The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Warmerdam, P. A. M.
Right arrow Articles by Collen, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Warmerdam, P. A. M.
Right arrow Articles by Collen, D.
The Journal of Immunology, 2002, 168: 155-161.
Copyright © 2002 by The American Association of Immunologists

Staphylokinase-Specific Cell-Mediated Immunity in Humans1

Petra A. M. Warmerdam2, Kristel Vanderlick, Petra Vandervoort, Heidi De Smedt, Stéphane Plaisance, Marc De Maeyer and Désiré Collen

Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylokinase is a highly fibrin-specific clot-dissolving agent that constitutes a promising drug for clinical development. It is of bacterial origin, and the majority of patients develop neutralizing Ab after its administration. Several antigenic regions, recognized by these Ab, have been identified, but the underlying immunogenic features of staphylokinase remain unknown. In this study, we show that staphylokinase is a T cell-dependent Ag, and that an immunological memory may be acquired, even without administration of staphylokinase. Thrombolysis with staphylokinase provokes the proliferation of staphylokinase-specific T lymphocytes, which remain elevated over 10 mo posttreatment. Interestingly, analysis of a large number of staphylokinase-specific T cell clones isolated from 10 unrelated donors revealed only six distinct immunogenic regions in the molecule. Moreover, five of the six regions are recognized by T lymphocytes from several individuals, indicating that these regions are not restricted to a single HLA-DR allele. Therefore, these new insights can guide the design of variants with a lower immunogenic profile in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Staphylokinase, a 136-aa protein, is secreted by certain Staphylococcus aureus strains. Its fibrinolytic potency has already been recognized half a century ago (1). The staphylokinase gene has been cloned, among others, from a bacteriophage (Sak42D) and from a lysogenic S. aureus DNA (SakSTAR) (2, 3). The encoded proteins, Sak42D and SakSTAR, have revealed a unique fibrin specificity (for review, see Ref. 4), which qualifies staphylokinase for clinical development. Pilot studies in patients with acute myocardial infarction or peripheral arterial occlusion showed a rapid recanalization of the obstructed artery (5, 6, 7). However, because of its nonhuman origin, most patients (>80%) developed a specific humoral response, and neutralizing IgG could be detected up to 1.5 years after treatment (6, 8, 9). These SakSTAR-specific Ab preclude its readministration, because they may reduce its efficacy, or induce acute allergic reactions.

Until now, studies to lower the immunological properties of the SakSTAR molecule have focused on SakSTAR-specific Ab. Three nonoverlapping immunodominant B cell epitopes have been identified (10, 11). Mutagenesis studies within these regions indicated that modification of several surface-exposed amino acids of the SakSTAR molecule is possible without substantial loss of its thrombolytic potency. Significantly less neutralizing Ab were found after administration of some variants as compared with patients treated with wild-type SakSTAR (12, 13).

Although B lymphocytes are directly responsible for the production of Ab, they can only do so with the help of specific T lymphocytes. Therefore, in a humoral response in which B and T cells meet, the T cells provide the restricting elements (14). In fact, if circulating Ab recognize an Ag (i.e., antigenic), the binding does not predict whether that particular Ag will stimulate subsequent immune reactions (i.e., immunogenic) (15). If T cell help is insufficient or absent, the formation of Ab is greatly impaired, and the development of an immunological memory is hampered (16, 17).

In this study, the underlying cellular aspects of the SakSTAR-specific humoral response were studied. SakSTAR-specific T lymphocytes were detected both in patients and in healthy individuals who never received staphylokinase treatment. Detailed analysis of SakSTAR-specific T lymphocytes, cloned from 10 unrelated individuals, revealed the presence of six distinct immunogenic regions. These regions were not restricted to a single HLA-DR molecule, but were more widely recognized. The limited number of functional T cell regions combined with their potential promiscuous behavior with respect to HLA-DR binding may allow the design of SakSTAR variants with a reduced immunogenic profile in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood donors and PBMC isolation

PBMC from healthy individuals were isolated from either blood or buffy coats (Red Cross Bloodbank, Leuven, Belgium), and from thromboembolic patients before and after (3–44 wk) SakSTAR treatment. The PBMC were isolated according to standard procedures using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation, and stored under liquid nitrogen until usage.

Ag and peptides

Two natural variants of recombinant staphylokinase were used, Sak42D and SakSTAR. The first was isolated and purified in Jena, Germany (18), and SakSTAR was expressed and purified as previously described (19). The purified preparation was further subjected to size exclusion chromatography on Superdex G75 (Pharmacia, Uppsala, Sweden), aliquoted, and stored at -20°C. Streptokinase (SK)3 was purchased from Behring (Hoechst, Brussels, Belgium), aliquoted, and stored at -20°C. The following SakSTAR-derived peptides, 17 mers overlapping 12 residues (1-SSSFDKGKYKKGDDASY-17, 6-KGKYKKGDDASYFEPTG-22, 11-KGDDASYFEPTGPYLMV-27, 16-SYFEPTGPYLMVNVTGV-32, 21-TGPYLMVNVTGVDSKGN-37, 26-MVNVTGVDSKGNELLSP-42, 31-GVDSKGNELLSPHYVEF-47, 36-GNELLSPHYVEFPIKPG-52, 41-SPHYVEFPIKPGTTLTK-57, 46-EFPIKPGTTLTKEKIEY-62, 51-PGTTLTKEKIEYYVEWA-67, 56-TKEKIEYYVEWALDATA-72, 61-EYYVEWALDATAYKEFR-77, 66-WALDATAYKEFRVVELD-82, 71-TAYKEFRVVELDPSAKI-87, 76-FRVVELDPSAKIEVTYY-92, 81-LDPSAKIEVTYYDKNKK-97, 86-KIEVTYYDKNKKKEETK-102, 91-YYDKNKKKEETKSKPIT-107, 96-KKKEETKSKPITEKGFV-112, 101-TKSKPITEKGFVVPDLS-117, 106-ITEKGFVVPDLSEHIKN-122, 110-FVVPDLSEHIKNPGFNL-127, 116-LSEHIKNPGFNLITKVV-132, 120-IKNPGFNLITKVVIEEKK-136), were synthesized and purified at Leiden University Hospital (Leiden, The Netherlands) using a fluorenylmethoxycarbonyl-protected amino acid-coupling procedure (20). The peptides were dissolved in DMSO at 5 mg/ml and stored at 4°C.

Cellular immune response; PBMC proliferation

PBMC were thawed, washed, and cultured in RPMI 1640 containing 5% heat-inactivated human pool serum (BioWhittaker, Walkersville, MD). Cells were resuspended at a concentration of 1.8 x 106/ml, and 2 ml was plated in a 24-well plate. The optimal concentration in the cultures for the Ags Sak42D and SakSTAR was determined to be 50 µg/ml and 250 U/ml for SK. The spontaneous proliferation was determined by culturing the PBMC in culture medium alone. The plates were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were pulsed with 5-bromo-2'-deoxyuridine (BrdU) for the last 20–24 h, and harvested on day 6 (determined to be the optimal day for SakSTAR-specific cellular immune response using PBMC from patients) or on day 7 (determined to be optimal for healthy donor PBMC). The cells were washed once in PBS and subsequently plated in serial dilutions in 96-well flat-bottom plates, and analyzed for their BrdU content using a BrdU ELISA kit (Boehringer Mannheim, Mannheim, Germany). The stimulation index (SI) was calculated by dividing the obtained OD from the PBMC in response to an Ag by that of the spontaneous proliferation. The given SI is the highest value obtained from the plated dilutions, and in which the OD was at least twice the background. The SI in this assay system has a maximum value of 30–40, because of the limited dynamic OD range. PBMC of some donors proliferated spontaneously in culture medium alone, precluding measurement of specific proliferation. Data from such donors were excluded from the results. A SI exceeding 2 was considered to be positive, indicating the presence of specific T lymphocytes to that particular Ag.

T cell cloning

Several healthy individuals, with a SakSTAR-specific cellular immune response, were typed for HLA-DR by the INNO-LiPa method (Innogenetics, Gent, Belgium) (21). Ten unrelated donors were selected, and their PBMC were used in cloning experiments. Briefly, approximately 15 x 106 PBMC were plated in a six-well plate and stimulated with optimal concentrations of SakSTAR or Sak42D for 9–11 days. The visible T cell clones were harvested under the microscope and individually restimulated in a 96-well flat-bottom plate with irradiated autologous PBMC as APC. These wells were supplemented on day 1 with 20 U/ml human rIL-2 (Boehringer Mannheim) and allowed to grow for 8–11 days. Restimulation was repeated every 8–11 days under the same conditions. After three to six passages, autologous PBMC were replaced by autologous EBV-transformed B lymphocytes, as APC.

EBV B cell lines

EBV was isolated from Marmoset B95-8 cells (ATCC CRL1612), according to standard centrifugation and filtration procedures (22). PBMC of the HLA-DR-typed donors were cultured with EBV-containing supernatants in RPMI 1640 supplemented with 10% FBS, 10 µg/ml gentamicin, and 1 µg/ml cyclosporin A. Established EBV B cell lines were then expanded conservatively, and after 3–4 wk of culture, aliquots were stored under liquid nitrogen. The cell lines could be maintained in culture for at least 1 year. These cell lines were tested for the expression of CD19, CD80, CD86, CD54, HLA-DR, and HLA-ABC. All EBV B cells expressed the indicated cell surface markers with minor intensity differences as determined by FACS analysis, and stained negative for CD3 and CD4. They were also tested for their Ag-presenting capacities, and showed to be functional for expanding T cell clones as well as in T cell epitope analysis.

Heterologous EBV B cell lines were obtained from the European Collection of Animal Cell Cultures, for their homozygous HLA-DR typing by the International Histocompatibility Workshop (IHW). For HLA-DR1, IHW9004 (B1*0101, B6*0101); for HLA-DR2, IHW9014 (B1*1501, B5*0101, B6*0201), IHW9010 (B1*1503, B5*0101, B6*0201), and IHW9009 (B1*1601, B5*02, B6*0202); for HLA-DR3, IHW9022 (B1*0301, B3*0101); for HLA-DR4, IHW9031 (B1*0401, B4*0101); for HLA-DR5, IHW9036 (B1*1101, B3*0202) and IHW9041 (B1*1104, B3*0202); for HLA-DR6, IHW9063 (B1*1302, B3*0301) and IHW9057 (B1*1401, B3*0201); for HLA-DR7, IHW9050 (B1*0701, B4*0101); and for HLA-DR8, IHW9068 (B1*0801) were obtained, and used as heterologous APC to study HLA-DR-restricted proliferation of the SakSTAR-specific T cell clones.

T cell clone analysis

All isolated T cell clones were tested twice for their specificity using the above 25 SakSTAR-derived peptides. Briefly, a specific T cell clone combined with mitomycin C-treated autologous EBV B cells (ratio 1:5) combined with Ag (no Ag, SK, negative controls; and SakSTAR, Sak42D, positive controls) or the SakSTAR-derived peptides were cultured in duplo in a 96-well round-bottom plate for 4 days. Subsequently, they were pulsed for 20–24 h with BrdU, harvested, and analyzed for their BrdU content. Proliferation of the T cell clones was found positive, if the SI was at least three in the independent experiments. Similar assays were performed, but with the IHW cell lines as APC, to study the HLA-DR restriction of a particular T cell clone. Blocking assays were done by preincubating the autologous APC with anti-HLA-DR Ab (clone G46-6 (L243, mIgG2a,{kappa}); BD Biosciences, Heidelberg, Germany), and subsequently cultured as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Staphylokinase-specific cellular immune response in normal individuals

Some patients develop SakSTAR-specific IgGs after treatment; therefore, the presence of circulating SakSTAR-specific T lymphocytes was studied. Classical proliferation assays in 96-well plates did not give reproducible data, because the frequency of SakSTAR-specific T lymphocytes in the periphery was found to be less than 1 in 200,000 PBMC. However, upscaling of the assay system, as described in Materials and Methods, resulted in the detection of a SakSTAR-specific cellular response. Fig. 1Go shows the proliferation responses of PBMC from 80 healthy normal individuals to SakSTAR, Sak42D, and a classical recall Ag SK. A positive response to SK was found for 97% of the donors, indicating that all assay conditions for proliferation were present. In contrast, a correlation was found between a positive staphylokinase-specific cellular response (SI > 2) and age. In the oldest age groups (over 40 years of age), >70% tested positive, whereas in the youngest age groups (under 30 years old), less than 30% of the donors showed a staphylokinase-specific cellular response.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. Proliferation responses to wild-type staphylokinase variants, SakSTAR (A) and Sak42D (B), and streptokinase (C) of PBMC from healthy individuals. PBMC isolated from 80 healthy individuals were cultured for 6 days with 50 µg/ml SakSTAR, Sak42D, or 250 U/ml SK. These Ag-stimulated PBMC were then pulsed with BrdU and harvested after 20–24 h. They were analyzed for their BrdU content, and the SI was calculated. A SI of >2 was considered to be positive cellular immune response. The 80 donors were divided into groups according to age. Each individual spot represents the measured cellular response from a single donor from the indicated age group.

 
Staphylokinase-specific cellular immune response in patients

The vast majority of peripheral arterial occlusion patients, selected for SakSTAR treatment, exceed the age of 55 years. Therefore, it was expected that many of the eligible patients had established an immunological memory against staphylokinase. For a single SakSTAR-treated patient, who developed a high neutralizing Ab titer, the cellular response could be monitored up to 10 mo after treatment. Fig. 2Go shows that this patient had a low, but detectable level of SakSTAR-specific T lymphocytes before treatment. Four weeks after treatment, the specific SakSTAR response reached the limits of our assay system (SI ± 30–40), indicating that the frequency of SakSTAR-specific T lymphocytes had largely increased. The SakSTAR-specific cellular response remained high for 10 mo after treatment.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. SakSTAR-specific proliferation responses of PBMC from a patient, who developed a high titer of neutralizing Ab after SakSTAR treatment. Blood samples were obtained before and at 4 and 8 wk and 10 mo after the thrombolytic treatment. PBMC were isolated and cultured for 5 days with 50 µg/ml SakSTAR. These Ag-stimulated PBMC were pulsed with BrdU, harvested after 20–24 h, and analyzed for their BrdU content, and the SI was calculated.

 
Subsequently, several patients were tested for the presence of SakSTAR-specific T lymphocytes before and 4 wk after treatment. In one patient who did not develop a humoral response, no SakSTAR-specific proliferation could be measured before nor 4 wk after treatment (Fig. 3GoA). Six patients who developed neutralizing Ab inactivating between 5 and 20 µg/ml (Fig. 3GoB) or >150 µg/ml (Fig. 3GoC) SakSTAR were also tested for a SakSTAR-specific cellular immune response. Before treatment, one of these patients tested negative for SakSTAR-specific proliferation, four patients were found with a low but detectable SakSTAR-specific cellular response, and one patient showed a relative high specific proliferation. The SakSTAR-specific cellular immune response may be increased 4 wk after treatment in all of these patients, and seemed to be higher for the group of patients who developed high titers of SakSTAR-neutralizing IgG (Fig. 3GoC) compared with those with intermediate levels of IgG (Fig. 3GoB).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. SakSTAR-specific proliferation responses of PBMC from patients before and after SakSTAR treatment. Patients with a peripheral arterial occlusion, who have been treated with SakSTAR, were monitored for the generation of neutralizing Ab. For one patient, no SakSTAR-specific IgG could be detected (A), and for six others neutralizing titers between 5–20 µg/ml (B) and >150 µg/ml (C) were measured after 4 wk. PBMC were obtained from these patients before and at 4 wk after thrombolytic therapy. The PBMC were cultured for 5 days with 50 µg/ml SakSTAR, subsequently pulsed with BrdU, and harvested after 20–24 h. The BrdU content was determined, and the SI was calculated. Each individual spot represents the SI found for a single patient at the indicated time of treatment.

 
Staphylokinase-specific T cell epitopes

Most T lymphocytes, supporting a humoral response, recognize a peptide in the context of a particular HLA-DR molecule. Therefore, 10 unrelated donors, covering approximately 95% of the HLA-DR haplotypes occurring in Europe and the U.S., were selected for the cloning of their staphylokinase-specific T cells. Table IGo summarizes the results. Of 283 isolated T cell clones, 109 could be maintained and were found to be SakSTAR specific and of the Th phenotype (CD3+CD4+). Their specificity was identified in proliferation assays using overlapping SakSTAR-derived peptides. The majority of the isolated clones proliferated on one or two overlapping peptides, revealing the localization of a T cell epitope. It could have been expected that such an extensive T cell screening resulted in different epitopes for each HLA-DR heterozygous donor. Interestingly, this was not the case, as is clearly depicted in Fig. 4Go. T cell clones from the unrelated donors recognized similar areas in the SakSTAR molecule, revealing six distinct immunogenic regions.


View this table:
[in this window]
[in a new window]
  		Table I. T cell cloning overview from 10 unrelated healthy individuals

 


View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. Alignment of the identified immunogenic regions recognized by T cell clones isolated from 10 unrelated healthy individuals with the SakSTAR sequence. Several T cell clones were isolated from each donor by repeated SakSTAR stimulation. The SakSTAR-specific T cell clones were then tested for their specificity using 25 17-mer peptides overlapping 12 residues, spanning the entire SakSTAR molecule. None of the individual T cell clones responded to more than two overlapping peptides. The regions of the SakSTAR molecule that induced proliferation of T cell clones from a particular donor are given, in which the amino acids from the corresponding peptides are depicted in lower case letters and the overlapping residues are depicted in upper case letters. The identified immunogenic areas are named, as indicated at the bottom line.

 
Two independent T cell clones isolated from a single donor recognize the A1 immunogenic region, located at the amino terminal. Interestingly, one of these clones did not only proliferate on SakSTAR, Sak42D, or a peptide including this particular region of the SakSTAR molecule, but also when SK is given as an Ag.

The first immunogenic cluster, named D1, is recognized by T cell clones isolated from four different donors (40, 41, 57, and 58), and is located within aa 16–32 of SakSTAR. Since these donors do not share a common HLA-DR molecule (see Table IGo), region D1-specific T cell clones were tested in proliferation assays using heterologous EBV B cells as APC (IHW cell lines). Specific proliferation could be measured for individual T cell clones, if an HLA-DR2, DR3, or DR7 homozygous IHW cell line presented SakSTAR or the peptide including the D1 region of the SakSTAR molecule (Table IIGo).


View this table:
[in this window]
[in a new window]
  		Table II. Proliferation of SakSTAR-specific T cell clones on HLA-DR-homozygous IHW cell lines as APC1

 
The immunogenic region F2 was identified by T cell clones isolated from donors 33, 59, and 66, and is located within the aa 56–72 of SakSTAR molecule. The HLA-DR restriction for this region is also not clear, because the donors do not share a common HLA-DR gene. HLA-DR1 and DR4 homozygous IHW cell lines supported the proliferation of region F2-specific T cell clones (Table IIGo).

The most noticeable immunogenic region is located within aa 71–87 (C3), as the vast majority of the isolated T cell clones recognized it. Moreover, region C3-specific T cell clones could be isolated from 9 of the 10 donors, and therefore the HLA-DR restriction, if any, is not obvious. Two region C3-specific T cell clones were tested for proliferation in the presence of peptide and anti-HLA-DR-preincubated APC. The proliferation of both clones could be inhibited in a dose-dependent manner (Fig. 5Go). Several clones from different donors were then tested for proliferation when using the IHW cell lines as APC. It was found that HLA-DR2, DR3, DR4, DR6, DR7, or DR8 homozygous IHW cell lines could support the proliferation of individual region C3-specific T cell clones (Table IIGo).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 5. Proliferation response of region C3-specific T cell clone in the presence or absence of anti-HLA-DR Ab. Two region C3-specific T cell clones ({circ} and •) were cocultured with the C3 peptide; irradiated autologous APC prebound with different concentrations of anti-HLA-DR Ab. After 4 days of culture, BrdU was added, and after 24 h harvested. The BrdU incorporation was analyzed, and the SI was calculated. The maximal proliferation was determined by the proliferation of the C3 peptide alone and was set at 100%. The proliferation found in the presence of different concentrations of anti-HLA-DR Ab is plotted relative to that of the C3 peptide alone.

 
T cell clones from the donors 38, 40, 44, 52, and 66, who all share HLA-DR3, revealed the immunogenic region D4, which is located within the aa 106–122 of the SakSTAR molecule. Several region D4-specific T cell clones seemed to be HLA-DR3 restricted, but others could proliferate on the SakSTAR peptide, including the D4 region presented by an HLA-DR6 homozygous IHW cell line (Table IIGo).

The final immunogenic region, named A5, is found at the carboxyl terminus of the SakSTAR molecule. Again the HLA-DR restriction of this region is not obvious, because of the different HLA-DR backgrounds of the five donors. HLA-DR restriction analysis of the region A5-specific T cell clones revealed that at least HLA-DR2, DR5, DR6, and DR8 homozygous IHW cell lines could present this region (Table IIGo).

Immunogenic region-specific cellular immune response in normal individuals

Human T cell clones identified the described immunogenic regions in SakSTAR. To relate their immunogenic importance for SakSTAR in humans, 50 new individuals over 45 years of age were tested for an immunogenic region-specific cellular response. Therefore, PBMC were primed with SakSTAR, followed by a restimulation with either SakSTAR or each of the immunogenic peptides. The results of the proliferation responses are summarized in Fig. 6Go. Of the 50 donors, 37 tested positive for SakSTAR. Only a single donor recognized the A1 region, whereas 7, 8, and 7 donors proliferated on the peptides, including the D1, F2, and A5 regions, respectively, which corresponds to approximately 20% of the SakSTAR+ donors. PBMC from 18 donors proliferated upon restimulation with the D4 region containing peptide, and 25 recognized the C3 region containing peptide, corresponding to approximately 50% and 70% of the SakSTAR+ donors, respectively.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 6. Proliferation responses of PBMC to SakSTAR or a SakSTAR-derived immunogenic peptide. PBMC from 50 new individuals older than 45 years of age were isolated and primed with SakSTAR. After 10 days of culture, the cells were restimulated with irradiated autologous CD3-depleted PBMC and Ag/peptides, as indicated. After 4 days of culture, the cells were pulsed with BrdU and harvested after 20–24 h. Their BrdU content was analyzed, and the SI was calculated. The number of donors with a positive cellular response is plotted, and the percentage given in the bars is the relative frequency of the region-specific response among the SakSTAR-positive donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thrombolytic treatment with nonhuman protein drugs, such as SK and SakSTAR, has been shown to result in the generation of neutralizing IgG. For SK, neutralizing Ab are generally found in humans before treatment (8, 23, 24). Infusion triggers a secondary immune response, as demonstrated by an increase of SK-specific IgG within 3–5 days, peaking within 2 wk (8, 23), and remains in the circulation for years (24). In contrast, SakSTAR-neutralizing Ab are virtually undetectable in healthy individuals (8). Administration of SakSTAR results in a humoral response in the majority of patients, which is characterized by intermediate and incidentally high titers of IgG, which start to increase 3 wk after treatment (6, 8, 25). Although these latter characteristics do not fit with a classical secondary immune response, staphylokinase is of bacterial origin and, therefore, the human immune system may have encountered this protein Ag before treatment.

Streptokinase is associated with group A streptococci (26), which explains its characteristics as a classical recall Ag (Fig. 1Go) (27). It has been reported that the presence of staphylokinase in a Staphylococcus strain is related to its lysogenic status (28), but reports on the percentage of staphylokinase-positive strains remain unclear. Of 100 consecutive cultures from a bacteriology laboratory, only four strains were reported to produce plasminogen activator activity (29), while significantly increased staphylokinase-specific Ab were reported in 3 of 17 patients with S. aureus bacteremia (8). Other studies with pathogenic strains have reported as many as 80% of their S. aureus isolates to produce staphylokinase (30, 31). Furthermore, it has been suggested that bacteria with a plasminogen-activating capacity are more invasive and virulent (26). We found an age-related SakSTAR-specific cellular response in healthy individuals, probably reflecting that not all S. aureus strains do express staphylokinase, and suggesting an increased probability for an individual to have encountered a positive strain over time.

Although the frequency of SakSTAR-specific T lymphocytes was determined to be low, the human immune system can acquire a cellular memory for staphylokinase without ever receiving SakSTAR treatment. The absence of a staphylokinase-specific cellular immune response may indicate that an individual never encountered staphylokinase, or that the amount of specific T cells dropped below detection limits, or that the individual is a nonresponder. Therefore, no predictions can be made on the outcome of the humoral response if such a person is treated with SakSTAR. Two patients tested negative for the presence of SakSTAR-specific T lymphocytes, before treatment. One patient was found to be a nonresponder, whereas the other generated high levels of neutralizing Ab and specific T cells were detected after treatment. In contrast, if an individual tested positive for SakSTAR-specific T lymphocytes, a secondary immune response may be expected upon thrombolytic therapy. This is observed in five patients showing increased levels of neutralizing IgG and a possible increased SakSTAR-specific cellular response after treatment. Furthermore, 70% of the older healthy donors were positive for a staphylokinase-specific cellular response. Consequently, at least as many of the SakSTAR-treated patients (the vast majority exceeds the age of 55) would be expected to develop a secondary immune response. This is in agreement with studies showing that >=80% of the SakSTAR-treated patients develop neutralizing Ab titers (6, 25). In conclusion, SakSTAR classifies as a T cell-dependent Ag, and thrombolytic treatment probably triggers a secondary immune response in most patients. However, the observed late onset of IgG production after treatment remains unexplained.

When 10 healthy HLA-DR heterozygous individuals were chosen for T cell cloning experiments, two to three T cell immunoreactive regions were identified per donor, as was expected, since T cell epitopes are genetically restricted. However, alignment of these T cell regions from all donors revealed the localization of only six SakSTAR immunogenic regions. Anti-HLA-DR Ab blocked the proliferation of two independent T cell clones, indicating that at least some of the SakSTAR-specific clones are HLA-DR restricted. Several SakSTAR-specific T cell clones could specifically proliferate on autologous EBV B cells, as well as on IHW cell lines. Although the HLA-DR homozygous IHW cell lines were matched with the HLA-DR B1 alleles from our donors, they are transformed human B cells, and consequently, they express other HLA molecules such as HLA-A, HLA-B, HLA-C, HLA-DP, and HLA-DQ. Thus, if a SakSTAR-specific T cell clone was found to proliferate on a particular IHW cell line, it points at the HLA-DR restriction for that T cell clone, although the involvement of another HLA molecule cannot be ruled out. Five of the SakSTAR immunogenic regions were recognized by T lymphocytes in the context of genetically distinct EBV B cells. Therefore, it is likely that the identified regions can accommodate more than a single HLA molecule, or may even be promiscuous in its HLA-DR binding, as was implicated for the C3 region.

Peptide cross-reactivity has been reported, and it is generally understood to be an essential feature of the TCR (32). Thus, a single TCR can recognize a number of peptides, but a cross-reactive Ag/peptide is often found by chance. Such a unique T cell clone was isolated and proliferated on the peptide comprising SakSTAR residues 1–17, SakSTAR, Sak42D, but also when SK was given as Ag. SakSTAR and SK do not share an overall sequence homology, and alignment of the SakSTAR amino-terminal part with SK reveals only a low sequence homology. However, TCR cross-reactivity is not necessarily due to a strong sequence homology. Moreover, a single TCR has been reported to recognize two different peptides in distinct MHC molecules (33). Although we did not confirm the proliferation with a SK peptide, the proliferation of the cross-reactive T cell clone in response to SakSTAR and SK implies that a cellular memory for SakSTAR may be acquired via a streptococcus infection in certain individuals.

A detailed analysis of the six T cell immunoreactive areas in SakSTAR is currently under study, to locate possible T cell epitopes and identify important residues for T cell reactivity. Recently, in the scope of developing peptide vaccines, several bioinformatic tools have emerged to discover good T cell epitope candidates (34, 35). These algorithms based on databases of candidate T cell epitopes and our own dead-end elimination computer program, evaluating the interaction energy between a HLA-DR peptide-binding groove and a peptide (36), will help to locate the T cell epitopes within the identified immunogenic regions. Studies combining computer models with functional assays using the SakSTAR-specific T cell clones may lead to the design of SakSTAR variants, aiming at the elimination of T cell reactivity, while retaining thrombolytic activity. Theoretically, the more widely recognized human T cell epitopes are eliminated from a protein, the lower the immunogenic potential for the human population.

In this study, the immunogenic regions of SakSTAR were identified by human T cell clones, and as they were obtained by selection and cloning procedures, they may not represent a normal distribution of their relative importance for the immunogenicity of SakSTAR. Therefore, 50 new individuals older than 45 years of age were evaluated for an immunogenic peptide-specific cellular immune response. Culturing PBMC with SakSTAR-derived peptides did not result in a specific proliferation, probably because the frequency of peptide-specific T lymphocytes is too low. However, this could be evaluated when PBMC were primed with SakSTAR, and subsequently restimulated with the immunogenic peptides. Similar as in the cellular assay for the age group over 45 years of age, 74% was found positive for SakSTAR. Only a single donor recognized the A1 region, whereas the D1, F2, and A5 regions were recognized by approximately 20% of the SakSTAR-positive donors, approximately 50% responded to the D4 region, and 70% to the C3 region. These data confirm the relative importance of five of the six identified immunogenic regions, and in particular the possible contribution of the C3 region to support a humoral response in humans when exposed to SakSTAR.

One earlier reported B cell epitope (19) overlaps with the most prominent C3 T cell immunoreactive region. Several SakSTAR variants, with multiple mutations in this area, were tried in patients with peripheral arterial occlusion. Interestingly, the Ab response was found significantly reduced compared with patients treated with wild-type SakSTAR (12, 13). The observed reduced humoral response may, however, be explained at least in part by the mutation of an important immunoreactive T cell region in these SakSTAR variants.

In conclusion, the identification of the clustered immunogenic regions in SakSTAR will guide rational studies to eliminate T cell reactivity of these areas, which may result in a SakSTAR variant with a reduced immunogenic profile, but with intact thrombolytic properties.


   Footnotes
 
1 This work was supported in part by a sponsored research agreement between the Flemish Institute for the Advancement of Scientific and Technological Research in the Industry (IWT) and Thromb-X NV, a spinoff company of the University of Leuven, in which D.C. has an equity interest. S.P. is a "Postdoctoraal Onderzoeker van het Fonds voor Wetenschappelijk Onderzoek-Vlaanderen." Back

2 Address correspondence and reprint requests to Dr. Petra A. M. Warmerdam, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Campus Gasthuisberg O&N, Herestraat 49, 3000 Leuven, Belgium. E-mail address: petra.warmerdam{at}med.kuleuven.ac.be Back

3 Abbreviations used in this paper: SK, streptokinase; BrdU, 5-bromo-2'-deoxyuridine; IHW, International Histocompatibility Workshop; SI, stimulation index. Back

Received for publication May 30, 2001. Accepted for publication October 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lack, C. H.. 1948. Staphylokinase: an activator of plasma protease. Nature 161:559.[Medline]
  2. Behnke, D., D. Gerlach. 1987. Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase: a bacterial plasminogen activator. Mol. Gen. Genet. 210:528.[Medline]
  3. Collen, D., A. Zhao, P. Holvoet, P. Marynen. 1992. Primary structure and gene structure of staphylokinase. Fibrinolysis 6:226.
  4. Collen, D.. 1998. Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent. Nat. Med. 4:279.[Medline]
  5. Collen, D., F. Van de Werf. 1993. Coronary thrombolysis with recombinant staphylokinase in patients with evolving myocardial infarction. Circulation 87:1850.[Abstract/Free Full Text]
  6. Vanderschueren, S., L. Stockx, G. Wilms, H. Lacroix, R. Verhaeghe, J. Vermylen, D. Collen. 1995. Thrombolytic therapy of peripheral arterial occlusion with recombinant staphylokinase. Circulation 92:2050.[Abstract/Free Full Text]
  7. Vanderschueren, S., L. Barrios, P. Kerdsinchai, P. Van den Heuvel, L. Hermans, M. Vrolix, F. De Man, E. Benit, L. Muyldermans, D. Collen, et al 1995. A randomized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction: the STAR Trial Group. Circulation 92:2044.[Abstract/Free Full Text]
  8. Declerck, P. J., S. Vanderschueren, J. Billiet, H. Moreau, D. Collen. 1994. Prevalence and induction of circulating antibodies against recombinant staphylokinase. Thromb. Haemostasis 71:129.[Medline]
  9. Vanderschueren, S. M., J. M. Stassen, D. Collen. 1994. On the immunogenicity of recombinant staphylokinase in patients and in animal models. Thromb. Haemostasis 72:297.[Medline]
  10. Collen, D., F. De Cock, E. Demarsin, S. Jenne, I. Lasters, Y. Laroche, P. Warmerdam, L. Jespers. 1997. Recombinant staphylokinase variants with altered immunoreactivity. III. Species variability of antibody binding patterns. Circulation 95:455.[Abstract/Free Full Text]
  11. Jenne, S., K. Brepoels, D. Collen, L. Jespers. 1998. High resolution mapping of the B cell epitopes of staphylokinase in humans using negative selection of a phage-displayed antigen library. J. Immunol. 161:3161.[Abstract/Free Full Text]
  12. Collen, D., H. Moreau, L. Stockx, S. Vanderschueren. 1996. Recombinant staphylokinase variants with altered immunoreactivity. II. Thrombolytic properties and antibody induction. Circulation 94:207.[Abstract/Free Full Text]
  13. Collen, D., L. Stockx, H. Lacroix, R. Suy, S. Vanderschueren. 1997. Recombinant staphylokinase variants with altered immunoreactivity. IV. Identification of variants with reduced antibody induction but intact potency. Circulation 95:463.[Abstract/Free Full Text]
  14. Amsen, D., A. M. Kruisbeek. 1998. Thymocyte selection: not by TCR alone. Immunol. Rev. 165:209.[Medline]
  15. Clark, M.. 2000. Antibody humanization: a case of the "Emperor’s new clothes"?. Immunol. Today 21:397.[Medline]
  16. Oscherwitz, J., F. M. Gotch, K. B. Cease, J. A. Berzofsky. 1999. New insights and approaches regarding B- and T-cell epitopes in HIV vaccine design. AIDS 13:S163.
  17. Hilleman, M. R.. 1999. Personal historical chronicle of six decades of basic and applied research in virology, immunology, and vaccinology. Immunol. Rev. 170:7.[Medline]
  18. Schlott, B., M. Hartmann, K. H. Guhrs, E. Birch-Hirschfeid, H. D. Pohl, S. Vanderschueren, F. Van de Werf, A. Michoel, D. Collen, D. Behnke. 1994. High yield production and purification of recombinant staphylokinase for thrombolytic therapy. Bio/Technology 12:185.[Medline]
  19. Collen, D., R. Bernaerts, P. Declerck, F. De Cock, E. Demarsin, S. Jenne, Y. Laroche, H. R. Lijnen, K. Silence, M. Verstreken. 1996. Recombinant staphylokinase variants with altered immunoreactivity. I. Construction and characterization. Circulation 94:197.[Abstract/Free Full Text]
  20. Hiemstra, H. S., G. Duinkerken, W. E. Benckhuijsen, R. Amons, R. R. de Vries, B. O. Roep, J. W. Drijfhout. 1997. The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries. Proc. Natl. Acad. Sci. USA 94:10313.[Abstract/Free Full Text]
  21. Buyse, I., R. Decorte, M. Baens, H. Cuppens, G. Semana, M. P. Emonds, P. Marynen, J. J. Cassiman. 1993. Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization. Tissue Antigens 41:1.[Medline]
  22. Miller, G., M. Lipman. 1973. Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci. USA 70:190.[Abstract/Free Full Text]
  23. Jalihal, S., G. K. Morris. 1990. Antistreptokinase titres after intravenous streptokinase. Lancet 335:184.[Medline]
  24. Squire, I. B., W. Lawley, S. Fletcher, E. Holme, W. S. Hillis, C. Hewitt, K. L. Woods. 1999. Humoral and cellular immune responses up to 7.5 years after administration of streptokinase for acute myocardial infarction. Eur. Heart J. 20:1245.[Abstract/Free Full Text]
  25. Vanderschueren, S., D. Collen, F. van de Werf. 1996. A pilot study on bolus administration of recombinant staphylokinase for coronary artery thrombolysis. Thromb. Haemostasis 76:541.[Medline]
  26. Coleman, J. L., J. L. Benach. 1999. Use of the plasminogen activation system by microorganisms. J. Lab. Clin. Med. 134:567.[Medline]
  27. Youkeles, L. H., M. Y. Soliman, D. L. Rosenstreich. 1991. Use of streptokinase as a recall antigen for lymphocyte proliferation testing in vitro. J. Allergy Clin. Immunol. 88:166.[Medline]
  28. Coleman, D. C., D. J. Sullivan, R. J. Russell, J. P. Arbuthnott, B. F. Carey, H. M. Pomeroy. 1989. Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of {beta}-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion. J. Gen. Microbiol. 135:1679.[Abstract/Free Full Text]
  29. Collen, D., K. Silence, E. Demarsin, M. De Mol, H. R. Lijnen. 1992. Isolation and characterization of natural and recombinant staphylokinase. Fibrinolysis 6:203.
  30. Humphreys, H., C. T. Keane, R. Hone, H. Pomeroy, R. J. Russell, J. P. Arbuthnott, D. C. Coleman. 1989. Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers. J. Med. Microbiol. 28:163.[Abstract/Free Full Text]
  31. Slobodnikova, L., D. Kotulova, I. Zahradnikova. 1995. Staphylococcus aureus in chronic and recurrent infections. Folia Microbiol. 40:655.
  32. Mason, D.. 1998. A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol. Today 19:395.[Medline]
  33. Basu, D., S. Horvath, I. Matsumoto, D. H. Fremont, P. M. Allen. 2000. Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR. J. Immunol. 164:5788.[Abstract/Free Full Text]
  34. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanovic. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213.[Medline]
  35. Sturniolo, T., E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, J. Hammer. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17:555.[Medline]
  36. Desmet, J., M. De Maeyer, B. Hazes, I. Lasters. 1992. The dead-end elimination theorem and its use in protein side-chain positioning. Nature 356:539.



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
F. A. Harding, A. D. Liu, M. Stickler, O. J. Razo, R. Chin, N. Faravashi, W. Viola, T. Graycar, V. P. Yeung, W. Aehle, et al.
A {beta}-lactamase with reduced immunogenicity for the targeted delivery of chemotherapeutics using antibody-directed enzyme prodrug therapy
Mol. Cancer Ther., November 1, 2005; 4(11): 1791 - 1800.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Warmerdam, P. A. M.
Right arrow Articles by Collen, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Warmerdam, P. A. M.
Right arrow Articles by Collen, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS