represented. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU. , P < 0.05 compared to nave animals by ANOVA; #, P < 0.05 compared to accepted vaccine strain (S19 or Rev 1) by ANOVA.
At 17 weeks postvaccination, recovery of the challenge organism 2308 from a single mouse vaccinated with BAasp24 was just above the limit of detection. The challenge organism was not recovered from the three remaining mice, which appeared to exhibit sterile immunity. Overall protection for this group was 4.7 U relative to nave controls (P < 0.0001). S19 protected mice to a similarly high degree at this time point as well, at 4.4 U (P < 0.0001). Mice exhibited 0.9 U protection when vaccinated with BAvirB2 (P = 0.002) or BAmanBA (P = 0.03), suggesting that persistent yet attenuated mutant strains provide better protection than rapidly cleared mutants (Fig. 2B). At this time point, the protection provided by BAasp24 against infection was superior to BAvirB2 and BAmanBA (P < 0.0001 and P = 0.0004, respectively), though there was no significant difference compared to S19 at this time point (P = 0.22). There was no significant difference between BAvirB2 and BAmanBA groups (P = 0.84), and both deletion mutants protected mice significantly less than S19 (P < 0.0001 and P = 0.0003, respectively).
At 21 weeks postvaccination, BAasp24 again provided superior protection at 4.5 U (P < 0.0001), followed by S19 at 4.3 U (P = 0.0001), BAvirB2 at 1.8 U (P = 0.001), and BAmanBA at 0.07 U (P = 0.86) relative to nave controls (Fig. 2C). Similar to earlier time points, BAasp24 provided significantly higher levels of protection than BAvirB2 and BAmanBA (P < 0.0001 for both strains) but no significant difference relative to S19 (P = 0.59). Vaccination with BAvirB2 elicited significantly better protection in mice than BAmanBA at this time point (P = 0.002), although neither protected as well as S19 (P = 0.0004 and P < 0.0001, respectively).
Efficacy of B. melitensis mutants against 16M challenge infection. At 13 weeks postvaccination, BMasp24-vaccinated mice exhibited the highest level of protection against virulent B. melitensis challenge relative to nave controls, at 3.7 U (P < 0.0001). Rev 1-vaccinated mice were protected at 2.4 U (P = 0.002), BMvirB2-vaccinated mice were protected at 1.9 U, and BMmanBA-vaccinated mice were protected at 1.5 U (P = 0.04 for both strains) compared to nave controls (Fig. 2D). At this time point, BMasp24 protected mice significantly better than Rev 1, BMvirB2, and BMmanBA (P = 0.02, P = 0.003, and P = 0.02, respectively). There was no significant difference between BMvirB2 or BMmanBA vaccinates (P = 0.67), and there was no significant difference in protection for these strains compared to Rev 1 (P = 0.16 and P = 0.45, respectively).
At 17 weeks postvaccination, BMasp24-vaccinated mice exhibited the highest level of protection at 5.2 U (P < 0.0001), Rev 1 at 3.7 U (P < 0.0001), BMvirB2 at 3.4 U (P < 0.0001), and BMmanBA at 3.4 U (P = 0.0002) against virulent B. melitensis compared to nave controls (Fig. 2E). At this time point, BMasp24 protected mice significantly greater than Rev 1 and BMvirB2 (P = 0.006 and P = 0.003, respectively), although there was no significant difference between BMasp24 and BMmanBA due to variation between mice (P = 0.07). There was no significant difference in protection between BMvirB2 or BMmanBA vaccinates (P = 0.92), and there was no difference for either of these strains compared to Rev 1(P = 0.35 and P = 0.72, respectively).
At 21 weeks postvaccination, BMasp24 provided the highest level of protection relative to nave mice at 4.7 U (P < 0.0001), followed by Rev 1 at 4.4 U (P = 0.0002) (Fig. 2F). The BMasp24 mutant also protected mice significantly better than mice vaccinated with BMvirB2 (P = 0.003). The BMmanBA mutant protected mice at 4.1 U and BMvirB2 protected at 2.8 U (P = 0.0001 and P < 0.0001). The protection provided by BMasp24 vaccination was equivalent to Rev 1 (P = 0.31) but was statistically indistinguishable from BMmanBA (P = 0.07) due to variations between mice. There was no significant difference in protection between BMvirB2 and BMmanBA (P = 0.14) or between Rev 1 and BMmanBA (P = 0.42), although Rev 1 vaccination elicited better protection than BMvirB2 (P = 0.04).
Efficacy of unmarked mutants against heterologous Brucella species. Mice were challenged with a heterologous wild-type strain to determine if the vaccine would be protective against another species of Brucella. When challenged with 2308, the BMasp24 mutant provided the greatest degree of protection compared to nave control groups, at 4.2 U (P < 0.0001) (Fig. 3A). BMvirB2 provided 3.3 U (P < 0.0001), and BMmanBA provided the least protection at 1.6 U (P = 0.02). These data indicate that, similar to the homologous efficacy data, an attenuated mutant that persists for a longer period in the host protects better than mutants that clear rapidly. Mice that were vaccinated with BMasp24 were protected better than BMvirB2, though this difference was not significant (P = 0.12). Both BMasp24 and BMvirB2 strains elicited significantly better protection than BMmanBA (P = 0.003 and P = 0.045, respectively).
FIG. 3. Heterologous efficacy of B. abortus and B. melitensis vaccine strains. Groups of 5 to 10 mice were vaccinated with 1 x 106 CFU of unmarked B. melitensis vaccine strain or PBS (A) or 1 x 106 CFU of unmarked B. abortus vaccine strains or the PBS control (B). Groups were allowed to rest for 16 weeks postvaccination, at which time they were subsequently challenged with 1 x 104 CFU 2308 (A) or 16M (B) and allowed to rest for 1 week. After 1 week, mice were euthanized via CO2 asphyxiation and spleens were collected. Data are reported as the log10 recovery of Brucella from spleens, with individual mice in each treatment group represented. The solid line at 0.69 logs represents the lower limit of detection, which is 5 CFU. , P < 0.05 compared to nave animals by ANOVA.
Vaccination with BAasp24 provided the highest level of protection against challenge with 16M at 5.1 U relative to nave mice (P < 0.0001) (Fig. 3B). Mice vaccinated with the BAvirB2 mutant were significantly protected at 2.1 U (P = 0.0001), and mice vaccinated with BAmanBA were protected at 1.4 U (P = 0.002) relative to nave mice. BAasp24-vaccinated mice were protected better relative to mice vaccinated with either BAvirB2 (P = 0.0002) or BAmanBA (P = 0.002). Although BAvirB2 elicited better protection than BAmanBA, the difference was not significant between these two groups (P = 0.95).
Production of cytokines from splenocytes of vaccinated mice. Mouse cytokines were evaluated at 21 weeks in an attempt to identify correlates of protective immunity associated with the different vaccine strains. Splenocytes were isolated from mice and stimulated as described above. Splenocytes from mice vaccinated with BMasp24 produced the highest amount of IFN- after stimulation with heat-killed B. melitensis (P < 0.0001) compared to nave mice stimulated with the same antigen (Fig. 4A). Splenocytes from the BMvirB2 vaccination group also produced a significant amount of IFN- compared to nave mice (P = 0.02). There was no significant difference in IFN- production between BMasp24 and BMvirB2 vaccine groups (P = 0.12). Splenocytes from mice vaccinated with BMmanBA did not produce significant amounts of IFN- relative to nave mice (data not shown).
FIG. 4. IFN- production from mouse splenocytes. Mice were vaccinated with 1 x 106 CFU of B. melitensis deletion mutants (A) or B. abortus deletion mutants (B) for 21 weeks, with nave mice kept as controls. Mice were then euthanized and splenocytes were isolated to measure in vitro IFN- responses. Splenocytes were stimulated with either ConA, 1 x 108 CFU heat-killed 16M (A) or 2308 (B) organisms, or unstimulated for a baseline cytokine concentration. IFN- production (in pg/ml) was detected after 3 days of growth in the presence of the stimulant and assayed as described in the text, via ELISA.
Splenocytes from BAasp24-vaccinated mice produced a significant amount of IFN- compared to nave mice when stimulated in the presence of heat-killed B. abortus (P = 0.002) (Fig. 4B). Production of this cytokine was also greater, though not significantly, than in BAvirB2 vaccinates (P = 0.37). Levels of IFN- from the BAvirB2 splenocytes, though elevated, were not significant compared to nave mice (P = 0.14).
Relative to nave controls, IL-10 production from splenocytes of BMasp24 and BMvirB2 vaccinates was significantly increased (P = 0.02 and P = 0.03, respectively), and these levels were not significantly different from one another (P = 0.09) (Fig. 5A). Levels of IL-10 from the BMmanBA group were not significantly different from nave mice (data not shown).
FIG. 5. IL-10 production from mouse splenocytes. Mice were vaccinated with 1 x 106 CFU of B. melitensis deletion mutants (A) or B. abortus deletion mutants (B) for 21 weeks, with nave mice kept as controls. Mice were then euthanized, and splenocytes were isolated to measure in vitro IL-10 responses. Splenocytes were stimulated with either ConA, 1 x 108 CFU heat-killed 16M (A) or 2308 (B), or unstimulated for a baseline cytokine concentration. IL-10 production (in pg/ml) was detected after 3 days of growth in the presence of the stimulant and assayed as described in the text, via ELISA.
IL-10 production was significantly increased compared to nave controls for BAasp24-vaccinated mice only (P = 0.05) (Fig. 5B). There was no significant increase in levels between any of the other vaccine groups stimulated with heat-killed Brucella.
DISCUSSION
Since it is not desirable to release an antibiotic resistance cassette into the field, particularly a transposon, removal of this resistance while retaining attenuation of the mutant is ideal. By utilizing homologous recombination between the bacterial chromosome and a plasmid carrying cloned chromosomal sequences and a selectable marker, unmarked, in-frame deletions can be created via allelic exchange. Mutants generated in this fashion were used in the mouse model to demonstrate a correlation between persistence of the vaccine candidate efficacy.
Genes of interest were chosen based upon differences in survival characteristics of the resulting mutants. Brucella abortus mutants deficient in O-antigen exhibit reduced virulence in the host and thus have been considered safe candidate live vaccines (1, 26, 27, 35, 44, 48). Rough mutants are taken up in greater numbers than smooth strains by macrophages and human monocytes, exhibit altered trafficking and host defense responses, and may cause necrotic cell death of the macrophages (27, 28, 42, 44). Due to their defect in O-antigen expression, rough mutants do not interfere with current diagnostic tests but have often proved mildly successful for protection of target livestock and wildlife species (1, 10, 12, 14, 26, 35, 48). A virB deletion was chosen because of its inability to persist within macrophages due to altered trafficking, despite the presence of O-antigen (7, 9, 13). The contribution of Asp24 to intracellular survival is unknown, but the upregulated expression of this gene in macrophages and in response to low pH has suggested a role in the establishment of an intracellular niche leading to chronic infection (33). Since deletions in asp24 possess both intact O-antigen and the type IV secretion system, it is an ideal candidate to evaluate the importance of both virulence criteria in vaccine efficacy.
The safety of selected vaccine candidates was evaluated based on clearance of unmarked deletion mutants using the mouse model. These experiments mimic utilization of the mutant as a vaccine in the field and may be used to estimate the duration of persistence of the vaccine strain under field conditions. Evaluation of colonization noncompetitively is important, since the presence of the wild-type organism can influence clearance of attenuated mutants from the host and may suggest enhanced rates of clearance (unpublished results). In these experiments, we constructed deletion mutants exhibiting a range of survival characteristics in the mouse model. The rough mutants, BAmanBA and BMmanBA, were significantly reduced in number by 1 week postvaccination but required 5 weeks postvaccination to be cleared completely. The virB2 mutants, BAvirB2 and BMvirB2, cleared more gradually than the manBA deletions, suggesting that their defect affects survival at a later stage of infection. Lastly, BAasp24 and BMasp24 cleared from mice at a much slower rate, with BAasp24 significantly reduced between 8 and 10 weeks postinfection and BMasp24 significantly reduced after 16 weeks, suggesting that Asp24 may only be required for persistence of the organism. The differences in survival exhibited by these mutants provided an opportunity to evaluate the relationship between protection and persistence of the vaccine strain.
Protection against homologous challenge (i.e., identical vaccine and challenge species) was most successful with the persistent BAasp24 and BMasp24 mutants. These mutants protected mice against infection at or near sterile levels at all time points examined. We consider these mutants to be the best candidates for novel attenuated vaccine strains, since they protect mice against infection better than current vaccine strains. In contrast, the rapid clearance of BAvirB2, BMvirB2, BAmanBA, and BMmanBA may enhance their safety as vaccine strains but provides significantly reduced levels of protection compared to asp24 mutants or currently available vaccine strains. Due to their enhanced safety, however, their use may be supported if delivered using controlled release, where the duration and presentation to the host can be extended to improve efficacy.
In protection studies against heterologous challenge, both BAasp24 and BMasp24 again protected mice significantly against challenge, with very few challenge organisms isolated. These results suggest that survival beyond 8 to 10 weeks does not enhance protection, and since prolonged survival may have side effects, the use of BAasp24 as a vaccine strain against other species of Brucella may be recommended. BAasp24 may be a protective vaccine strain in the event of a bioterrorist attack or from natural exposure to nonvaccinated, infected animals, providing cross-species protection.
The degree of protection provided by different vaccine candidates was compared with the cytokine profiles elicited from splenocytes in order to identify any correlates of protective immunity that may be used to predict potency or efficacy. The experiments described were designed to evaluate long-term immune response, mimicking field conditions, in which vaccination is expected to protect against challenge exposure occurring at undefined later dates.
IFN-, a Th1 cytokine involved in pathogen clearance from activated macrophages, has been well defined as a critical factor in the control of Brucella abortus infections in mice via enhanced activation of macrophages and intracellular killing (3, 21, 40). Infection in mice is more severe when the animals are treated with antibody to IFN-, and brucellosis becomes a fatal infection in IFN- knockout mice, confirming the critical role for IFN- in the early stages of infection (3, 36, 37, 40).
Vaccine candidates asp24 and virB2 produced significant levels of IFN- in response to stimulation with heat-killed Brucella relative to nave mice. However, significant differences in efficacy determined by reduced recovery of the challenge strain were not matched by significant differences in IFN- production. As such, there did not seem to be an absolute correlation of protection with IFN- in this study, since mice vaccinated with asp24 mutants demonstrated significantly higher protection than mice vaccinated with virB2 mutants at all time points tested. Although increased levels of this cytokine may reflect an improved memory response with subsequent protection against challenge, it appears that IFN-, although necessary, is not sufficient to provide protection against infection. IFN- was not induced significantly in mice vaccinated with a rough mutant, manBA. In other studies evaluating cytokine responses from splenocytes from mice vaccinated with rough mutants, a similar result was published (1).
IL-10 is generally considered an antiinflammatory molecule, thought to reduce negative effects caused by proinflammatory cytokines such as IFN- (47). Evidence suggests that inhibition of IL-10 activity results in improved clearance of B. abortus from the spleens of infected mice (16, 20). Since BALB/c mice are known to mount a very strong inflammatory response early in Brucella infection, Th2 cytokines such as IL-10 may be necessary to prevent overwhelming the host and to limit the influx of phagocytic cells and potential spread of the disease (3, 19, 41). Recently, it has been shown that IL-10 develops proinflammatory qualities during active inflammatory responses (47). The fact that IL-10 was generally increased in these experiments compared to nave controls for BMasp24, BAasp24, and BMvirB2 may be related to the observed levels of protection, suggesting a proinflammatory role in the mouse model.
In this study, mice were vaccinated for 20 weeks, and since previous research has never considered such late time points in the mouse, direct comparisons cannot be made to published work. Splenocytes may not be the ideal tissue to evaluate long-term cytokine responses in animals vaccinated for extended periods, and another tissue such as lymph nodes may need to be considered to identify correlates of immunity associated with protection.
Overall, the mouse is a sensitive and popular model for evaluating brucellosis. This model has been invaluable for characterization of genetic components necessary for virulence and is an affordable and consistent method for evaluation of potential vaccine candidates (15, 18, 25). Mutants considered safe and protective in the mouse model are often used as candidates for vaccines in target animal species. The mouse model cannot be used, however, to gauge the effect of persistence on the colonization of reproductive tissues, thus, the need for other animal models after the initial murine screen. Since the mouse is not an exact model of disease in target livestock and wildlife species, vaccine candidates from the mouse will need to be reevaluated in a suitable animal model. The asp24 deletion mutants were the most protective in the mouse screen against both homologous and heterologous challenge infections, and perhaps their protective ability in target species may be superior to the other candidates in that model as well.
ACKNOWLEDGMENTS
Melissa Kahl-McDonagh was supported by USDA Food and Agricultural Sciences National Needs Graduate Fellowship Grant 98-38420-5806 (CSREES) and an NIH Training Grant Predoctoral Fellowship Award. Research was funded by USDA/CSREES-NRICGP 99-35204-7550 and NIH 1RO1 AI48496-01.
pEX18Ap was kindly given to the Ficht lab by Herbert Schweizer.
FOOTNOTES
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