A Mutant of Staphylococcal Enterotoxin C Devoid of Bacterial Superantigenic Activity Elicits a Th2 Immune Response for Protection against St
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感染与免疫杂志 2005年第1期
Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki
Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University, Morioka, Iwate
Department of Immunology, National Institute of Animal Health, Tsukuba, Japan
Department of Bio-Engineering, Dalian Nationalities University, Dalian, People's Republic of China
ABSTRACT
Staphylococcal enterotoxin C (SEC), a bacterial superantigenic exotoxin, is commonly produced by invasive Staphylococcus aureus isolates, especially methicillin-resistant strains and isolates from animal diseases. We constructed and expressed a nontoxic mutant SEC (mSEC) and investigated whether immunization with mSEC, which is devoid of superantigenic activity, can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. The bacterial counts in the organs of mSEC-immunized mice were significantly lower and the survival rate was higher than the corresponding values for the control group. Immunization with mSEC strongly induced the production of T-helper 2 type antibodies, immunoglobulin G1, and immunoglobulin G2b. The production of interleukin-10 (IL-10) and IL-4 was significantly greater in immunized mice challenged with S. aureus than in the control mice, whereas the production of gamma interferon (IFN-) was significantly decreased in the immunized mice. The cytokine response in a spleen cell culture that was stimulated with heat-killed S. aureus or SEC showed that immunization with mSEC inhibited IFN- production and up-regulated IL-10 production in vitro. Furthermore, IFN- and tumor necrosis factor alpha production in vitro was significantly inhibited by sera from mSEC-immunized mice but not by sera from control mice. These results suggest that immunization with mSEC devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by SEC-specific neutralizing antibodies.
INTRODUCTION
Staphylococcus aureus is an important bacterial pathogen in human infections and animal diseases (5, 28, 39). The emergence of antibiotic resistance among clinical isolates has made treatment of staphylococcal infections difficult. To prevent S. aureus infection, a variety of whole staphylococcal preparations, including live, heat-killed, and formalin-fixed preparations of S. aureus cells, have been investigated as vaccines in clinical and veterinary trials. None of these has shown a convincing benefit in patients or farm animals (15, 23, 28). The mechanism for protecting hosts against staphylococcal infections is still not fully understood.
Staphylococcal enterotoxins (SEs), which are bacterial superantigenic proteins produced by S. aureus, play important roles in establishing and maintaining infections (7). Staphylococcal enterotoxin C (SEC) is commonly produced by invasive S. aureus isolates, especially methicillin-resistant S. aureus (MRSA) strains, and can cause severe pathologies. Previous studies have shown that the majority of MRSA in the United States produce SEC or SEB at very high concentrations (37). The majority of S. aureus isolates from bovine mastitis also produce large amounts of SEC (8, 9, 11). These toxins have a significant economic impact on health care and the dairy industry. There is a considerable need for development of vaccines and therapeutic approaches capable of eliminating the toxicity of these compounds (37).
Fields et al. (10) reported the crystal structure of an SEC complex with a T-cell receptor (TCR) -chain and showed that SEC2 and SEC3 bind in the same way to the TCR -chain. Recent studies demonstrated that several residues of SEC, including T20, N23, Y90, K103, and Q210, are important for binding to TCR and are also important for superantigenicity (10, 20, 22, 35). Several reports described the toxicities and biological activities of wild-type and mutant SEA, SEB, and toxic shock syndrome toxin 1 (TSST-1) and showed that genetically altered SEA and SEB were immunogenic in mice and rhesus monkeys (1, 31, 41, 43). Immunization with recombinant or mutant SEA, SEB, and TSST-1 could elicit neutralizing antibodies against wild-type SEs and protect mice or rabbits against lethal shock induced by the wild-type superantigenic toxins (1, 24, 25, 41, 42).
In the present study, we constructed and expressed a single mutant SEC (mSEC), in which residue N23 was changed to A23 and which was devoid of superantigenic activity, and we investigated whether vaccination with mSEC could protect animals against systemic S. aureus infection in a mouse model. The results demonstrated that immunization with mSEC provided protection against the bacterial infection. In addition, our studies showed that the protection was mediated by SEC-specific neutralizing antibodies.
MATERIALS AND METHODS
Animals. Six- to eight-week-old BALB/c mice were purchased from Clea Japan, Inc., Tokyo, Japan. The mice were housed in plastic cages under specific-pathogen-free conditions at the Institute for Experiments, Hirosaki University School of Medicine. The daily cycle consisted of 12 h of light and 12 h of darkness, and food and water were available at all times. All animal experiments were carried out in accordance with the Guidelines for Animal Experimentation of Hirosaki University.
Bacterial strains and culture condition. For infection, S. aureus strain 834, a clinical septic isolate that expresses SEC2 and TSST-1 (30), was cultured at 37°C in tryptic soy broth (Becton, Dickinson, Sparks, Md.) for 15 h and then collected by centrifugation and washed with sterile 0.01 M phosphate-buffered saline (PBS). The washed bacteria were diluted with PBS, and the concentration was adjusted spectrophotometrically at 550 nm to the appropriate value. For genomic DNA preparation, S. aureus FRI 361 expressing SEC2 was inoculated into 5 ml of soybean-casein digest broth (Nissui, Tokyo, Japan) and grown overnight at 37°C with shaking (110 rpm). Escherichia coli DH5 (Toyobo Biochemicals, Osaka, Japan) and E. coli NM522 mutS (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) were routinely grown in Luria broth (Becton) at 37°C with shaking (110 rpm). The antibiotic concentration used to maintain plasmids in E. coli was 100 μg of ampicillin per ml. E. coli DH5 derivatives were grown in 2x YTA medium containing 100 μg of ampicillin per ml at 37°C with shaking.
Expression of rSEC in E. coli. Genomic DNA containing the sec2 gene was isolated from S. aureus FRI 361 by standard procedures (33). In order to construct recombinant SEC (rSEC) expression plasmids, PCR primers were designed to amplify the gene fragment encoding the mature form of SEC (SEC2/GST+ [5'-CCCCGGATTCGAGAGCCAACCAGACCCTACG], whose sequence includes a 5' BamHI site; and SEC2/GST– [5'-CCCCGAATTCTTATCCATTCTTTGTTGTAAGGTGG], whose sequence includes a 5' EcoRI site). The sec2 gene was amplified by PCR by using Pyrobest DNA polymerase (Takara, Shiga, Japan), and the sec2 fragment was then subcloned into pGEM3Zf(+), yielding pKOC2. The nucleotide sequence of the sec2 gene in pKOC2 was verified by using an ABI 310 automatic DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The sec2 fragment of pKOC2 was then digested with BamHI and EcoRI and was subcloned into the pGEX-6P-1 (Amersham Pharmacia Biotech, Inc.) glutathione S-transferase (GST) fusion expression vector. The resultant plasmid containing sec2 was designated pKC2X1. Expression, purification of GST-fused rSEC, and the cleavage and removal of the GST tag from rSEC were performed as described by Omoe et al. (33). The resulting mature rSEC had five additional amino acid residues, GPLGS, at its N terminus.
Construction and expression of mSEC. A selection primer (5'-GCGTGACACCACGATGCCCGCGGCAATGGCAACAACG) and a mutagenic primer (5'-CTGGTACGATGGGTGCTATGAAATATTTATATG) were designed to change oligonucleotide AAT (coding for asparagine 23 in the N terminus of the SEC2 molecule) to GCT (coding for alanine). Site-directed mutagenesis was performed as described by Hu et al. (18). The mutant DNA sequence was confirmed as described above. The asparagine-to-alanine mutant plasmid was designated pGXmSEC and transformed into E. coli DH5. Expression and purification of GST-fused mSEC and cleavage and removal of the GST tag from mSEC were performed as described above for rSEC.
rSEC and mSEC toxicity assay. The toxic effects of rSEC and mSEC on BALB/c mice were tested by using a lipopolysaccharide (LPS)-potentiated mouse lethality model. Mice were first inoculated intraperitoneally with rSEC or mSEC diluted in PBS (0.1 to 20 μg per mouse). The mice were then inoculated with 75 μg of LPS from E. coli O55:B5 (Sigma) 4 h later. The controls included animals given either SEC or LPS alone, and lethality was recorded over 72 h.
Immunodiffusion assay. To determine the immunological reactivity of mSEC, gel double-immunodiffusion assays of rSEC and mSEC with anti-rSEC antibody were performed as described previously (18). Briefly, 1.2% Noble agar (Becton) in PBS was poured into plastic petri dishes. Wells that were the same size (diameter, 8.0 mm) were cut, and the agar plugs were removed. Rabbit anti-rSEC serum (1:16 dilution) and samples containing recombinant or mutant toxins were added to wells (75 μl per well) and incubated in a humidified box at 37°C for 24 h. The gel was stained with 0.1% Coomassie brilliant blue R-250 in 10% acetic acid-40% methanol in distilled water, and this was followed by destaining in the solvent.
Immunization and S. aureus infection. For vaccination of mice, purified rSEC or mSEC was dissolved in PBS and emulsified 1:1 in alum adjuvant (Pierce, Rockford, Ill.). Two-hundred-microliter portions of the emulsion containing 10 μg of rSEC or mSEC or alum alone were injected at two subcutaneous sites on the backs of the mice. Booster immunizations were administered 2 and 4 weeks after the initial vaccination. The mice were challenged on day 7 after the last booster with a lethal dose of S. aureus 834 by intravenous injection. Blood samples were obtained before and after the bacterial challenge. The bacteria in the spleen, liver, and kidneys were enumerated on day 3 after infection by preparing homogenates of these organs in PBS and by plating 10-fold serial dilutions on tryptic soy agar (Becton). Colonies were counted after 24 h of incubation at 37°C. The death of mice was recorded for 15 days.
Serum antibodies. The production of anti-SEC antibodies was measured in serum samples by enzyme-linked immunosorbent assays (ELISAs) as described previously (18). Serum samples were diluted with PBS (1:100), and then serial twofold dilutions were prepared. The levels of immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3, and IgM were expressed as the maximal dilutions of sera.
Cell culture. Spleens were removed aseptically from naive or immunized mice, and spleen cells were obtained by squeezing the organs in RPMI 1640 medium (Nissui). Each cell suspension was filtered through stainless steel mesh (size, 100). After lysis of erythrocytes with 0.85% NH4Cl, the cells were washed three times and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin G per ml, and 100 μg of streptomycin per ml and then placed in a 24-well tissue culture plate at a density of 1 x 106 cells/well in the presence of rSEC, mSEC, or heat-killed S. aureus (HKSA). After 72 h of incubation at 37°C in a 5% CO2 incubator, the supernatants were collected and stored at –80°C until the cytokine assays were performed.
Neutralization assay. For determination of the neutralizing activity of anti-mSEC serum against cytokine production induced by rSEC in vitro, anti-mSEC serum or control serum was preincubated with rSEC at 37°C for 1 h before rSEC was added to spleen cell cultures. After 72 h of incubation at 37°C in a 5% CO2 incubator, the supernatants were collected, and the cytokine assays were performed as described below.
Determination of cytokines. The amounts of gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), interleukin-4 (IL-4), and IL-10 in the supernatants of cell cultures and sera of mice were determined by double-sandwich ELISAs as described previously (29, 30). Organ extracts were prepared by centrifuging 10% (wt/vol) spleen and were homogenized in RPMI 1640 medium containing 1% (wt/vol) 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulfate (CHAPS) (Wako Pure Chemical Co., Osaka, Japan) at 2,000 x g for 20 min. The IL-2 content was determined with a mouse IL-2 ELISA kit (BioSource International, Camarillo, Calif.).
Cell proliferation assays. Spleen cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 μM sodium pyruvate (Wako), and 50 μM 2-mercaptoethanol (Wako). Spleen cells (1 x 106 cells per ml) were incubated with various amounts of mSEC or rSEC in round-bottom microplates at 37°C for 48 h. The cultures were pulsed for 24 h with 20 kBq of [3H]thymidine (ICN Biomedicals, Inc., Irvine, Calif.) per well and then harvested on glass fiber filters. The amount of incorporated [3H]thymidine was measured by liquid scintillation counting.
Statistical analysis. Data were expressed as means ± standard deviations, and the Mann-Whitney U test was used to determine the significance of the differences in bacterial counts in the organs and in cytokine titers between control and experimental groups. For survival experiments, the Kaplan-Meier method was used to obtain the survival fractions, and significance was determined by a log rank test.
RESULTS
Characteristics of mSEC. It has been suggested that several residues of SEC2 or SEC3, including T20, N23, Y90, K103. and Q210, for binding to TCR are important for superantigenicity (10, 20, 22, 35). In this study, we changed the N23 residue of SEC2 molecule to A23 (Fig. 1A), and we designated the mutant gene product mSEC. mSEC was compared with wild-type rSEC on a Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and the results revealed the presence of a readily detectable purified protein band that comigrated with purified rSEC. The immunological reactivities of mSEC and rSEC with polyclonal rabbit anti-rSEC antibody were assayed by using gel immunodiffusion. The antibody reacted readily with purified mSEC and rSEC, and the precipitation lines between mSEC and rSEC were united (Fig. 1B). These results indicate that mSEC has the same matched antibody-binding epitopes as wild-type rSEC. To confirm that the superantigenic activity of mSEC was deleted, the proliferation (Fig. 1C) and cytokine production (Fig. 1D to G) induced by mSEC and rSEC in mouse spleen cells were determined. Substantial amounts of cytokines were induced in the cell cultures at all concentrations of rSEC used, and the results revealed higher proliferation activity. In contrast, for mSEC there was no detectable IFN-, TNF-, and IL-2 production or the amounts produced were less, and there was lower proliferation activity (Fig. 1). These results indicate that mSEC is significantly devoid of superantigenic activity. To further examine whether the toxicity of the mSEC protein could also be detected in vivo, mice were inoculated with LPS plus rSEC or mSEC, and the survival was observed. In striking contrast to the 78% mortality rate of mice inoculated with 15 μg of rSEC plus LPS, none of the mice that were given an equivalent dose of mSEC plus LPS died (data not shown).
Protective effect of immunization with mSEC. Mice were immunized subcutaneously with mSEC or rSEC plus alum or with alum alone, and they were then challenged intravenously with 5 x 107 CFU of S. aureus strain 834 per mouse. Three days after inoculation, the numbers of bacterial cells in the spleens, livers, and kidneys were determined. There were significantly fewer bacterial cells in the spleens and livers of mSEC-immunized mice than in the organs of control mice (Fig. 2A), whereas there was no significant difference in the bacterial counts in the kidneys between the immunized mice and the control mice (data not shown). To further confirm the effect of immunization with mSEC and rSEC on the survival of mice, mice were immunized three times and then intravenously challenged with 5 x 107 CFU of S. aureus per mouse. On day 8 after challenge, 62.5% of the mice immunized with mSEC were alive, and 50% of the mSEC-immunized mice were still alive on day 15 after infection (P < 0.05, as determined by a log rank test). Conversely, all of the mice inoculated with alum alone died 8 days after bacterial challenge (Fig. 2B). In addition, we immunized mice with rSEC in the same way. The results also showed that there was a protective effect, but on day 12 only 37.5% of the mice were alive. These experiments indicate that immunization with mSEC provides efficient protection against lethal S. aureus infection.
Antibody production in immunized mice. Antibody responses were evaluated to analyze the protection provided by antibodies resulting from mSEC and rSEC immunizations. A strong IgG1 antibody response to SEC was seen in the sera obtained from mice immunized with mSEC or rSEC (Fig. 3). In contrast, sera from mice inoculated with only alum failed to react significantly to SEC. Interestingly, there were higher levels of IgG2b in the sera obtained from mSEC-immunized mice than in the sera obtained from rSEC-immunized mice (P < 0.05). In contrast, the sera from mSEC-immunized mice had a lower level of IgG3 (P < 0.05) than the sera from rSEC-immunized mice (Fig. 3). The difference in production of these antibodies might have been due to deletion of the superantigenicity of mSEC.
Cytokine responses in immunized mice after S. aureus challenge. To analyze the role of endogenous cytokines in mice immunized with mSEC against S. aureus infection, IFN- production, IL-4 production, and IL-10 production in the sera were determined by sandwich ELISAs at 2, 4, 6, 12, and 24 h after infection (Fig. 4). The endogenous IFN- production in the sera of mSEC-immunized mice peaked at 12 h and decreased at 24 h of infection (Fig. 4A). The levels of IFN- production were significantly lower in the sera from mSEC-immunized mice than in the sera from control mice. The sera from rSEC-immunized mice also exhibited lower levels of IFN- production. On the other hand, the titers of IL-10 and IL-4 in the sera of mSEC-vaccinated mice increased from 4 and 6 h and were significantly higher than those of control mice (Fig. 4B and C). These results indicated that immunization with mSEC induced strong IL-10 and IL-4 production and inhibited IFN- production.
Cytokine production in spleen cells of mSEC-immunized mice in vitro. To determine the cytokine response in vitro, spleen cells were prepared from mSEC-immunized mice, rSEC-immunized mice, and control mice and stimulated with HKSA, mSEC, or rSEC. The IFN-, TNF- and IL-10 titers in the supernatants of the cell cultures were determined by sandwich ELISAs. The IFN- production was significantly lower in the cell culture from mSEC-immunized mice than in the cell culture from control mice when the mice were stimulated with rSEC (Fig. 5A). There was no significant difference between immunized and control mice stimulated with HKSA or mSEC. Interestingly, the titers of IL-10 in the supernatants of the cell cultures from mSEC-vaccinated mice were significantly higher than the titers of IL-10 in the supernatants of the cell cultures from control mice when they were stimulated with HKSA (P < 0.05) or mSEC (P < 0.05) but not when they were stimulated with rSEC (Fig. 5B). The titers of IL-10 in the cell cultures from rSEC-vaccinated mice were also significantly higher than the titers of IL-10 in the cell cultures from control mice when they stimulated with HKSA (P < 0.05) but not when they were stimulated with mSEC or rSEC (Fig. 5B). The TNF- production in the cell culture from immunized mice and the TNF- production in the cell culture from the control group were not significantly different (Fig. 5C). These results indicated that immunization with mSEC up-regulated IL-10 production and inhibited IFN- production in spleen cells.
Neutralizing effect of anti-mSEC antibodies on cytokine production induced by SEC in vitro. We further examined the effect of serum samples from mSEC-vaccinated mice on SEC-induced production of IFN- and TNF-, which are commonly associated with superantigenic activity. Serum samples from the mice immunized with mSEC plus alum effectively inhibited IFN- and TNF- production from murine spleen cells by SEC compared to serum samples from the alum-injected controls and nonsuperantigenic mutant TSST-1-immunized controls (P < 0.05) (Fig. 6). These results suggest that inhibition of inflammatory cytokine production may be involved in the protective effect obtained from immunization with mSEC.
DISCUSSION
S. aureus expresses a repertoire of factors, including exotoxins such as SEs, TSST-1, exoenzymes, adhesins, and numerous cell-associated components, that play important roles in establishing and maintaining infections (7, 13). SEC, produced by pathogenic strains of S. aureus, especially clinic isolates of MRSA, is known for its involvement in toxic shock syndrome (TSS), persistent infections, and bovine mastitis (6, 8, 11, 34). It is important to consider the fact that the majority of MRSA in the United States produce SEC or SEB at very high concentrations. In Japan, the majority of MRSA produce large amounts of SEC and TSST-1. Since two physicians injected themselves with 3 to 5 μg of superantigenic toxin and subsequently developed TSS, it is probable that a very slight infection could induce TSS (37).
Several residues of SEC for binding to TCR that are important for superantigenicity have been identified (10, 20, 22, 35). In the present study, we changed N23 of the SEC molecule to A23 and obtained a single-site mutant SEC devoid of superantigenic activity. We were unable to detect IFN- and IL-2 production and proliferative responses in murine spleen cells with this mutant at concentrations up to 1,000 ng/ml (Fig. 1), while the mutant still exhibited immunological activity. Our results showed that this mutant toxin, mSEC, is highly effective in inducing toxin-specific antibodies capable of neutralizing superantigenicity, decreasing bacterial growth in organs, and protecting animals from lethal S. aureus infection. Recent studies have examined the development of toxoid vaccines that may protect against the immunobiological effects of pyrogenic toxic superantigens, including SEs, TSST-1, and streptococcal pyrogenic exotoxin, presumably through neutralization by antibodies (14, 26, 32, 38, 40). Gampfer et al. (14) demonstrated that vaccination with nonsuperantigenic SEB and TSST-1 resulted in antibody responses against these toxins and protected against challenge with lethal doses of superantigen potentiated with LPS. Nilsson et al. (32) and another previous study (18) demonstrated that immunization with nonsuperantigenic SEA and TSST-1 protected against S. aureus-induced lethal septic shock. The mechanism of protection after vaccination with these nonsuperantigenic toxins remains unclear.
Mice immunized with mSEC produced high titers of SEC-specific IgG1 and significantly higher levels of IgG2b antibodies than mice in the rSEC-immunized group produced, whereas the animals immunized with mSEC produced significantly lower levels of IgG3 than the rSEC-immunized mice produced (Fig. 3). Immunization with rSEC induced production of both Th1- and Th2-induced immunoglobulin subclasses compared with the control group. The difference in induction of the antibody subclass responses may be due to the lack of superantigenic activity of mSEC. Furthermore, serum samples from mSEC-immunized mice also significantly inhibited IFN- and TNF- production in murine spleen cells by SEC (Fig. 6). These results indicated that SEC-specific antibodies might play an important role in host resistance against S. aureus infection and neutralization of superantigenic activity. The mechanism of action of serum antibodies in S. aureus infections remains elusive. Previous studies identified cross-reactive antibodies for staphylococcal enterotoxins and streptococcal pyrogenic exotoxin A (4, 27). Mice vaccinated with TSST-1 survived when they were challenged with SEA, SEB, or SEC (2). Recently, it was reported that anti-TSST-1 monoclonal antibody also cross-inhibited SEA-induced mitogenic activity and TNF- production in vitro and protected against SEA-induced lethality in a mouse model (19). The demonstrated effects of antibodies may be due to anti-inflammatory activity (21, 37) and neutralization of the toxicity of S. aureus surface components and secreted products (32, 37).
In the present study, serum samples obtained from mSEC-immunized mice produced significantly higher titers of IL-10 and IL-4 and lower titers of IFN- in response to S. aureus infection than serum samples obtained from the control mice produced (Fig. 4). Furthermore, the IFN- production in a spleen cell culture from mSEC-immunized mice was significantly lower than the production in a spleen cell culture from control mice when the cultures were stimulated with HKSA or rSEC in vitro. In contrast, the titers of IL-10 in the supernatants were significantly higher than the titers of IL-10 in the supernatants from control mice (Fig. 5). IL-10 is known to have anti-inflammatory activity in various inflammatory diseases (3, 12, 17). Previous studies demonstrated that IL-10 plays a beneficial role in protecting the host from shock due to endotoxin (17), septic shock (3), and staphylococcal enterotoxin shock (12, 16). A previous study showed that administration of anti-IL-10 monoclonal antibody to mice inhibited the elimination of S. aureus from the organs and suggested that IL-10 might play a beneficial role in host resistance to S. aureus infection (36). It is possible that this cytokine may regulate excess inflammatory responses in S. aureus infection. On the other hand, another previous study showed that IFN- plays a detrimental role in S. aureus infection in mice (30). Administration of anti-IFN- monoclonal antibody resulted in suppression of bacterial growth in the organs and protected mice from the lethal effects of S. aureus infection (30). In addition, IFN- receptor-deficient mice developed severe sepsis with high mortality after S. aureus infection (44). Recently, it was shown that IFN- plays an important role in the pathogenesis of S. aureus infection, because there was an increase in the survival rate, a decrease in the bacterial numbers in the kidneys, and an amelioration of histological changes observed in the kidneys in IFN-–/– mice compared with IFN-+/+ mice (36). In the present study, the results suggest that mSEC vaccination may polarize Th0 toward Th2 in vivo and that the regulation of cytokine production might be involved in acquisition of protection in mSEC-immunized mice. These results, together with previous data, indicated that the protection might be mediated by SEC-specific antibodies that neutralize the S. aureus-produced SEC, as well as by IL-10 and IL-4 induction, and that down-regulate IFN- production induced by superantigenic toxins and S. aureus.
In conclusion, a nontoxic mutant bacterial superantigen, mSEC, was constructed and expressed. Immunization with mSEC profoundly altered the course of disease, including bacterial growth in the organs and survival, by neutralizing the pyrogenic superantigen. mSEC and its specific antibodies should be useful in treatment of toxic shock syndrome and control of S. aureus infection.
ACKNOWLEDGMENTS
We thank Shouji Tsutaya, Hirosaki University Hospital, for assistance with the DNA sequence analysis.
This work was supported in part by a grant-in-aid from the Bovine Mastitis Project, Ministry of Agriculture, Forestry and Fisheries, Japan, and by the National Science and Technology Tackling Research Grant of China for the Key Technique Research on Cow Mastitis Prevention and Control Project (2002 BA518A04).
REFERENCES
1. Bavari, S., B. Dyas, and R. G. Ulrich. 1996. Superantigen vaccines: a comparative study of genetically attenuated receptor-binding mutants of staphylococcal enterotoxin A. J. Infect. Dis. 174:338-345.
2. Bavari, S., R. G. Ulrich, and R. D. LeClaire. 1999. Cross-reactive antibodies prevent the lethal effects of Staphylococcus aureus superantigens. J. Infect. Dis. 180:1365-1369.
3. Berg, D. J., R. Kuhn, K. Rajewsky, W. Muller, S. Menon, N. Davidson, G. Grunig, and D. Rennick. 1995. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. Investig. 96:2339-2347.
4. Bohach, G. A., C. J. Hovde, J. P. Handley, and P. M. Schlievert. 1988. Cross-neutralization of staphylococcal and streptococcal pyrogenic toxins by monoclonal and polyclonal antibodies. Infect. Immun. 56:400-404.
5. Bone, R. C. 1994. Gram-positive organisms and sepsis. Arch. Intern. Med. 154:26-34.
6. Booth, M. C., L. M. Pence, P. Mahasreshti, M. C. Callegan, and M. S. Gilmore. 2001. Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect. Immun. 69:345-352.
7. Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16-34.
8. Edwards, V. M., J. R. Deringer, S. D. Callantine, C. F. Deobald, P. H. Berger, V. Kapur, C. V. Stauffacher, and G. A. Bohach. 1997. Characterization of the canine type C enterotoxin produced by Staphylococcus intermedius pyoderma isolates. Infect. Immun. 65:2346-2352.
9. Ferens, W. A., W. C. Davis, M. J. Hamilton, Y. H. Park, C. F. Deobald, L. Fox, and G. Bohach. 1998. Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C. Infect. Immun. 66:573-580.
10. Fields, B. A., E. L. Malchiodi, H. Li, X. Ysern, C. V. Stauffacher, P. M. Schlievert, K. Karjalainen, and R. A. Mariuzza. 1996. Crystal structure of a T-cell receptor -chain complexed with a superantigen. Nature 384:188-192.
11. Fitzgerald, J. R., S. R. Monday, T. J. Foster, G. A. Bohach, P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J. Bacteriol. 183:63-70.
12. Florquin, S., Z. Amraoui, D. Abramowicz, and M. Goldman. 1994. Systemic release and protective role of IL-10 in staphylococcal enterotoxin B-induced shock in mice. J. Immunol. 153:2618-2623.
13. Foster, T. J. 1991. Potential for vaccination against infections caused by Staphylococcus aureus. Vaccine 9:221-227.
14. Gampfer, J., V. Thon, H. Gulle, H. M. Wolf, and M. M. Eibl. 2002. Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome. Vaccine 20:1354-1364.
15. Greenberg, D. P., J. I. Ward, and A. S. Bayer. 1987. Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits. Infect. Immun. 55:3030-3034.
16. Hasko, G., L. Virag, G. Egnaczyk, A. L. Salzman, and C. Szabo. 1998. The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B. Eur. J. Immunol. 28:1417-1425.
17. Howard, M., T. Muchamuel, S. Andrade, and S. Menon. 1993. Interleukin 10 protects mice from lethal endotoxemia. J. Exp. Med. 177:1205-1208.
18. Hu, D. L., K. Omoe, S. Sasaki, H. Sashinami, H. Sakuraba, Y. Yokomizo, K. Shinagawa, and A. Nakane. 2003. Vaccination with nontoxic mutant toxic shock syndrome toxin 1 protects against Staphylococcus aureus infection. J. Infect. Dis. 188:743-752.
19. Kum, W. W., and A. W. Chow. 2001. Inhibition of staphylococcal enterotoxin A-induced superantigenic and lethal activities by a monoclonal antibody to toxic shock syndrome toxin-1. J. Infect. Dis. 183:1739-1748.
20. Lamphear, J. G., G. A. Bohach, and R. R. Rich. 1998. Structural dichotomy of staphylococcal enterotoxin C superantigens leading to MHC class II-independent activation of T lymphocytes. J. Immunol. 160:2107-2114.
21. LeClaire, R. D., R. E. Hunt, and S. Bavari. 2002. Protection against bacterial superantigen staphylococcal enterotoxin B by passive vaccination. Infect. Immun. 70:2278-2281.
22. Leder, L., A. Llera, P. M. Lavoie, M. I. Lebedeva, H. Li, R. P. Sekaly, G. A. Bohach, P. J. Gahr, P. M. Schlievert, K. Karjalainen, and R. A. Mariuzza. 1998. A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor chain and major histocompatibility complex class II. J. Exp. Med. 187:823-833.
23. Lee, J. C. 1996. The prospects for developing a vaccine against Staphylococcus aureus. Trends Microbiol. 4:162-166.
24. Lowell, G. H., C. Colleton, D. Frost, R. W. Kaminski, M. Hughes, J. Hatch, C. Hooper, J. Estep, L. Pitt, M. Topper, R. E. Hunt, W. Baker, and W. B. Baze. 1996. Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines. Infect. Immun. 64:4686-4693.
25. Lowell, G. H., R. W. Kaminski, S. Grate, R. E. Hunt, C. Charney, S. Zimmer, and C. Colleton. 1996. Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. Infect. Immun. 64:1706-1713.
26. McCormick, J. K., H. Hirt, G. M. Dunny, and P. M. Schlievert. 2000. Pathogenic mechanisms of enterococcal endocarditis. Curr. Infect. Dis. Rep. 2:315-321.
27. Meyer, R. F., L. Miller, R. W. Bennett, and J. D. MacMillan. 1984. Development of a monoclonal antibody capable of interacting with five serotypes of Staphylococcus aureus enterotoxin. Appl. Environ. Microbiol. 47:283-287.
28. Michie, C. A. 2002. Staphylococcal vaccines. Trends Immunol. 23:461-463.
29. Nakane, A., S. Nishikawa, S. Sasaki, T. Miura, M. Asano, M. Kohanawa, K. Ishiwata, and T. Minagawa. 1996. Endogenous interleukin-4, but not interleukin-10, is involved in suppression of host resistance against Listeria monocytogenes infection in gamma interferon-depleted mice. Infect. Immun. 64:1252-1258.
30. Nakane, A., M. Okamoto, M. Asano, M. Kohanawa, and T. Minagawa. 1995. Endogenous gamma interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice. Infect. Immun. 63:1165-1172.
31. Neill, R. J., M. Jett, R. Crane, J. Wootres, C. Welch, D. Hoover, and P. Gemski. 1996. Mitogenic activities of amino acid substitution mutants of staphylococcal enterotoxin B in human and mouse lymphocyte cultures. Infect. Immun. 64:3007-3015.
32. Nilsson, I. M., J. M. Patti, T. Bremell, M. Hook, and A. Tarkowski. 1998. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J. Clin. Investig. 101:2640-2649.
33. Omoe, K., M. Ishikawa, Y. Shimoda, D. L. Hu, S. Ueda, and K. Shinagawa. 2002. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. J. Clin. Microbiol. 40:857-862.
34. Rasheed, J. K., R. J. Arko, J. C. Feeley, F. W. Chandler, C. Thornsberry, R. J. Gibson, M. L. Cohen, C. D. Jeffries, and C. V. Broome. 1985. Acquired ability of Staphylococcus aureus to produce toxic shock-associated protein and resulting illness in a rabbit model. Infect. Immun. 47:598-604.
35. Rossle, S. C., P. M. Bisch, Y. C. Lone, J. P. Abastado, P. Kourilsky, and M. Bellio. 2002. Mutational analysis and molecular modeling of the binding of Staphylococcus aureus enterotoxin C2 to a murine T cell receptor V10 chain. Eur. J. Immunol. 32:2172-2178.
36. Sasaki, S., S. Nishikawa, T. Miura, M. Mizuki, K. Yamada, H. Madarame, Y. I. Tagawa, Y. Iwakura, and A. Nakane. 2000. Interleukin-4 and interleukin-10 are involved in host resistance to Staphylococcus aureus infection through regulation of gamma interferon. Infect. Immun. 68:2424-2430.
37. Schlievert, P. M. 2001. Use of intravenous immunoglobulin in the treatment of staphylococcal and streptococcal toxic shock syndromes and related illnesses. J. Allergy Clin. Immunol. 108:S107-110.
38. Schlievert, P. M., L. M. Jablonski, M. Roggiani, I. Sadler, S. Callantine, D. T. Mitchell, D. H. Ohlendorf, and G. A. Bohach. 2000. Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals. Infect. Immun. 68:3630-3634.
39. Soares, M. J., N. H. Tokumaru-Miyazaki, A. L. Noleto, and A. M. Figueiredo. 1997. Enterotoxin production by Staphylococcus aureus clones and detection of Brazilian epidemic MRSA clone (III::B:A) among isolates from food handlers. J. Med. Microbiol. 46:214-221.
40. Stiles, B. G., A. R. Garza, R. G. Ulrich, and J. W. Boles. 2001. Mucosal vaccination with recombinantly attenuated staphylococcal enterotoxin B and protection in a murine model. Infect. Immun. 69:2031-2036.
41. Ulrich, R. G., M. A. Olson, and S. Bavari. 1998. Development of engineered vaccines effective against structurally related bacterial superantigens. Vaccine 16:1857-1864.
42. Woody, M. A., T. Krakauer, and B. G. Stiles. 1997. Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model. Vaccine 15:133-139.
43. Woody, M. A., T. Krakauer, R. G. Ulrich, and B. G. Stiles. 1998. Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors. J. Infect. Dis. 177:1013-1022.
44. Zhao, Y. X., and A. Tarkowski. 1995. Impact of interferon-gamma receptor deficiency on experimental Staphylococcus aureus septicemia and arthritis. J. Immunol. 155:5736-5742.(Dong-Liang Hu, Jing-Chun )
Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University, Morioka, Iwate
Department of Immunology, National Institute of Animal Health, Tsukuba, Japan
Department of Bio-Engineering, Dalian Nationalities University, Dalian, People's Republic of China
ABSTRACT
Staphylococcal enterotoxin C (SEC), a bacterial superantigenic exotoxin, is commonly produced by invasive Staphylococcus aureus isolates, especially methicillin-resistant strains and isolates from animal diseases. We constructed and expressed a nontoxic mutant SEC (mSEC) and investigated whether immunization with mSEC, which is devoid of superantigenic activity, can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. The bacterial counts in the organs of mSEC-immunized mice were significantly lower and the survival rate was higher than the corresponding values for the control group. Immunization with mSEC strongly induced the production of T-helper 2 type antibodies, immunoglobulin G1, and immunoglobulin G2b. The production of interleukin-10 (IL-10) and IL-4 was significantly greater in immunized mice challenged with S. aureus than in the control mice, whereas the production of gamma interferon (IFN-) was significantly decreased in the immunized mice. The cytokine response in a spleen cell culture that was stimulated with heat-killed S. aureus or SEC showed that immunization with mSEC inhibited IFN- production and up-regulated IL-10 production in vitro. Furthermore, IFN- and tumor necrosis factor alpha production in vitro was significantly inhibited by sera from mSEC-immunized mice but not by sera from control mice. These results suggest that immunization with mSEC devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by SEC-specific neutralizing antibodies.
INTRODUCTION
Staphylococcus aureus is an important bacterial pathogen in human infections and animal diseases (5, 28, 39). The emergence of antibiotic resistance among clinical isolates has made treatment of staphylococcal infections difficult. To prevent S. aureus infection, a variety of whole staphylococcal preparations, including live, heat-killed, and formalin-fixed preparations of S. aureus cells, have been investigated as vaccines in clinical and veterinary trials. None of these has shown a convincing benefit in patients or farm animals (15, 23, 28). The mechanism for protecting hosts against staphylococcal infections is still not fully understood.
Staphylococcal enterotoxins (SEs), which are bacterial superantigenic proteins produced by S. aureus, play important roles in establishing and maintaining infections (7). Staphylococcal enterotoxin C (SEC) is commonly produced by invasive S. aureus isolates, especially methicillin-resistant S. aureus (MRSA) strains, and can cause severe pathologies. Previous studies have shown that the majority of MRSA in the United States produce SEC or SEB at very high concentrations (37). The majority of S. aureus isolates from bovine mastitis also produce large amounts of SEC (8, 9, 11). These toxins have a significant economic impact on health care and the dairy industry. There is a considerable need for development of vaccines and therapeutic approaches capable of eliminating the toxicity of these compounds (37).
Fields et al. (10) reported the crystal structure of an SEC complex with a T-cell receptor (TCR) -chain and showed that SEC2 and SEC3 bind in the same way to the TCR -chain. Recent studies demonstrated that several residues of SEC, including T20, N23, Y90, K103, and Q210, are important for binding to TCR and are also important for superantigenicity (10, 20, 22, 35). Several reports described the toxicities and biological activities of wild-type and mutant SEA, SEB, and toxic shock syndrome toxin 1 (TSST-1) and showed that genetically altered SEA and SEB were immunogenic in mice and rhesus monkeys (1, 31, 41, 43). Immunization with recombinant or mutant SEA, SEB, and TSST-1 could elicit neutralizing antibodies against wild-type SEs and protect mice or rabbits against lethal shock induced by the wild-type superantigenic toxins (1, 24, 25, 41, 42).
In the present study, we constructed and expressed a single mutant SEC (mSEC), in which residue N23 was changed to A23 and which was devoid of superantigenic activity, and we investigated whether vaccination with mSEC could protect animals against systemic S. aureus infection in a mouse model. The results demonstrated that immunization with mSEC provided protection against the bacterial infection. In addition, our studies showed that the protection was mediated by SEC-specific neutralizing antibodies.
MATERIALS AND METHODS
Animals. Six- to eight-week-old BALB/c mice were purchased from Clea Japan, Inc., Tokyo, Japan. The mice were housed in plastic cages under specific-pathogen-free conditions at the Institute for Experiments, Hirosaki University School of Medicine. The daily cycle consisted of 12 h of light and 12 h of darkness, and food and water were available at all times. All animal experiments were carried out in accordance with the Guidelines for Animal Experimentation of Hirosaki University.
Bacterial strains and culture condition. For infection, S. aureus strain 834, a clinical septic isolate that expresses SEC2 and TSST-1 (30), was cultured at 37°C in tryptic soy broth (Becton, Dickinson, Sparks, Md.) for 15 h and then collected by centrifugation and washed with sterile 0.01 M phosphate-buffered saline (PBS). The washed bacteria were diluted with PBS, and the concentration was adjusted spectrophotometrically at 550 nm to the appropriate value. For genomic DNA preparation, S. aureus FRI 361 expressing SEC2 was inoculated into 5 ml of soybean-casein digest broth (Nissui, Tokyo, Japan) and grown overnight at 37°C with shaking (110 rpm). Escherichia coli DH5 (Toyobo Biochemicals, Osaka, Japan) and E. coli NM522 mutS (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) were routinely grown in Luria broth (Becton) at 37°C with shaking (110 rpm). The antibiotic concentration used to maintain plasmids in E. coli was 100 μg of ampicillin per ml. E. coli DH5 derivatives were grown in 2x YTA medium containing 100 μg of ampicillin per ml at 37°C with shaking.
Expression of rSEC in E. coli. Genomic DNA containing the sec2 gene was isolated from S. aureus FRI 361 by standard procedures (33). In order to construct recombinant SEC (rSEC) expression plasmids, PCR primers were designed to amplify the gene fragment encoding the mature form of SEC (SEC2/GST+ [5'-CCCCGGATTCGAGAGCCAACCAGACCCTACG], whose sequence includes a 5' BamHI site; and SEC2/GST– [5'-CCCCGAATTCTTATCCATTCTTTGTTGTAAGGTGG], whose sequence includes a 5' EcoRI site). The sec2 gene was amplified by PCR by using Pyrobest DNA polymerase (Takara, Shiga, Japan), and the sec2 fragment was then subcloned into pGEM3Zf(+), yielding pKOC2. The nucleotide sequence of the sec2 gene in pKOC2 was verified by using an ABI 310 automatic DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The sec2 fragment of pKOC2 was then digested with BamHI and EcoRI and was subcloned into the pGEX-6P-1 (Amersham Pharmacia Biotech, Inc.) glutathione S-transferase (GST) fusion expression vector. The resultant plasmid containing sec2 was designated pKC2X1. Expression, purification of GST-fused rSEC, and the cleavage and removal of the GST tag from rSEC were performed as described by Omoe et al. (33). The resulting mature rSEC had five additional amino acid residues, GPLGS, at its N terminus.
Construction and expression of mSEC. A selection primer (5'-GCGTGACACCACGATGCCCGCGGCAATGGCAACAACG) and a mutagenic primer (5'-CTGGTACGATGGGTGCTATGAAATATTTATATG) were designed to change oligonucleotide AAT (coding for asparagine 23 in the N terminus of the SEC2 molecule) to GCT (coding for alanine). Site-directed mutagenesis was performed as described by Hu et al. (18). The mutant DNA sequence was confirmed as described above. The asparagine-to-alanine mutant plasmid was designated pGXmSEC and transformed into E. coli DH5. Expression and purification of GST-fused mSEC and cleavage and removal of the GST tag from mSEC were performed as described above for rSEC.
rSEC and mSEC toxicity assay. The toxic effects of rSEC and mSEC on BALB/c mice were tested by using a lipopolysaccharide (LPS)-potentiated mouse lethality model. Mice were first inoculated intraperitoneally with rSEC or mSEC diluted in PBS (0.1 to 20 μg per mouse). The mice were then inoculated with 75 μg of LPS from E. coli O55:B5 (Sigma) 4 h later. The controls included animals given either SEC or LPS alone, and lethality was recorded over 72 h.
Immunodiffusion assay. To determine the immunological reactivity of mSEC, gel double-immunodiffusion assays of rSEC and mSEC with anti-rSEC antibody were performed as described previously (18). Briefly, 1.2% Noble agar (Becton) in PBS was poured into plastic petri dishes. Wells that were the same size (diameter, 8.0 mm) were cut, and the agar plugs were removed. Rabbit anti-rSEC serum (1:16 dilution) and samples containing recombinant or mutant toxins were added to wells (75 μl per well) and incubated in a humidified box at 37°C for 24 h. The gel was stained with 0.1% Coomassie brilliant blue R-250 in 10% acetic acid-40% methanol in distilled water, and this was followed by destaining in the solvent.
Immunization and S. aureus infection. For vaccination of mice, purified rSEC or mSEC was dissolved in PBS and emulsified 1:1 in alum adjuvant (Pierce, Rockford, Ill.). Two-hundred-microliter portions of the emulsion containing 10 μg of rSEC or mSEC or alum alone were injected at two subcutaneous sites on the backs of the mice. Booster immunizations were administered 2 and 4 weeks after the initial vaccination. The mice were challenged on day 7 after the last booster with a lethal dose of S. aureus 834 by intravenous injection. Blood samples were obtained before and after the bacterial challenge. The bacteria in the spleen, liver, and kidneys were enumerated on day 3 after infection by preparing homogenates of these organs in PBS and by plating 10-fold serial dilutions on tryptic soy agar (Becton). Colonies were counted after 24 h of incubation at 37°C. The death of mice was recorded for 15 days.
Serum antibodies. The production of anti-SEC antibodies was measured in serum samples by enzyme-linked immunosorbent assays (ELISAs) as described previously (18). Serum samples were diluted with PBS (1:100), and then serial twofold dilutions were prepared. The levels of immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3, and IgM were expressed as the maximal dilutions of sera.
Cell culture. Spleens were removed aseptically from naive or immunized mice, and spleen cells were obtained by squeezing the organs in RPMI 1640 medium (Nissui). Each cell suspension was filtered through stainless steel mesh (size, 100). After lysis of erythrocytes with 0.85% NH4Cl, the cells were washed three times and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin G per ml, and 100 μg of streptomycin per ml and then placed in a 24-well tissue culture plate at a density of 1 x 106 cells/well in the presence of rSEC, mSEC, or heat-killed S. aureus (HKSA). After 72 h of incubation at 37°C in a 5% CO2 incubator, the supernatants were collected and stored at –80°C until the cytokine assays were performed.
Neutralization assay. For determination of the neutralizing activity of anti-mSEC serum against cytokine production induced by rSEC in vitro, anti-mSEC serum or control serum was preincubated with rSEC at 37°C for 1 h before rSEC was added to spleen cell cultures. After 72 h of incubation at 37°C in a 5% CO2 incubator, the supernatants were collected, and the cytokine assays were performed as described below.
Determination of cytokines. The amounts of gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), interleukin-4 (IL-4), and IL-10 in the supernatants of cell cultures and sera of mice were determined by double-sandwich ELISAs as described previously (29, 30). Organ extracts were prepared by centrifuging 10% (wt/vol) spleen and were homogenized in RPMI 1640 medium containing 1% (wt/vol) 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulfate (CHAPS) (Wako Pure Chemical Co., Osaka, Japan) at 2,000 x g for 20 min. The IL-2 content was determined with a mouse IL-2 ELISA kit (BioSource International, Camarillo, Calif.).
Cell proliferation assays. Spleen cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 μM sodium pyruvate (Wako), and 50 μM 2-mercaptoethanol (Wako). Spleen cells (1 x 106 cells per ml) were incubated with various amounts of mSEC or rSEC in round-bottom microplates at 37°C for 48 h. The cultures were pulsed for 24 h with 20 kBq of [3H]thymidine (ICN Biomedicals, Inc., Irvine, Calif.) per well and then harvested on glass fiber filters. The amount of incorporated [3H]thymidine was measured by liquid scintillation counting.
Statistical analysis. Data were expressed as means ± standard deviations, and the Mann-Whitney U test was used to determine the significance of the differences in bacterial counts in the organs and in cytokine titers between control and experimental groups. For survival experiments, the Kaplan-Meier method was used to obtain the survival fractions, and significance was determined by a log rank test.
RESULTS
Characteristics of mSEC. It has been suggested that several residues of SEC2 or SEC3, including T20, N23, Y90, K103. and Q210, for binding to TCR are important for superantigenicity (10, 20, 22, 35). In this study, we changed the N23 residue of SEC2 molecule to A23 (Fig. 1A), and we designated the mutant gene product mSEC. mSEC was compared with wild-type rSEC on a Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and the results revealed the presence of a readily detectable purified protein band that comigrated with purified rSEC. The immunological reactivities of mSEC and rSEC with polyclonal rabbit anti-rSEC antibody were assayed by using gel immunodiffusion. The antibody reacted readily with purified mSEC and rSEC, and the precipitation lines between mSEC and rSEC were united (Fig. 1B). These results indicate that mSEC has the same matched antibody-binding epitopes as wild-type rSEC. To confirm that the superantigenic activity of mSEC was deleted, the proliferation (Fig. 1C) and cytokine production (Fig. 1D to G) induced by mSEC and rSEC in mouse spleen cells were determined. Substantial amounts of cytokines were induced in the cell cultures at all concentrations of rSEC used, and the results revealed higher proliferation activity. In contrast, for mSEC there was no detectable IFN-, TNF-, and IL-2 production or the amounts produced were less, and there was lower proliferation activity (Fig. 1). These results indicate that mSEC is significantly devoid of superantigenic activity. To further examine whether the toxicity of the mSEC protein could also be detected in vivo, mice were inoculated with LPS plus rSEC or mSEC, and the survival was observed. In striking contrast to the 78% mortality rate of mice inoculated with 15 μg of rSEC plus LPS, none of the mice that were given an equivalent dose of mSEC plus LPS died (data not shown).
Protective effect of immunization with mSEC. Mice were immunized subcutaneously with mSEC or rSEC plus alum or with alum alone, and they were then challenged intravenously with 5 x 107 CFU of S. aureus strain 834 per mouse. Three days after inoculation, the numbers of bacterial cells in the spleens, livers, and kidneys were determined. There were significantly fewer bacterial cells in the spleens and livers of mSEC-immunized mice than in the organs of control mice (Fig. 2A), whereas there was no significant difference in the bacterial counts in the kidneys between the immunized mice and the control mice (data not shown). To further confirm the effect of immunization with mSEC and rSEC on the survival of mice, mice were immunized three times and then intravenously challenged with 5 x 107 CFU of S. aureus per mouse. On day 8 after challenge, 62.5% of the mice immunized with mSEC were alive, and 50% of the mSEC-immunized mice were still alive on day 15 after infection (P < 0.05, as determined by a log rank test). Conversely, all of the mice inoculated with alum alone died 8 days after bacterial challenge (Fig. 2B). In addition, we immunized mice with rSEC in the same way. The results also showed that there was a protective effect, but on day 12 only 37.5% of the mice were alive. These experiments indicate that immunization with mSEC provides efficient protection against lethal S. aureus infection.
Antibody production in immunized mice. Antibody responses were evaluated to analyze the protection provided by antibodies resulting from mSEC and rSEC immunizations. A strong IgG1 antibody response to SEC was seen in the sera obtained from mice immunized with mSEC or rSEC (Fig. 3). In contrast, sera from mice inoculated with only alum failed to react significantly to SEC. Interestingly, there were higher levels of IgG2b in the sera obtained from mSEC-immunized mice than in the sera obtained from rSEC-immunized mice (P < 0.05). In contrast, the sera from mSEC-immunized mice had a lower level of IgG3 (P < 0.05) than the sera from rSEC-immunized mice (Fig. 3). The difference in production of these antibodies might have been due to deletion of the superantigenicity of mSEC.
Cytokine responses in immunized mice after S. aureus challenge. To analyze the role of endogenous cytokines in mice immunized with mSEC against S. aureus infection, IFN- production, IL-4 production, and IL-10 production in the sera were determined by sandwich ELISAs at 2, 4, 6, 12, and 24 h after infection (Fig. 4). The endogenous IFN- production in the sera of mSEC-immunized mice peaked at 12 h and decreased at 24 h of infection (Fig. 4A). The levels of IFN- production were significantly lower in the sera from mSEC-immunized mice than in the sera from control mice. The sera from rSEC-immunized mice also exhibited lower levels of IFN- production. On the other hand, the titers of IL-10 and IL-4 in the sera of mSEC-vaccinated mice increased from 4 and 6 h and were significantly higher than those of control mice (Fig. 4B and C). These results indicated that immunization with mSEC induced strong IL-10 and IL-4 production and inhibited IFN- production.
Cytokine production in spleen cells of mSEC-immunized mice in vitro. To determine the cytokine response in vitro, spleen cells were prepared from mSEC-immunized mice, rSEC-immunized mice, and control mice and stimulated with HKSA, mSEC, or rSEC. The IFN-, TNF- and IL-10 titers in the supernatants of the cell cultures were determined by sandwich ELISAs. The IFN- production was significantly lower in the cell culture from mSEC-immunized mice than in the cell culture from control mice when the mice were stimulated with rSEC (Fig. 5A). There was no significant difference between immunized and control mice stimulated with HKSA or mSEC. Interestingly, the titers of IL-10 in the supernatants of the cell cultures from mSEC-vaccinated mice were significantly higher than the titers of IL-10 in the supernatants of the cell cultures from control mice when they were stimulated with HKSA (P < 0.05) or mSEC (P < 0.05) but not when they were stimulated with rSEC (Fig. 5B). The titers of IL-10 in the cell cultures from rSEC-vaccinated mice were also significantly higher than the titers of IL-10 in the cell cultures from control mice when they stimulated with HKSA (P < 0.05) but not when they were stimulated with mSEC or rSEC (Fig. 5B). The TNF- production in the cell culture from immunized mice and the TNF- production in the cell culture from the control group were not significantly different (Fig. 5C). These results indicated that immunization with mSEC up-regulated IL-10 production and inhibited IFN- production in spleen cells.
Neutralizing effect of anti-mSEC antibodies on cytokine production induced by SEC in vitro. We further examined the effect of serum samples from mSEC-vaccinated mice on SEC-induced production of IFN- and TNF-, which are commonly associated with superantigenic activity. Serum samples from the mice immunized with mSEC plus alum effectively inhibited IFN- and TNF- production from murine spleen cells by SEC compared to serum samples from the alum-injected controls and nonsuperantigenic mutant TSST-1-immunized controls (P < 0.05) (Fig. 6). These results suggest that inhibition of inflammatory cytokine production may be involved in the protective effect obtained from immunization with mSEC.
DISCUSSION
S. aureus expresses a repertoire of factors, including exotoxins such as SEs, TSST-1, exoenzymes, adhesins, and numerous cell-associated components, that play important roles in establishing and maintaining infections (7, 13). SEC, produced by pathogenic strains of S. aureus, especially clinic isolates of MRSA, is known for its involvement in toxic shock syndrome (TSS), persistent infections, and bovine mastitis (6, 8, 11, 34). It is important to consider the fact that the majority of MRSA in the United States produce SEC or SEB at very high concentrations. In Japan, the majority of MRSA produce large amounts of SEC and TSST-1. Since two physicians injected themselves with 3 to 5 μg of superantigenic toxin and subsequently developed TSS, it is probable that a very slight infection could induce TSS (37).
Several residues of SEC for binding to TCR that are important for superantigenicity have been identified (10, 20, 22, 35). In the present study, we changed N23 of the SEC molecule to A23 and obtained a single-site mutant SEC devoid of superantigenic activity. We were unable to detect IFN- and IL-2 production and proliferative responses in murine spleen cells with this mutant at concentrations up to 1,000 ng/ml (Fig. 1), while the mutant still exhibited immunological activity. Our results showed that this mutant toxin, mSEC, is highly effective in inducing toxin-specific antibodies capable of neutralizing superantigenicity, decreasing bacterial growth in organs, and protecting animals from lethal S. aureus infection. Recent studies have examined the development of toxoid vaccines that may protect against the immunobiological effects of pyrogenic toxic superantigens, including SEs, TSST-1, and streptococcal pyrogenic exotoxin, presumably through neutralization by antibodies (14, 26, 32, 38, 40). Gampfer et al. (14) demonstrated that vaccination with nonsuperantigenic SEB and TSST-1 resulted in antibody responses against these toxins and protected against challenge with lethal doses of superantigen potentiated with LPS. Nilsson et al. (32) and another previous study (18) demonstrated that immunization with nonsuperantigenic SEA and TSST-1 protected against S. aureus-induced lethal septic shock. The mechanism of protection after vaccination with these nonsuperantigenic toxins remains unclear.
Mice immunized with mSEC produced high titers of SEC-specific IgG1 and significantly higher levels of IgG2b antibodies than mice in the rSEC-immunized group produced, whereas the animals immunized with mSEC produced significantly lower levels of IgG3 than the rSEC-immunized mice produced (Fig. 3). Immunization with rSEC induced production of both Th1- and Th2-induced immunoglobulin subclasses compared with the control group. The difference in induction of the antibody subclass responses may be due to the lack of superantigenic activity of mSEC. Furthermore, serum samples from mSEC-immunized mice also significantly inhibited IFN- and TNF- production in murine spleen cells by SEC (Fig. 6). These results indicated that SEC-specific antibodies might play an important role in host resistance against S. aureus infection and neutralization of superantigenic activity. The mechanism of action of serum antibodies in S. aureus infections remains elusive. Previous studies identified cross-reactive antibodies for staphylococcal enterotoxins and streptococcal pyrogenic exotoxin A (4, 27). Mice vaccinated with TSST-1 survived when they were challenged with SEA, SEB, or SEC (2). Recently, it was reported that anti-TSST-1 monoclonal antibody also cross-inhibited SEA-induced mitogenic activity and TNF- production in vitro and protected against SEA-induced lethality in a mouse model (19). The demonstrated effects of antibodies may be due to anti-inflammatory activity (21, 37) and neutralization of the toxicity of S. aureus surface components and secreted products (32, 37).
In the present study, serum samples obtained from mSEC-immunized mice produced significantly higher titers of IL-10 and IL-4 and lower titers of IFN- in response to S. aureus infection than serum samples obtained from the control mice produced (Fig. 4). Furthermore, the IFN- production in a spleen cell culture from mSEC-immunized mice was significantly lower than the production in a spleen cell culture from control mice when the cultures were stimulated with HKSA or rSEC in vitro. In contrast, the titers of IL-10 in the supernatants were significantly higher than the titers of IL-10 in the supernatants from control mice (Fig. 5). IL-10 is known to have anti-inflammatory activity in various inflammatory diseases (3, 12, 17). Previous studies demonstrated that IL-10 plays a beneficial role in protecting the host from shock due to endotoxin (17), septic shock (3), and staphylococcal enterotoxin shock (12, 16). A previous study showed that administration of anti-IL-10 monoclonal antibody to mice inhibited the elimination of S. aureus from the organs and suggested that IL-10 might play a beneficial role in host resistance to S. aureus infection (36). It is possible that this cytokine may regulate excess inflammatory responses in S. aureus infection. On the other hand, another previous study showed that IFN- plays a detrimental role in S. aureus infection in mice (30). Administration of anti-IFN- monoclonal antibody resulted in suppression of bacterial growth in the organs and protected mice from the lethal effects of S. aureus infection (30). In addition, IFN- receptor-deficient mice developed severe sepsis with high mortality after S. aureus infection (44). Recently, it was shown that IFN- plays an important role in the pathogenesis of S. aureus infection, because there was an increase in the survival rate, a decrease in the bacterial numbers in the kidneys, and an amelioration of histological changes observed in the kidneys in IFN-–/– mice compared with IFN-+/+ mice (36). In the present study, the results suggest that mSEC vaccination may polarize Th0 toward Th2 in vivo and that the regulation of cytokine production might be involved in acquisition of protection in mSEC-immunized mice. These results, together with previous data, indicated that the protection might be mediated by SEC-specific antibodies that neutralize the S. aureus-produced SEC, as well as by IL-10 and IL-4 induction, and that down-regulate IFN- production induced by superantigenic toxins and S. aureus.
In conclusion, a nontoxic mutant bacterial superantigen, mSEC, was constructed and expressed. Immunization with mSEC profoundly altered the course of disease, including bacterial growth in the organs and survival, by neutralizing the pyrogenic superantigen. mSEC and its specific antibodies should be useful in treatment of toxic shock syndrome and control of S. aureus infection.
ACKNOWLEDGMENTS
We thank Shouji Tsutaya, Hirosaki University Hospital, for assistance with the DNA sequence analysis.
This work was supported in part by a grant-in-aid from the Bovine Mastitis Project, Ministry of Agriculture, Forestry and Fisheries, Japan, and by the National Science and Technology Tackling Research Grant of China for the Key Technique Research on Cow Mastitis Prevention and Control Project (2002 BA518A04).
REFERENCES
1. Bavari, S., B. Dyas, and R. G. Ulrich. 1996. Superantigen vaccines: a comparative study of genetically attenuated receptor-binding mutants of staphylococcal enterotoxin A. J. Infect. Dis. 174:338-345.
2. Bavari, S., R. G. Ulrich, and R. D. LeClaire. 1999. Cross-reactive antibodies prevent the lethal effects of Staphylococcus aureus superantigens. J. Infect. Dis. 180:1365-1369.
3. Berg, D. J., R. Kuhn, K. Rajewsky, W. Muller, S. Menon, N. Davidson, G. Grunig, and D. Rennick. 1995. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. Investig. 96:2339-2347.
4. Bohach, G. A., C. J. Hovde, J. P. Handley, and P. M. Schlievert. 1988. Cross-neutralization of staphylococcal and streptococcal pyrogenic toxins by monoclonal and polyclonal antibodies. Infect. Immun. 56:400-404.
5. Bone, R. C. 1994. Gram-positive organisms and sepsis. Arch. Intern. Med. 154:26-34.
6. Booth, M. C., L. M. Pence, P. Mahasreshti, M. C. Callegan, and M. S. Gilmore. 2001. Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect. Immun. 69:345-352.
7. Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16-34.
8. Edwards, V. M., J. R. Deringer, S. D. Callantine, C. F. Deobald, P. H. Berger, V. Kapur, C. V. Stauffacher, and G. A. Bohach. 1997. Characterization of the canine type C enterotoxin produced by Staphylococcus intermedius pyoderma isolates. Infect. Immun. 65:2346-2352.
9. Ferens, W. A., W. C. Davis, M. J. Hamilton, Y. H. Park, C. F. Deobald, L. Fox, and G. Bohach. 1998. Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C. Infect. Immun. 66:573-580.
10. Fields, B. A., E. L. Malchiodi, H. Li, X. Ysern, C. V. Stauffacher, P. M. Schlievert, K. Karjalainen, and R. A. Mariuzza. 1996. Crystal structure of a T-cell receptor -chain complexed with a superantigen. Nature 384:188-192.
11. Fitzgerald, J. R., S. R. Monday, T. J. Foster, G. A. Bohach, P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J. Bacteriol. 183:63-70.
12. Florquin, S., Z. Amraoui, D. Abramowicz, and M. Goldman. 1994. Systemic release and protective role of IL-10 in staphylococcal enterotoxin B-induced shock in mice. J. Immunol. 153:2618-2623.
13. Foster, T. J. 1991. Potential for vaccination against infections caused by Staphylococcus aureus. Vaccine 9:221-227.
14. Gampfer, J., V. Thon, H. Gulle, H. M. Wolf, and M. M. Eibl. 2002. Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome. Vaccine 20:1354-1364.
15. Greenberg, D. P., J. I. Ward, and A. S. Bayer. 1987. Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits. Infect. Immun. 55:3030-3034.
16. Hasko, G., L. Virag, G. Egnaczyk, A. L. Salzman, and C. Szabo. 1998. The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B. Eur. J. Immunol. 28:1417-1425.
17. Howard, M., T. Muchamuel, S. Andrade, and S. Menon. 1993. Interleukin 10 protects mice from lethal endotoxemia. J. Exp. Med. 177:1205-1208.
18. Hu, D. L., K. Omoe, S. Sasaki, H. Sashinami, H. Sakuraba, Y. Yokomizo, K. Shinagawa, and A. Nakane. 2003. Vaccination with nontoxic mutant toxic shock syndrome toxin 1 protects against Staphylococcus aureus infection. J. Infect. Dis. 188:743-752.
19. Kum, W. W., and A. W. Chow. 2001. Inhibition of staphylococcal enterotoxin A-induced superantigenic and lethal activities by a monoclonal antibody to toxic shock syndrome toxin-1. J. Infect. Dis. 183:1739-1748.
20. Lamphear, J. G., G. A. Bohach, and R. R. Rich. 1998. Structural dichotomy of staphylococcal enterotoxin C superantigens leading to MHC class II-independent activation of T lymphocytes. J. Immunol. 160:2107-2114.
21. LeClaire, R. D., R. E. Hunt, and S. Bavari. 2002. Protection against bacterial superantigen staphylococcal enterotoxin B by passive vaccination. Infect. Immun. 70:2278-2281.
22. Leder, L., A. Llera, P. M. Lavoie, M. I. Lebedeva, H. Li, R. P. Sekaly, G. A. Bohach, P. J. Gahr, P. M. Schlievert, K. Karjalainen, and R. A. Mariuzza. 1998. A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor chain and major histocompatibility complex class II. J. Exp. Med. 187:823-833.
23. Lee, J. C. 1996. The prospects for developing a vaccine against Staphylococcus aureus. Trends Microbiol. 4:162-166.
24. Lowell, G. H., C. Colleton, D. Frost, R. W. Kaminski, M. Hughes, J. Hatch, C. Hooper, J. Estep, L. Pitt, M. Topper, R. E. Hunt, W. Baker, and W. B. Baze. 1996. Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines. Infect. Immun. 64:4686-4693.
25. Lowell, G. H., R. W. Kaminski, S. Grate, R. E. Hunt, C. Charney, S. Zimmer, and C. Colleton. 1996. Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. Infect. Immun. 64:1706-1713.
26. McCormick, J. K., H. Hirt, G. M. Dunny, and P. M. Schlievert. 2000. Pathogenic mechanisms of enterococcal endocarditis. Curr. Infect. Dis. Rep. 2:315-321.
27. Meyer, R. F., L. Miller, R. W. Bennett, and J. D. MacMillan. 1984. Development of a monoclonal antibody capable of interacting with five serotypes of Staphylococcus aureus enterotoxin. Appl. Environ. Microbiol. 47:283-287.
28. Michie, C. A. 2002. Staphylococcal vaccines. Trends Immunol. 23:461-463.
29. Nakane, A., S. Nishikawa, S. Sasaki, T. Miura, M. Asano, M. Kohanawa, K. Ishiwata, and T. Minagawa. 1996. Endogenous interleukin-4, but not interleukin-10, is involved in suppression of host resistance against Listeria monocytogenes infection in gamma interferon-depleted mice. Infect. Immun. 64:1252-1258.
30. Nakane, A., M. Okamoto, M. Asano, M. Kohanawa, and T. Minagawa. 1995. Endogenous gamma interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice. Infect. Immun. 63:1165-1172.
31. Neill, R. J., M. Jett, R. Crane, J. Wootres, C. Welch, D. Hoover, and P. Gemski. 1996. Mitogenic activities of amino acid substitution mutants of staphylococcal enterotoxin B in human and mouse lymphocyte cultures. Infect. Immun. 64:3007-3015.
32. Nilsson, I. M., J. M. Patti, T. Bremell, M. Hook, and A. Tarkowski. 1998. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J. Clin. Investig. 101:2640-2649.
33. Omoe, K., M. Ishikawa, Y. Shimoda, D. L. Hu, S. Ueda, and K. Shinagawa. 2002. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. J. Clin. Microbiol. 40:857-862.
34. Rasheed, J. K., R. J. Arko, J. C. Feeley, F. W. Chandler, C. Thornsberry, R. J. Gibson, M. L. Cohen, C. D. Jeffries, and C. V. Broome. 1985. Acquired ability of Staphylococcus aureus to produce toxic shock-associated protein and resulting illness in a rabbit model. Infect. Immun. 47:598-604.
35. Rossle, S. C., P. M. Bisch, Y. C. Lone, J. P. Abastado, P. Kourilsky, and M. Bellio. 2002. Mutational analysis and molecular modeling of the binding of Staphylococcus aureus enterotoxin C2 to a murine T cell receptor V10 chain. Eur. J. Immunol. 32:2172-2178.
36. Sasaki, S., S. Nishikawa, T. Miura, M. Mizuki, K. Yamada, H. Madarame, Y. I. Tagawa, Y. Iwakura, and A. Nakane. 2000. Interleukin-4 and interleukin-10 are involved in host resistance to Staphylococcus aureus infection through regulation of gamma interferon. Infect. Immun. 68:2424-2430.
37. Schlievert, P. M. 2001. Use of intravenous immunoglobulin in the treatment of staphylococcal and streptococcal toxic shock syndromes and related illnesses. J. Allergy Clin. Immunol. 108:S107-110.
38. Schlievert, P. M., L. M. Jablonski, M. Roggiani, I. Sadler, S. Callantine, D. T. Mitchell, D. H. Ohlendorf, and G. A. Bohach. 2000. Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals. Infect. Immun. 68:3630-3634.
39. Soares, M. J., N. H. Tokumaru-Miyazaki, A. L. Noleto, and A. M. Figueiredo. 1997. Enterotoxin production by Staphylococcus aureus clones and detection of Brazilian epidemic MRSA clone (III::B:A) among isolates from food handlers. J. Med. Microbiol. 46:214-221.
40. Stiles, B. G., A. R. Garza, R. G. Ulrich, and J. W. Boles. 2001. Mucosal vaccination with recombinantly attenuated staphylococcal enterotoxin B and protection in a murine model. Infect. Immun. 69:2031-2036.
41. Ulrich, R. G., M. A. Olson, and S. Bavari. 1998. Development of engineered vaccines effective against structurally related bacterial superantigens. Vaccine 16:1857-1864.
42. Woody, M. A., T. Krakauer, and B. G. Stiles. 1997. Staphylococcal enterotoxin B mutants (N23K and F44S): biological effects and vaccine potential in a mouse model. Vaccine 15:133-139.
43. Woody, M. A., T. Krakauer, R. G. Ulrich, and B. G. Stiles. 1998. Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors. J. Infect. Dis. 177:1013-1022.
44. Zhao, Y. X., and A. Tarkowski. 1995. Impact of interferon-gamma receptor deficiency on experimental Staphylococcus aureus septicemia and arthritis. J. Immunol. 155:5736-5742.(Dong-Liang Hu, Jing-Chun )