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Infection and Immunity, October 2002, p. 5822-5826, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5822-5826.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cellular Localization of Babesia bovis Merozoite Rhoptry-Associated Protein 1 and Its Erythrocyte-Binding Activity

Naoaki Yokoyama, Boonchit Suthisak, Haruyuki Hirata, Tomohide Matsuo, Noboru Inoue, Chihiro Sugimoto, and Ikuo Igarashi^*

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

Received 30 April 2002/ Returned for modification 7 June 2002/ Accepted 28 June 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca²⁺ accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Babesia bovis is a hemoprotozoan parasite that causes great economic losses to the cattle industry worldwide. It is transmitted by tick vectors and has an asexual intraerythrocytic cycle in the infected cattle (5, 9). Understanding the basic molecular mechanism(s) of the asexual intraerythrocytic cycle, particularly the process of merozoite invasion into and escape from infected erythrocytes (RBC), may accelerate the development of an effective vaccine. Extracellular merozoites are directly exposed to the host humoral immune components. Consequently, efforts to identify potential components for the development of a vaccine are primarily directed at the merozoite stage.

Apicomplexans utilize several rhoptry proteins in their invasion into and development within the host cell (16, 18). Extracellular merozoites attach to the host RBC and reorient to bring the apical organelles close to the attachment interface, and through the interaction of protozoan ligands with several surface receptors, the rhoptry products are released at the point of membrane invagination. Although the morphological events during host cell recognition and penetration appear to be similar among the apicomplexans, some molecular events mediated by each secretory component of the rhoptry vary and are unique (16, 18).

The rhoptry-associated protein 1 (RAP-1) of B. bovis merozoites bears substantial sequence homology to the RAP-1 of other Babesia parasites (3, 4) and contains immunogenic B-cell epitopes (20), and the purified recombinant RAP-1 has proven effective in inducing protective immunity in the vaccinated cattle (22). All of these earlier findings point to the biological and immunological characteristics of RAP-1 that would justify its inclusion in a recombinant vaccine to prevent B. bovis infection. However, the role or roles of the rhoptries in merozoite binding to host RBC are still not fully understood. In view of the recently reported RAP-1 expression in sporozoites and the subsequent inhibition of sporozoite attachment to host cells by using a specific antibody (Ab) (12), we postulated a similar host erythrocytic binding role of RAP-1 expressed in B. bovis merozoites. In this paper, we present our findings on the cellular localization and binding affinity of RAP-1 with bovine RBC (Bo-RBC) and the neutralizing effect of the anti-RAP-1-specific monoclonal Ab (MAb) on merozoite attachment to Bo-RBC, including its growth-inhibitory effect on the parasite proliferation in an in vitro culture.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parasite. The Texas T2B strain of B. bovis (19) was maintained in purified Bo-RBC by the microaerophilus stationary-phase culture system (10). Cell cultures that had between 5 and 10% parasitemia were washed three times with cold phosphate-buffered saline (PBS), and the pellets were stored at -80°C for later use in DNA extraction and protein analysis.

Cloning of RAP-1 gene. Parasite-infected Bo-RBC were suspended in a DNA extraction buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10 mM EDTA) and digested with 0.1% sodium dodecyl sulfate (SDS) and 100 µg of protease K per ml at 55°C for 2 h. Genomic DNA was then extracted with phenol-chloroform and precipitated with ethanol. The DNA pellet was suspended in a Tris-EDTA (TE) buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and used as a template for PCR cloning. The primers 5'-acggatccGACAATGAGAATCATT-3' and 5'-acggatccAAACGCATCTCATCAG-3' (lowercase letters form BamHI restriction site linkers) were designed on the basis of the nucleotide sequence of the B. bovis RAP-1 gene (21) and used to amplify a 1,698-bp DNA fragment according to a protocol described previously (13). The amplified DNA product was digested with BamHI, purified with the QIAquick gel extraction kit (Qiagen, Inc., Hilden, Germany), and then ligated into the BamHI site of pBluescript SK(+). The plasmid was designated as pBS/RAP-1, and the nucleotide sequence of the inserted RAP-1 gene was confirmed with the ABI PRISM 377 DNA sequencer (Perkin-Elmer, Norwalk, Conn.).

Preparation of the anti-RAP-1 Ab. The BamHI fragment of the RAP-1 gene in pBS/RAP-1 was inserted into the BamHI site of a pGEMEX E. coli expression vector (Amersham Pharmacia Biotech, Piscataway, N.J.). The resultant vector, pGEMEX/RAP-1, was used to produce the insoluble RAP-1 gene product fused with a bacteriophage T7 gene 10-leader peptide in Escherichia coli. The transformed E. coli cells were washed three times with cold PBS, lysed by sonication, and centrifuged at 1,800 x g for 10 min at 4°C. The pellet was resuspended in PBS to a final concentration of 10 mg/ml and used as an antigen. Eight-week-old female BALB/c mice were intraperitoneally (i.p.) immunized with 2 mg of the antigen premixed with complete Freund's adjuvant (Difco, Detroit, Mich.). On days 14 and 28 post-initial immunization, the mice were i.p. injected with the same amount of the antigen in incomplete Freund's adjuvant (Difco). Sera from the immunized mice were collected 10 days after the last booster.

Anti-B. bovis MAb was prepared with approximately 10⁷ free merozoite-injected BALB/c mice, according to an immunization protocol similar to that described for the production of the anti-RAP-1 gene product Ab. The spleen from the mouse that registered the highest Ab titer was processed by the protocol described earlier (13). One relevant antibody-producing hybridoma was identified, and the MAb was designated as MAb 1C1 of the isotype immunoglobulin G1 (IgG1) (Amersham Pharmacia Biotech). MAb 1C1 and mouse IgG (for use as a control in subsequent experiments) were then purified (1).

Construction of a recombinant baculovirus. A previously described technique (23) was used to generate a recombinant baculovirus. The recombinant donor plasmid, pRAP-1/FBD, was generated by insertion of the BamHI fragment of the RAP-1 gene in pBS/RAP-1 into the BamHI site downstream of the polyhedrin promoter region in pFastBac-Dual (Life Technologies). pRAP-1/FBD was transformed into DH10Bac competent cells (Life Technologies), and the resultant transposed bacmid containing the RAP-1 gene was selected and transfected into Sf9 insect cells. A recombinant baculovirus obtained from the supernatant of transfected cells was designated as AcRAP-1.

IFAT. The indirect immunofluorescence antibody test (IFAT) was performed as follows. Smears of infected Bo-RBC were prepared on slides, dried, and fixed in 50% acetone-50% methanol solution for 5 min at -20°C. MAb 1C1 was applied as the first antibody on the fixed RBC and incubated for 30 min at 37°C. After three washes with PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (ICN Pharmaceuticals, Irvine, Calif.) was applied as a second Ab and incubated for 30 min at 37°C. The slides were washed three times with PBS, incubated with 25 µg of propidium iodide (PI) per ml (Molecular Probes) and 50 µg of RNase A per ml for 10 min at 37°C, and then mounted in 50% glycerol-PBS. The slides were then observed by confocal laser scanning microscopy (CLM) (TCS NT; Leica).

Smears of infected RBC preincubated with the MAb 1C1 were also prepared for IFAT. In brief, 20 µl of the infected RBC (5% parasitemia) was suspended in 500 µl of 1 mg of MAb 1C1-PBS per ml and 10% fetal calf serum-PBS. The suspension was incubated for 6 h at 4°C with gentle shaking. After three washes with cold PBS, thin smears of the treated RBC were prepared on slides and fixed. The slides were incubated with the FITC-conjugated goat anti-mouse IgG and stained with PI for observation by CLM.

IEM. For immunoelectron microscopy (IEM), infected Bo-RBC (5% parasitemia) were fixed in 4% paraformaldehyde-PBS for 2 h at 4°C, embedded in 2% agarose, and then frozen in an optimal cutting temperature compound (Tissue-Tek). Frozen sections (about 10 µm thick) were cut on a Leica CM 3050 cryostat and placed on poly-L-lysine-coated glass slides. Sections were incubated with the MAb 1C1 or mouse IgG (control) for 2 h at room temperature and visualized with 3,3'-diaminobenzidine (DAB; Dako Envision kit). DAB-stained sections were fixed in 1% OsO₄-PBS, dehydrated in an ethanol series, and then mounted in Epon 812 resin-filled capsules (TAAB). After polymerization, the immunostained sections were embedded in Epon blocks, cut into sections that were about 80 nm thick, counterstained with uranyl acetate-lead citrate, and examined with a Hitachi H-7500 transmission electron microscope.

RBC-binding assay. For the purification of B. bovis merozoites, infected Bo-RBC were treated with 0.83% NH₄Cl solution for 10 min at 37°C and then washed three times with cold PBS (13). Similarly, Sf9 insect cells were seeded on an 80-cm² culture flask (Nunc, Roskilde, Denmark) and infected with the recombinant baculovirus, AcRAP-1, at a multiplicity of infection of 5 PFU per cell. At 72 h postinfection, infected and noninfected cells were harvested and washed three times with cold PBS. The pellets containing approximately 10⁷ parasites or infected Sf9 cells were suspended in 1 ml of a lysis buffer (50 mM Tris-HCl [pH 7.6], 0.1% Triton X-100, 150 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT], 10 µg [each] of pepstatin A and leupeptin per ml), incubated on ice for 20 min, and then centrifuged at 18,000 x g for 30 min at 4°C. These clarified lysates were dialyzed overnight against 1 liter of a dialysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM PMSF, 1 mM DTT). The dialysates were centrifuged at 18,000 x g for 30 min at 4°C again, and the supernatants were frozen in liquid nitrogen and stored at -80°C until use.

The cell extracts were equally divided into microcentrifuge tubes, and the aliquots were incubated with 10 µl of Bo-RBC or equine RBC for 1 h at 4°C with gentle shaking. In order to test the inhibition due to the MAb or the Ca²⁺ dependence for the binding, the aliquots were preincubated with the indicated amount of each agent for 1 h at 4°C with gentle shaking before addition of RBC. The treated RBC were precipitated by centrifugation at 250 x g for 5 min at 4°C, suspended in 1 ml of a dialysis buffer, and then incubated for 15 min at 4°C with gentle shaking. This step was repeated three times. Each of the precipitated pools of RBC was suspended in 50 µl of an SDS sample buffer (62.5 mM Tris-HCl [pH 6.8]), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.01% bromphenol blue) and heated at 100°C for 5 min. After centrifugation at 18,000 x g for 5 min at room temperature, the supernatants were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) with 10% polyacrylamide gel, and Western blot analysis (23) was carried out with the anti-RAP-1 polyclonal Ab.

In vitro growth-inhibitory assay. For the in vitro growth-inhibitory assay, 20 µl of infected Bo-RBC (5% parasitemia) was suspended in 500 µl of M199 growth medium supplemented with 40% bovine serum, and MAb 1C1 or control mouse IgG was added to a final concentration of 1 mg/ml. The mixture was incubated for 6 h at 4°C with gentle shaking. The culture was diluted to 1% parasitemia and 10% hematocrit with a growth medium containing fresh Bo-RBC, and 200 µl of the mixture was dispensed into 96-well plates (Nunc), incubated at 37°C in a humidified multigas water-jacketed incubator for 4 days, and replaced daily with 200 µl of fresh medium containing 1 mg of the indicated IgG per ml. Parasite growth was monitored in Giemsa-stained smears based on approximately 1,000 observed RBC.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cellular localization of B. bovis RAP-1. We generated two kinds of Ab specific for B. bovis RAP-1: the MAb 1C1 and the polyclonal Ab raised in mice immunized with the recombinant RAP-1 gene product expressed in E. coli. The specificities of these Abs were confirmed by Western blot analyses with the merozoite extract or the recombinant RAP-1 gene product produced by a baculoviral expression system (data not shown).

In IFAT with the MAb 1C1, RAP-1 was detectable in all of the developmental stages of merozoites as well as in extracellular parasites (Fig. 1). In the early phase of merozoite development (the ring and subsequent pear-shaped forms), RAP-1 appeared as a dense accumulation in the parasite cytoplasm (Fig. 1A), which later thinned out and blanketed the host cell cytoplasm (Fig. 1B), but retained a denser mass or accumulation around the nuclei of extracellular merozoite (Fig. 1C). In the host cell, RAP-1 showed preferential accumulation on the inner cell membrane, and near the merozoite, it was observed by IEM bordering the parasite's outer surface with the MAb 1C1 (Fig. 2A).

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FIG. 1. Methanol-acetone-fixed smears of B. bovis-infected RBC incubated with anti-RAP-1 MAb 1C1 and then observed with CLM. The MAb-antigen reaction (green) and nucleus (red) were visualized with the FITC-conjugated secondary Ab and PI staining, respectively. Bars, 5 µm.

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FIG. 2. Localization of RAP-1 (arrows) in infected Bo-RBC by IEM. Reaction with anti-RAP-1 MAb 1C1 (A) and control mouse IgG (B). Note the absence of RAP-1 in the control. Bars, 2 µm.

Erythrocyte-binding activity of B. bovis RAP-1. An in vivo erythrocyte-binding assay was carried out to determine the binding of RAP-1 to Bo-RBC. Bo-RBC coprecipitated the 60-kDa RAP-1 from the merozoite lysate (Fig. 3A). An inhibitory test with MAb 1C1 was also conducted to confirm RAP-1 binding specificity with Bo-RBC. MAb 1C1 prevented the interaction of RAP-1 with Bo-RBC in a dose-dependent manner (Fig. 3B). Interestingly, RAP-1 also exhibited binding affinity with equine RBC (Fig. 3C).

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FIG. 3. In vivo erythrocyte-binding activity of B. bovis RAP-1 in the presence of merozoite extract. (A) Binding of RAP-1 to Bo-RBC mixed with the extract (lane 2) and control buffer (lane 3). Precipitated proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-RAP-1 polyclonal Ab. Input sample (merozoite extract) prior to RBC precipitation (lane 1). (B) Inhibition of B. bovis RAP-1-erythrocyte binding with the addition of MAb 1C1. Merozoite extracts were preincubated with 1 mg (lane 2), 0.2 mg (lane 3), 0.04 mg (lane 4), and 0.008 mg (lane 5) of MAb 1C1 and 1 mg of control mouse IgG (lane 1). Note complete inhibition with higher Ab titers (lanes 2 and 3). (C) Binding of B. bovis RAP-1 to bovine (lane 2) and equine (lane 4) RBC in the presence of merozoite extract. Lanes 3 and 5 show the reaction of bovine (lane 3) and equine (lane 5) RBC plus control buffer. Lane 1 contained input sample (merozoite extract) prior to RBC precipitation.

The recombinant RAP-1 gene product of 60 kDa visualized by Western blot analysis was highly expressed in the AcRAP-1-infected insect cells. The reproducibility of RAP-1 and the Bo-RBC interaction were similarly assessed with the recombinant RAP-1 gene product in the infected insect cell lysate. Binding of recombinant RAP-1 to Bo-RBC was weak (Fig. 4A), and the addition of increasing concentrations of Ca²⁺ accentuated its binding activity (Fig. 4B).

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FIG. 4. In vitro erythrocyte binding activity of recombinant RAP-1 expressed in Sf9 cells. (A) Recombinant RAP-1 binding to Bo-RBC. RBC were mixed with AcRAP-1-infected (lane 2) and noninfected (lane 3) cell extracts. Precipitated proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-RAP-1 polyclonal Ab. (B) Ca2+-dependent Bo-RBC-binding activity of recombinant RAP-1 in the presence of 0 (lane 2), 10 (lane 3), or 100 (lane 4) mM CaCl2. Lane 1 (A and B) shows input sample prior to RBC precipitation.

In vitro growth inhibition with the anti-RAP-1 MAb. Supplementation of the growth medium with 1 mg of MAb 1C1 per ml significantly inhibited the proliferation of B. bovis (Fig. 5). To examine which stage of RAP-1 is critical for the inhibition of parasite growth by the MAb in the culture, we carried out another IFAT by using the infected Bo-RBC smear preincubated with the MAb 1C1 before fixing. MAb 1C1 recognized RAP-1, appearing as a localized mass adjacent to newly released merozoites (Fig. 6A), as well as RAP-1 exposed on the cell membrane of broken infected RBC (Fig. 6B). However, the localization of reactive RAP-1 antigen was undetectable in the parasites within Bo-RBC (Fig. 6C). These results suggest that MAb 1C1 could not penetrate intact infected RBC.

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FIG. 5. Growth-inhibitory effect of MAb 1C1 on B. bovis in vitro. Cultures were initiated at 1% parasitemia containing 1 mg of MAb 1C1 or mouse IgG (control) per ml and replaced daily with 200 µl of fresh medium plus the indicated Ab. Each point represents the mean ± standard deviation. Asterisks indicate a significant difference (P < 0.05) between the experimental and control groups.

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FIG. 6. Localization of RAP-1 in B. bovis-infected RBC preincubated with anti-RAP-1 MAb 1C1 before fixing and then examined with CLM. The MAb-antigen reaction (green) and nucleus (red) were visualized with FITC-conjugated secondary antibody and PI staining, respectively. RAP-1 was detectable around extracellular merozoites (A) and on broken infected Bo-RBC (B), but not in live intact infected Bo-RBC (C). Bars, 5 µm.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Earlier studies have identified and characterized a 60-kDa RAP-1 protein that has an apical location on the surface of B. bovis merozoites (3, 7, 15, 17) and was detectable as a rhoptry component by IEM (20). In the present study, we have demonstrated that RAP-1 localized in the different developmental stages of merozoites (the ring and subsequent pear-shaped forms) and extracellular merozoites. RAP-1 also seemed to have interacted with the cytoskeleton and/or membrane of RBC at the later phase of parasite development, which is associated with merozoite maturation and release and host cell rupture, as suggested in malaria parasites (18). Since RAP-1 has a signal sequence (3), it may be logical to assume then that the RAP-1 had been secreted from merozoites. The RAP-1 family of proteins does not possess any significant homologies with any known proteases (3). However, the proteolytic activity of RAP-1, which is noncovalently associated with protease of a similar size and functions as a cofactor, has been proposed earlier (2). In view of these earlier reports, the biochemical characterization of the recombinant B. bovis RAP-1 gene product should help clarify the protease and/or protease-associated protein function of RAP-1.

The binding of RAP-1 to intact Bo-RBC demonstrated in an in vivo erythrocyte-binding assay reinforces our other observations with CLM and IEM of its role in host cell attachment. RAP-1 recognized equine RBC as well, implying the presence of surface receptors common to both bovine and equine RBC. The in vivo erythrocyte-binding assay with merozoite lysate revealed RAP-1 binding to be independent of the presence of Ca²⁺, since the incorporation of EDTA did not eliminate its interaction with Bo-RBC. In the in vitro erythrocyte-binding assay with the lysate from the AcRAP-1-infected insect cells in the presence of EDTA, binding was weaker than that in the native RAP-1. With an increased concentration of Ca²⁺, however, the binding became more evident. Recently, native Py235, a high-molecular-weight rhoptry protein of Plasmodium yoelii subsp. yoelii, was reported to bind to murine RBC in a Ca²⁺-independent manner (14). For now, we postulate that the native RAP-1 derived from the merozoite lysate becomes fully functional to ensure complete entry into host cells during parasite maturation in the presence of Ca²⁺, considering that calcium is essential for successful invasion of protozoan parasites into host cells (11). Additional work on the identification of the RBC surface receptors recognized by RAP-1 is important to elucidate the biological basis of RAP-1 and erythrocyte interaction.

A reduction in the growth of the virulent P. yoelii subsp. yoelii strain has been reported in mice either administered anti-Py235 MAb or immunized with the affinity-purified protein (6, 8). In the present study, the growth of B. bovis parasites was significantly inhibited in the presence of MAb 1C1, and the interaction of RAP-1 and the MAb was nonetheless evident adjacent to the extracellular merozoites. In contrast, the MAb could not penetrate intact cells and recognize the merozoites in the cells. These results suggest the possibility that the Ab inhibition of B. bovis replication occurred during the extracellular phase of the parasite's life cycle due to the neutralization or immobilization of functional RAP-1 covering the merozoite surface with the MAb, thus hindering parasite binding to new host cells. Although 1 mg of MAb 1C1 completely blocked the binding of RAP-1 to Bo-RBC in vitro, it only reduced the developmental parasitemia by about 20% at the dose of 1 mg/ml. These data suggest the existence of additional mechanisms by which B. bovis binds to and penetrates host RBC as seen in other apicomplexan (16, 18). Since the MAb also reacted to the surface of broken infected RBC membrane, another possibility, the blockage of mature merozoite release by the presence of RAP-1-MAb 1C1 complexes on the RBC membrane, could not be excluded. In order to understand the multiple functions of RAP-1, further study is required, not only of the invasion of merozoites into RBC, but also the escape of newly formed merozoites from host cells.

 
We are grateful to Guy H. Palmer, Washington State University, for kindly providing the B. bovis Texas T2B strain and for critically reading the manuscript and offering suggestions.

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

 
* Corresponding author. Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5641. Fax: 81-155-49-5643. E-mail: igarcpmi{at}obihiro.ac.jp.

Editor: W. A. Petri, Jr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, October 2002, p. 5822-5826, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5822-5826.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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