Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia
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感染与免疫杂志 2005年第10期
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2H7, Canada
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, AB T2N 4N1, Canada
ABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.
INTRODUCTION
The enterohemorrhagic group of Escherichia coli (EHEC) causes hemorrhagic colitis and, in anywhere from 5 to 15% of infected individuals, primarily very young and elderly subjects, a serious clinical complication called hemolytic-uremic syndrome (HUS) (8, 23, 45). HUS is characterized by a triad of clinical features, including hemolytic anemia, thrombocytopenia, and ultimately, acute renal failure. As well, in the most severe cases, various degrees of central nervous system involvement can become apparent. EHEC is also referred to as Shiga toxigenic E. coli because this organism expresses exotoxins that are biochemically related to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (43). Once EHEC has colonized the intestines, it is possible for Shiga toxins to be translocated into the submucosal compartment of the gut (3, 19). From there, the toxins can be transported, possibly on the surface of polymorphonuclear leukocytes (23, 46, 47), to extraintestinal organs and tissues, primarily the kidneys, where Shiga toxin-mediated damage to endothelial cells in the glomerular capillaries induces a cascade of microangiopathic events leading ultimately to HUS (45).
The Shiga toxins produced by EHEC are classified into two families, Stx1 and Stx2, also commonly referred to as verotoxin or verocytotoxin 1 and 2, according to their genetic and antigenic relatedness to the prototypic Stx produced by S. dysenteriae. In this classification scheme, Stx1 is virtually identical to the prototypic S. dysenteriae Stx (38). In contrast, Stx2 is more distantly related to Stx, and at least 10 variant species of Stx2 (reviewed in references 8, 45, and 49) have now been described in various EHEC strains and serotypes isolated from humans and animals.
Regardless of their relationship to one another, the Shiga toxins all display a classic AB5 structure in which one enzymatically active A subunit is combined with five identical B subunits which form a homopentamer displaying fivefold radial symmetry around a central pore (12, 13). In the Stx family, the A and B subunits of prototypic Stx1 and Stx2 are 52% and 60% identical at the primary amino acid sequence level, respectively. With the exception of one of the Stx2 variants (Stx2e), the B pentamers of the Shiga toxins recognize the glycan sequence of globotriaosylceramide (Gb3) receptors found on many eukaryotic cell surfaces (22, 29, 42), including renal endothelial cells (28). Upon receptor ligation, the toxin is internalized by the host cell, and the A subunit's RNA N-glycosidase activity becomes activated, resulting in the catalytic removal of a specific adenine from the eukaryotic 28S rRNA component of the 60S ribosomal subunit (11, 40). This Stx A-subunit-mediated rRNA depurination activity causes eukaryotic cell death by, depending on the cell type, a number of possible mechanisms, including apoptosis.
The universal expression of the Shiga toxins by antigenically diverse EHEC strains and serotypes as well as their central role in severe pathogenesis makes these exotoxins compelling targets for vaccine development. Moreover, there is evidently a much greater correlation between EHEC isolates expressing prototypic Stx2 and a more severe course of illness (16, 24, 39, 41). Considering these epidemiological findings, we previously (32) proposed that an acellular vaccine consisting of the nontoxic B subunit from prototypic Stx2 might provide safe and effective protection against the most severe complications of EHEC infections in a majority of the at-risk population. In support of this conjecture, we previously reported (32) that rabbits immunized with a recombinant preparation of the prototypic Stx2 B subunit were protected from a subsequent challenge with a lethal dose (LD) of Stx2 holotoxin. However, effective vaccination in this study was found to be unpredictable unless lipopolysaccharide (LPS) was included with the antigen. Others have also found that inducing an effective immune response to the Stx2 B subunit is difficult to achieve (1, 7). Since the presence of a high concentration of LPS would be unacceptable in a human vaccine preparation, the object of the studies described herein was to investigate ways of effectively augmenting, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml.
MATERIALS AND METHODS
Toxin purification and cell lines. The recombinant Stx2 B subunit and Stx2 holotoxin were expressed and affinity purified as described previously (32, 34). Endotoxin was removed from all the preparations by using a Detoxi-gel (Pierce, Rockford, IL) LPS affinity column, as recommended by the manufacturer and by employing the modifications described in our previous article (32). The colorimetric Limulus amebocyte lysate assay (QCL-100; BioWhittaker, Walkersville, MD) indicated that the purified Stx2 B-subunit preparations contained <1 endotoxin unit/ml. The lack of holotoxin contamination in the recombinant Stx2 B-subunit preparation was confirmed by assaying it for cytotoxic activity in Vero and Ramos Burkitt's lymphoma B cells (30-32). Whereas Stx2 displays 50% cytolethal doses of 380 pg/105 Vero cells and 20 pg/105 Ramos B cells, the Stx2 B-subunit preparation, at a concentration of 1 mg/105 cells, was found to be completely nontoxic in these two cell lines.
Conjugation of Stx2 B subunit to KLH. Imject mariculture keyhole limpet hemocyanin (KLH), high purity research grade (Pierce Biotechnology, Rockford, IL), was conjugated to the Stx2 B subunit using 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Pierce Biotechnology) per the manufacturer's instructions. Briefly, 2 mg of Stx2 B subunit was admixed with 2 mg of KLH in conjugation buffer [0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), 0.1 M NaCl, pH 4.7] with 250 μg of EDC and incubated at room temperature for 2 h. To remove excess EDC and any unconjugated Stx2 B subunit, the conjugation reaction mixture was exhaustively dialyzed at 4°C over a period of 4 days against 0.05 M NaPO4 buffer (pH 7.2) containing 0.15 M NaCl using a 300,000-molecular-weight exclusion membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA). Conjugation efficiency was analyzed qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-PROTEAN II cell system (Bio-Rad, Mississauga, ON, Canada).
Mouse immunization and Stx2 holotoxin challenge protocols. The mouse immunization and Stx2 holotoxin challenge experiments were conducted in a randomized double-blind manner with adherence to the recommendations of the Canadian Council on Animal Care. The adjuvants tested in the study were Quil-A (saponin; Cedarlane Laboratories Ltd., Hornby, ON, Canada), Quil-A plus LPS from E. coli O111:B4 (Sigma-Aldrich, Oakville, ON, Canada), the Ribi adjuvant system containing synthetic trehalose dicorynomycolate (RAS-TDM; Cedarlane), RAS-TDM plus monophosphoryl lipid A from Salmonella enterica serovar Minnesota R595 (MPL; Corixa, Hamilton, MT), 2% Alhydrogel (Cedarlane), or 2% Alhydrogel plus MPL. LPS was included as one of the adjuvants in the pilot study described herein because it had to be included to induce rabbit immunity to the Stx2 B subunit, as reported in our previous article (32). It was therefore used in the present study to provide a point of reference against which we could relate the activity of the non-LPS-based adjuvants. Six-week-old, 20-g female BALB/c mice were used in all the experiments. The mice were ear notched for identification. Preimmunization blood samples were obtained from all the mice via the jugular vein. The mice subsequently received two 0.1-ml anterior dorsal subcutaneous injections containing a total of 30 μg of Stx2 B subunit administered with each of the adjuvant formulations. One group of mice was sham immunized with pyrogen-free 0.9% NaCl irrigation solution (USP; Baxter Corporation, Toronto, ON, Canada). Alternatively, the mice were immunized with 30 μg of Stx2 B-subunit-KLH conjugate or KLH alone administered with RAS-TDM or 2% Alhydrogel. The mice were immunized at 3-week intervals a maximum of three times. Seven days postimmunization, the mice were bled from the jugular vein to obtain test sera. Fourteen days after receiving their last injection, the mice received a single anterior dorsal subcutaneous LD (0.2 ng/g of body weight) injection of a cocktail consisting of Stx2 holotoxin plus 7.5 μg Quil-A in 100 μl phosphate-buffered saline (PBS). The lethal challenge was administered with Quil-A because this provides a depot from which the toxins are more slowly released into the circulation than if they were administered in PBS. The rationale was to create a situation that would more accurately mimic the release of the toxins from the intestines of an infected individual. Beginning on the third day after initiation of the Stx2 challenge, the mice were monitored every 2 to 4 h and immediately euthanized by CO2 asphyxia when signs (obvious lethargy or anterior paralysis) of Shigatoxemia became apparent (35).
Analysis of mouse sera in the Ramos B-cell cytotoxicity neutralization assay. Undiluted mouse serum (5 μl) was preincubated at room temperature with 0.05 ng of Stx2 holotoxin in 5 μl of saline for 20 min and then transferred to 0.5 ml of RPMI 1640 containing 2.5 x 105 cultured Ramos B cells. After 2 h of incubation at 37°C in an atmosphere of 5% CO2-95% air, the Ramos B cells were washed two times by low-speed centrifugation and finally resuspended in fresh RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. After an additional 16-h incubation period, the Ramos B cells were labeled with annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen, Mississauga, ON, Canada) and propidium iodide (Sigma-Aldrich), as described by the suppliers. The percentage of apoptotic cells was recorded by flow cytometry using a FACscan flow cytometer (Becton Dickinson, Mountain View, CA).
Determination of mouse IgG1 and IgG2a titers by the enzyme-linked immunosorbent assay (ELISA). One hundred microliters/well of a 2.5-μg/ml Stx2 PBS solution was incubated overnight at 4°C in 96-well enzyme immunoassay-radioimmunoassay plates. The plates were then washed five times with PBS Tween (PBST). The remaining protein binding sites were saturated using 2.5% skim milk in PBST for 2 h at 37°C. One hundred microliters of serial dilutions of sera in PBST was added to the wells, and the plates were incubated overnight at 4°C. Following this step, the plates were washed five times with PBST, and 100 μl/well of a 1/2,000 dilution of peroxidase-conjugated goat anti-mouse immunoglobulin G1 (IgG1)- or IgG2a-specific antibodies (Southern Biotechnology, Birmingham, AL) was added for 2 h at 37°C. The plates were again washed five times with PBST and subsequently developed for 20 min with 100 μl/well of 10 mM citrate buffer (pH 4.2), 0.06% H2O2, and 0.055% 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Boehringer-Mannheim, Indianapolis, IN). The resulting absorbance data were recorded using a Spectramax 340 microtiter plate reader set at a wavelength of 410 nm.
RESULTS
In a pilot study, groups of mice were immunized a total of three times over a 2-month period with the recombinant Stx2 B-subunit preparation (<1 endotoxin unit/ml) in admixture with one of the six adjuvants listed in the legend to Fig. 1. With the exception of mice immunized with the Stx2 B subunit mixed with RAS-TDM, none of the animals immunized with the Stx2 B subunit in admixture with any of the other adjuvants survived a subsequent challenge with an LD of Stx2 holotoxin. However, because of the small sample size, this result was not significant (P = 1.00, Fisher's exact test). Accordingly, to further investigate if RAS-TDM could enhance the immunogenicity of the Stx2 B subunit, the experiment was repeated with a larger group of animals. The results presented in Fig. 1B demonstrate that RAS-TDM did have a significant (P = 0.006, Fisher's exact test) promotional effect on the immunogenicity of the Stx2 B subunit but, once again, protection was less than optimal, with the survival rate being only 42%. As a result of these observations, we decided to investigate whether the murine immune response to the recombinant Stx2 B subunit could be further improved by chemically coupling the B subunit to a carrier protein such as KLH (15).
As is evident in the SDS-PAGE results presented in Fig. 2, the EDC conjugation process resulted in the production of very-high-molecular-weight complexes of KLH and Stx2 B subunit, which barely penetrated the separating gel. There is no evidence in Fig. 2, lane D, of the gel of unconjugated KLH or Stx2 B subunit, but they are readily detected in lanes B and C, respectively.
Groups of 10 mice were then immunized with unconjugated KLH or the Stx2 B-subunit-KLH conjugate preparation administered with either Alhydrogel or RAS-TDM. The ELISA data presented in Fig. 3 demonstrate that after only two injections, the postimmunization sera from individual mice receiving the Stx2 B-subunit-KLH conjugate preparation administered with RAS-TDM displayed the highest Stx2-specific IgG1 responses (Fig. 3B). In Fig. 3, the differences between the responses of the control mice immunized with unconjugated KLH and those immunized with the Stx2 B-subunit-KLH conjugate administered with RAS-TDM (Fig. 3B) were highly significant (P < 0.001, Student's t test). For the IgG2a titers (Fig.3D), these differences were also significant (P = 0.009, Student's t test). For the IgG1 titers, the differences between the responses of the control mice immunized with unconjugated KLH and those immunized with the Stx2 B-subunit-KLH conjugate administered with Alhydrogel (Fig. 3A) were also highly significant (P < 0.001, Student's t test). For the IgG2a titers (Fig. 3C), however, these differences did not achieve significance (P = 0.198, Student's t test). As well, the differences between the response of mice immunized with the Stx2 B-subunit-KLH conjugate vaccine administered with RAS-TDM (Fig. 3B) and that of mice immunized with the conjugate vaccine plus Alhydrogel (Fig. 3A) were also significant (P = 0.002, Student's t test). The differences, however, between the IgG2a titers in mice immunized with the Stx2 B-subunit-KLH conjugate vaccine administered with RAS-TDM (Fig. 3D) and those of mice immunized with the conjugate vaccine plus Alhydrogel (Fig. 3C) were not (P = 0.077, Student's t test). The ELISA data correlated with the Ramos B-cell apoptosis neutralization results in that the greatest neutralization was obtained using sera from mice immunized with the Stx2 B-subunit-KLH conjugate preparation administered with RAS-TDM (Fig. 4).
When the mice immunized with the Stx2 B-subunit-KLH conjugates were challenged with an LD of Stx2 holotoxin, all 10 of the mice vaccinated with the preparation administered with RAS-TDM survived whereas only 8 of the 10 mice immunized with the Stx2 B-subunit-KLH conjugate preparation mixed with Alhydrogel survived the challenge (Fig. 5). However, the difference between these two groups was not significant (P = 0.474, Fisher's exact test).
DISCUSSION
Over the preceding decades, large outbreaks and continuing sporadic cases of EHEC infections in North and South America, Japan, Europe, and Australia have prompted a global research effort directed at minimizing or eliminating the threat that these organisms pose to human health. The more recent inclusion of the diarrheagenic E. coli on the NIAID Biodefense Research Priority Pathogens Category B list has also spurred efforts in countermeasure research directed at these organisms. Essentially, three approaches have been advocated, including therapeutic interventions (4, 37), preventing the organisms from entering the food or water supply (17, 20, 33), and vaccination strategies aimed at protecting humans or eliminating the organisms from their major zoonotic reservoir, mainly cattle (18, 21). In the present study, we have continued our research into the feasibility of producing a safe, chemically defined, and effective vaccine for protecting humans from the serious complications of EHEC infections.
Although O polysaccharide-based EHEC vaccines have been described (9, 10, 25-27, 44), such vaccines suffer from the limitation that the resulting immunity is serogroup specific, and a large number of different EHEC serogroups have now been implicated in human cases of hemorrhagic colitis and HUS (20). Therefore, to provide broad-spectrum protection, we and others (2, 5-7, 14, 32, 50) have proposed vaccine strategies focused on the Shiga toxins, the only virulence factors common to all EHEC strains and serogroups.
Given that the amino acid sequences of the Shiga toxins are 60% identical, it is feasible that polyclonal antibodies to the Stx2 B subunit might confer cross-protection against Stx1. Similarly, Stx2 B-subunit-specific polyclonal antibodies might also confer protection against the more closely related variant forms of Stx2. However, prototypic Stx2 is the most prevalent of all the Shiga toxins expressed by EHEC isolates obtained from subjects who develop HUS (16, 24, 39). Consequently, a vaccine composed of the Stx2 B subunit should provide broad-spectrum protection against the extraintestinal complications of EHEC infections regardless of its ability to provide cross-protective immunity against Stx1 or any of the Stx2 variants.
Although RAS-TDM promoted a protective immune response to the hypoimmunogenic Stx2 B subunit, this required a primary injection followed by two boosters, and we found that the protection elicited was only partial. We therefore elected to investigate whether coupling the Stx2 B subunit to a carrier protein would improve the protection provided by a Stx2 B-subunit-based vaccine. We chose KLH for the study because it is approved for human use and has been used as a hapten carrier for stimulating an immune response to small immunogens such as drugs, hormones, peptides, polysaccharides, lipids, and oligonucleotides (15, 36, 48, 51). Due to its mitogenic activity, carbohydrate content, highly organized quaternary structure, high molecular mass, and propensity to aggregate, KLH elicits a vigorous immune response to itself (15). This immune response involves activated antigen-specific B and T lymphocytes and the expression of transactivating cytokines and lymphokines, which can also invigorate the response of B and T lymphocytes activated by poor immunogens which are coupled to KLH.
Although ELISA clearly revealed that a Stx2-specific immune response was induced in mice immunized with the Stx2 B-subunit-KLH preparation, the Ramos B-cell neutralization data suggested the possibility that the protective activity of the Stx2 B-subunit-KLH vaccine preparations may not have been related to a specific immune response to the Stx2 B subunit. However, it is conceivable that antibodies generated to the Stx2 B subunit in the immunized mice may not have been of sufficient affinity to neutralize the apoptogenic activity of Stx2 holotoxin in Ramos B-cells. In this regard, cytotoxicity assays represent a two-step process involving first the binding and then the subsequent internalization of the bound toxins into the cytoplasmic compartment of the cell. In these assays, toxin binding to specific receptors on the cell surface is reversible, as is antigen binding to specific antibodies. Once the toxin-receptor complexes have been internalized, however, the process is irreversible, and the cell is destined to die. Antibodies, which remain external to the cell, lose their neutralizing function once toxin internalization has occurred.
During the 2-h incubation with the antigen-antibody complexes, it is possible that an exchange reaction may have occurred in which loosely complexed Stx2 was able to access Gb3 receptors and become irreversibly internalized into the Ramos B-cells. Such an exchange reaction would be favored in a situation involving antibodies with a relatively low affinity for their antigen, antibodies which would have been produced if the immune response was still in the maturing phase in mice which had only received two doses of the vaccine. In the challenge phase of the experiment, however, any Stx2 holotoxin-antibody complexes that formed, regardless of their affinity, would have been exposed to a disposal mechanism involving cellular internalization which would avoid cytotoxicity. Such a disposal mechanism may have been sufficiently effective at eliminating the Stx2 challenge dose in the mice before the circulating holotoxin had time to exchange with Gb3 receptors on target cells. An alternative but more remote explanation is that the two injection immunization protocols may have simply primed the murine immune system for it to respond in a booster-like fashion to the challenge dose of Stx2 holotoxin and quickly enough to produce de novo antibodies with an affinity that was high enough to be protective. These de novo antibodies would not have been present in the postimmunization but prechallenge serum samples which were evaluated in the Ramos B-cell apoptosis neutralization assays.
Several reports have indicated that the Stx1 B subunit, which lacks the enzymatic activity of the A subunit, activates the apoptosis program in certain tissue culture cells. This B-subunit-specific apoptogenic activity was seen only at relatively high concentrations, much higher than that needed by the holotoxin to achieve the same end. Nonetheless, such activity would still raise safety concerns about any vaccine preparation containing the Stx2 B subunit. Although we previously reported (30) that the Stx2 B subunit caused apoptosis in Ramos B-cells, we subsequently discovered (31) that this activity was in fact due to minute amounts of Stx2 holotoxin in the B-subunit preparations used in those studies. As a consequence, the recombinant Stx2 B-subunit preparations used in the present immunization studies were found to be completely free of any cytotoxic activity at a concentration which was 2.6 x 106 to 5 x 107 times greater than a 50% cytolethal dose for Stx2 in Vero and Ramos B-cells, respectively.
In summary, by conjugating the Stx 2 B subunit to KLH, it appears that the hyporesponsiveness of the endotoxin-poor Stx2 B-subunit preparation can thereby be overcome following only a primary injection and a single booster injection, inducing a protective immune response to Stx 2 holotoxin in mice. Further, the Stx2 B-subunit preparation used in these studies was found to contain below-detectable levels of apoptogenic activity in Ramos B cells. These results support additional evaluation of an endotoxin-poor Stx2 B-subunit-based EHEC vaccine using adjuvants and carrier proteins which have been approved for clinical use in humans. The vaccine could be used alone or in combination with other virulence factors to provide protection from the devastating consequences of foodborne or waterborne EHEC infections in human subjects. The availability of such vaccine preparations would also serve as a deterrent to using EHEC to compromise the safety of food or water intended for human consumption.
ACKNOWLEDGMENTS
This work was supported by operating grants MWS 56081 from the Canadian Institutes for Health Research (CIHR) and VP 17 from the Canadian Bacterial Diseases Network to G.D.A. P.M. was supported by doctoral scholarships from the CIHR and Alberta Heritage Foundation for Medical Research.
We thank Stefanie Wee for her technical assistance.
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Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, AB T2N 4N1, Canada
ABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.
INTRODUCTION
The enterohemorrhagic group of Escherichia coli (EHEC) causes hemorrhagic colitis and, in anywhere from 5 to 15% of infected individuals, primarily very young and elderly subjects, a serious clinical complication called hemolytic-uremic syndrome (HUS) (8, 23, 45). HUS is characterized by a triad of clinical features, including hemolytic anemia, thrombocytopenia, and ultimately, acute renal failure. As well, in the most severe cases, various degrees of central nervous system involvement can become apparent. EHEC is also referred to as Shiga toxigenic E. coli because this organism expresses exotoxins that are biochemically related to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (43). Once EHEC has colonized the intestines, it is possible for Shiga toxins to be translocated into the submucosal compartment of the gut (3, 19). From there, the toxins can be transported, possibly on the surface of polymorphonuclear leukocytes (23, 46, 47), to extraintestinal organs and tissues, primarily the kidneys, where Shiga toxin-mediated damage to endothelial cells in the glomerular capillaries induces a cascade of microangiopathic events leading ultimately to HUS (45).
The Shiga toxins produced by EHEC are classified into two families, Stx1 and Stx2, also commonly referred to as verotoxin or verocytotoxin 1 and 2, according to their genetic and antigenic relatedness to the prototypic Stx produced by S. dysenteriae. In this classification scheme, Stx1 is virtually identical to the prototypic S. dysenteriae Stx (38). In contrast, Stx2 is more distantly related to Stx, and at least 10 variant species of Stx2 (reviewed in references 8, 45, and 49) have now been described in various EHEC strains and serotypes isolated from humans and animals.
Regardless of their relationship to one another, the Shiga toxins all display a classic AB5 structure in which one enzymatically active A subunit is combined with five identical B subunits which form a homopentamer displaying fivefold radial symmetry around a central pore (12, 13). In the Stx family, the A and B subunits of prototypic Stx1 and Stx2 are 52% and 60% identical at the primary amino acid sequence level, respectively. With the exception of one of the Stx2 variants (Stx2e), the B pentamers of the Shiga toxins recognize the glycan sequence of globotriaosylceramide (Gb3) receptors found on many eukaryotic cell surfaces (22, 29, 42), including renal endothelial cells (28). Upon receptor ligation, the toxin is internalized by the host cell, and the A subunit's RNA N-glycosidase activity becomes activated, resulting in the catalytic removal of a specific adenine from the eukaryotic 28S rRNA component of the 60S ribosomal subunit (11, 40). This Stx A-subunit-mediated rRNA depurination activity causes eukaryotic cell death by, depending on the cell type, a number of possible mechanisms, including apoptosis.
The universal expression of the Shiga toxins by antigenically diverse EHEC strains and serotypes as well as their central role in severe pathogenesis makes these exotoxins compelling targets for vaccine development. Moreover, there is evidently a much greater correlation between EHEC isolates expressing prototypic Stx2 and a more severe course of illness (16, 24, 39, 41). Considering these epidemiological findings, we previously (32) proposed that an acellular vaccine consisting of the nontoxic B subunit from prototypic Stx2 might provide safe and effective protection against the most severe complications of EHEC infections in a majority of the at-risk population. In support of this conjecture, we previously reported (32) that rabbits immunized with a recombinant preparation of the prototypic Stx2 B subunit were protected from a subsequent challenge with a lethal dose (LD) of Stx2 holotoxin. However, effective vaccination in this study was found to be unpredictable unless lipopolysaccharide (LPS) was included with the antigen. Others have also found that inducing an effective immune response to the Stx2 B subunit is difficult to achieve (1, 7). Since the presence of a high concentration of LPS would be unacceptable in a human vaccine preparation, the object of the studies described herein was to investigate ways of effectively augmenting, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml.
MATERIALS AND METHODS
Toxin purification and cell lines. The recombinant Stx2 B subunit and Stx2 holotoxin were expressed and affinity purified as described previously (32, 34). Endotoxin was removed from all the preparations by using a Detoxi-gel (Pierce, Rockford, IL) LPS affinity column, as recommended by the manufacturer and by employing the modifications described in our previous article (32). The colorimetric Limulus amebocyte lysate assay (QCL-100; BioWhittaker, Walkersville, MD) indicated that the purified Stx2 B-subunit preparations contained <1 endotoxin unit/ml. The lack of holotoxin contamination in the recombinant Stx2 B-subunit preparation was confirmed by assaying it for cytotoxic activity in Vero and Ramos Burkitt's lymphoma B cells (30-32). Whereas Stx2 displays 50% cytolethal doses of 380 pg/105 Vero cells and 20 pg/105 Ramos B cells, the Stx2 B-subunit preparation, at a concentration of 1 mg/105 cells, was found to be completely nontoxic in these two cell lines.
Conjugation of Stx2 B subunit to KLH. Imject mariculture keyhole limpet hemocyanin (KLH), high purity research grade (Pierce Biotechnology, Rockford, IL), was conjugated to the Stx2 B subunit using 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Pierce Biotechnology) per the manufacturer's instructions. Briefly, 2 mg of Stx2 B subunit was admixed with 2 mg of KLH in conjugation buffer [0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), 0.1 M NaCl, pH 4.7] with 250 μg of EDC and incubated at room temperature for 2 h. To remove excess EDC and any unconjugated Stx2 B subunit, the conjugation reaction mixture was exhaustively dialyzed at 4°C over a period of 4 days against 0.05 M NaPO4 buffer (pH 7.2) containing 0.15 M NaCl using a 300,000-molecular-weight exclusion membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA). Conjugation efficiency was analyzed qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-PROTEAN II cell system (Bio-Rad, Mississauga, ON, Canada).
Mouse immunization and Stx2 holotoxin challenge protocols. The mouse immunization and Stx2 holotoxin challenge experiments were conducted in a randomized double-blind manner with adherence to the recommendations of the Canadian Council on Animal Care. The adjuvants tested in the study were Quil-A (saponin; Cedarlane Laboratories Ltd., Hornby, ON, Canada), Quil-A plus LPS from E. coli O111:B4 (Sigma-Aldrich, Oakville, ON, Canada), the Ribi adjuvant system containing synthetic trehalose dicorynomycolate (RAS-TDM; Cedarlane), RAS-TDM plus monophosphoryl lipid A from Salmonella enterica serovar Minnesota R595 (MPL; Corixa, Hamilton, MT), 2% Alhydrogel (Cedarlane), or 2% Alhydrogel plus MPL. LPS was included as one of the adjuvants in the pilot study described herein because it had to be included to induce rabbit immunity to the Stx2 B subunit, as reported in our previous article (32). It was therefore used in the present study to provide a point of reference against which we could relate the activity of the non-LPS-based adjuvants. Six-week-old, 20-g female BALB/c mice were used in all the experiments. The mice were ear notched for identification. Preimmunization blood samples were obtained from all the mice via the jugular vein. The mice subsequently received two 0.1-ml anterior dorsal subcutaneous injections containing a total of 30 μg of Stx2 B subunit administered with each of the adjuvant formulations. One group of mice was sham immunized with pyrogen-free 0.9% NaCl irrigation solution (USP; Baxter Corporation, Toronto, ON, Canada). Alternatively, the mice were immunized with 30 μg of Stx2 B-subunit-KLH conjugate or KLH alone administered with RAS-TDM or 2% Alhydrogel. The mice were immunized at 3-week intervals a maximum of three times. Seven days postimmunization, the mice were bled from the jugular vein to obtain test sera. Fourteen days after receiving their last injection, the mice received a single anterior dorsal subcutaneous LD (0.2 ng/g of body weight) injection of a cocktail consisting of Stx2 holotoxin plus 7.5 μg Quil-A in 100 μl phosphate-buffered saline (PBS). The lethal challenge was administered with Quil-A because this provides a depot from which the toxins are more slowly released into the circulation than if they were administered in PBS. The rationale was to create a situation that would more accurately mimic the release of the toxins from the intestines of an infected individual. Beginning on the third day after initiation of the Stx2 challenge, the mice were monitored every 2 to 4 h and immediately euthanized by CO2 asphyxia when signs (obvious lethargy or anterior paralysis) of Shigatoxemia became apparent (35).
Analysis of mouse sera in the Ramos B-cell cytotoxicity neutralization assay. Undiluted mouse serum (5 μl) was preincubated at room temperature with 0.05 ng of Stx2 holotoxin in 5 μl of saline for 20 min and then transferred to 0.5 ml of RPMI 1640 containing 2.5 x 105 cultured Ramos B cells. After 2 h of incubation at 37°C in an atmosphere of 5% CO2-95% air, the Ramos B cells were washed two times by low-speed centrifugation and finally resuspended in fresh RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. After an additional 16-h incubation period, the Ramos B cells were labeled with annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen, Mississauga, ON, Canada) and propidium iodide (Sigma-Aldrich), as described by the suppliers. The percentage of apoptotic cells was recorded by flow cytometry using a FACscan flow cytometer (Becton Dickinson, Mountain View, CA).
Determination of mouse IgG1 and IgG2a titers by the enzyme-linked immunosorbent assay (ELISA). One hundred microliters/well of a 2.5-μg/ml Stx2 PBS solution was incubated overnight at 4°C in 96-well enzyme immunoassay-radioimmunoassay plates. The plates were then washed five times with PBS Tween (PBST). The remaining protein binding sites were saturated using 2.5% skim milk in PBST for 2 h at 37°C. One hundred microliters of serial dilutions of sera in PBST was added to the wells, and the plates were incubated overnight at 4°C. Following this step, the plates were washed five times with PBST, and 100 μl/well of a 1/2,000 dilution of peroxidase-conjugated goat anti-mouse immunoglobulin G1 (IgG1)- or IgG2a-specific antibodies (Southern Biotechnology, Birmingham, AL) was added for 2 h at 37°C. The plates were again washed five times with PBST and subsequently developed for 20 min with 100 μl/well of 10 mM citrate buffer (pH 4.2), 0.06% H2O2, and 0.055% 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Boehringer-Mannheim, Indianapolis, IN). The resulting absorbance data were recorded using a Spectramax 340 microtiter plate reader set at a wavelength of 410 nm.
RESULTS
In a pilot study, groups of mice were immunized a total of three times over a 2-month period with the recombinant Stx2 B-subunit preparation (<1 endotoxin unit/ml) in admixture with one of the six adjuvants listed in the legend to Fig. 1. With the exception of mice immunized with the Stx2 B subunit mixed with RAS-TDM, none of the animals immunized with the Stx2 B subunit in admixture with any of the other adjuvants survived a subsequent challenge with an LD of Stx2 holotoxin. However, because of the small sample size, this result was not significant (P = 1.00, Fisher's exact test). Accordingly, to further investigate if RAS-TDM could enhance the immunogenicity of the Stx2 B subunit, the experiment was repeated with a larger group of animals. The results presented in Fig. 1B demonstrate that RAS-TDM did have a significant (P = 0.006, Fisher's exact test) promotional effect on the immunogenicity of the Stx2 B subunit but, once again, protection was less than optimal, with the survival rate being only 42%. As a result of these observations, we decided to investigate whether the murine immune response to the recombinant Stx2 B subunit could be further improved by chemically coupling the B subunit to a carrier protein such as KLH (15).
As is evident in the SDS-PAGE results presented in Fig. 2, the EDC conjugation process resulted in the production of very-high-molecular-weight complexes of KLH and Stx2 B subunit, which barely penetrated the separating gel. There is no evidence in Fig. 2, lane D, of the gel of unconjugated KLH or Stx2 B subunit, but they are readily detected in lanes B and C, respectively.
Groups of 10 mice were then immunized with unconjugated KLH or the Stx2 B-subunit-KLH conjugate preparation administered with either Alhydrogel or RAS-TDM. The ELISA data presented in Fig. 3 demonstrate that after only two injections, the postimmunization sera from individual mice receiving the Stx2 B-subunit-KLH conjugate preparation administered with RAS-TDM displayed the highest Stx2-specific IgG1 responses (Fig. 3B). In Fig. 3, the differences between the responses of the control mice immunized with unconjugated KLH and those immunized with the Stx2 B-subunit-KLH conjugate administered with RAS-TDM (Fig. 3B) were highly significant (P < 0.001, Student's t test). For the IgG2a titers (Fig.3D), these differences were also significant (P = 0.009, Student's t test). For the IgG1 titers, the differences between the responses of the control mice immunized with unconjugated KLH and those immunized with the Stx2 B-subunit-KLH conjugate administered with Alhydrogel (Fig. 3A) were also highly significant (P < 0.001, Student's t test). For the IgG2a titers (Fig. 3C), however, these differences did not achieve significance (P = 0.198, Student's t test). As well, the differences between the response of mice immunized with the Stx2 B-subunit-KLH conjugate vaccine administered with RAS-TDM (Fig. 3B) and that of mice immunized with the conjugate vaccine plus Alhydrogel (Fig. 3A) were also significant (P = 0.002, Student's t test). The differences, however, between the IgG2a titers in mice immunized with the Stx2 B-subunit-KLH conjugate vaccine administered with RAS-TDM (Fig. 3D) and those of mice immunized with the conjugate vaccine plus Alhydrogel (Fig. 3C) were not (P = 0.077, Student's t test). The ELISA data correlated with the Ramos B-cell apoptosis neutralization results in that the greatest neutralization was obtained using sera from mice immunized with the Stx2 B-subunit-KLH conjugate preparation administered with RAS-TDM (Fig. 4).
When the mice immunized with the Stx2 B-subunit-KLH conjugates were challenged with an LD of Stx2 holotoxin, all 10 of the mice vaccinated with the preparation administered with RAS-TDM survived whereas only 8 of the 10 mice immunized with the Stx2 B-subunit-KLH conjugate preparation mixed with Alhydrogel survived the challenge (Fig. 5). However, the difference between these two groups was not significant (P = 0.474, Fisher's exact test).
DISCUSSION
Over the preceding decades, large outbreaks and continuing sporadic cases of EHEC infections in North and South America, Japan, Europe, and Australia have prompted a global research effort directed at minimizing or eliminating the threat that these organisms pose to human health. The more recent inclusion of the diarrheagenic E. coli on the NIAID Biodefense Research Priority Pathogens Category B list has also spurred efforts in countermeasure research directed at these organisms. Essentially, three approaches have been advocated, including therapeutic interventions (4, 37), preventing the organisms from entering the food or water supply (17, 20, 33), and vaccination strategies aimed at protecting humans or eliminating the organisms from their major zoonotic reservoir, mainly cattle (18, 21). In the present study, we have continued our research into the feasibility of producing a safe, chemically defined, and effective vaccine for protecting humans from the serious complications of EHEC infections.
Although O polysaccharide-based EHEC vaccines have been described (9, 10, 25-27, 44), such vaccines suffer from the limitation that the resulting immunity is serogroup specific, and a large number of different EHEC serogroups have now been implicated in human cases of hemorrhagic colitis and HUS (20). Therefore, to provide broad-spectrum protection, we and others (2, 5-7, 14, 32, 50) have proposed vaccine strategies focused on the Shiga toxins, the only virulence factors common to all EHEC strains and serogroups.
Given that the amino acid sequences of the Shiga toxins are 60% identical, it is feasible that polyclonal antibodies to the Stx2 B subunit might confer cross-protection against Stx1. Similarly, Stx2 B-subunit-specific polyclonal antibodies might also confer protection against the more closely related variant forms of Stx2. However, prototypic Stx2 is the most prevalent of all the Shiga toxins expressed by EHEC isolates obtained from subjects who develop HUS (16, 24, 39). Consequently, a vaccine composed of the Stx2 B subunit should provide broad-spectrum protection against the extraintestinal complications of EHEC infections regardless of its ability to provide cross-protective immunity against Stx1 or any of the Stx2 variants.
Although RAS-TDM promoted a protective immune response to the hypoimmunogenic Stx2 B subunit, this required a primary injection followed by two boosters, and we found that the protection elicited was only partial. We therefore elected to investigate whether coupling the Stx2 B subunit to a carrier protein would improve the protection provided by a Stx2 B-subunit-based vaccine. We chose KLH for the study because it is approved for human use and has been used as a hapten carrier for stimulating an immune response to small immunogens such as drugs, hormones, peptides, polysaccharides, lipids, and oligonucleotides (15, 36, 48, 51). Due to its mitogenic activity, carbohydrate content, highly organized quaternary structure, high molecular mass, and propensity to aggregate, KLH elicits a vigorous immune response to itself (15). This immune response involves activated antigen-specific B and T lymphocytes and the expression of transactivating cytokines and lymphokines, which can also invigorate the response of B and T lymphocytes activated by poor immunogens which are coupled to KLH.
Although ELISA clearly revealed that a Stx2-specific immune response was induced in mice immunized with the Stx2 B-subunit-KLH preparation, the Ramos B-cell neutralization data suggested the possibility that the protective activity of the Stx2 B-subunit-KLH vaccine preparations may not have been related to a specific immune response to the Stx2 B subunit. However, it is conceivable that antibodies generated to the Stx2 B subunit in the immunized mice may not have been of sufficient affinity to neutralize the apoptogenic activity of Stx2 holotoxin in Ramos B-cells. In this regard, cytotoxicity assays represent a two-step process involving first the binding and then the subsequent internalization of the bound toxins into the cytoplasmic compartment of the cell. In these assays, toxin binding to specific receptors on the cell surface is reversible, as is antigen binding to specific antibodies. Once the toxin-receptor complexes have been internalized, however, the process is irreversible, and the cell is destined to die. Antibodies, which remain external to the cell, lose their neutralizing function once toxin internalization has occurred.
During the 2-h incubation with the antigen-antibody complexes, it is possible that an exchange reaction may have occurred in which loosely complexed Stx2 was able to access Gb3 receptors and become irreversibly internalized into the Ramos B-cells. Such an exchange reaction would be favored in a situation involving antibodies with a relatively low affinity for their antigen, antibodies which would have been produced if the immune response was still in the maturing phase in mice which had only received two doses of the vaccine. In the challenge phase of the experiment, however, any Stx2 holotoxin-antibody complexes that formed, regardless of their affinity, would have been exposed to a disposal mechanism involving cellular internalization which would avoid cytotoxicity. Such a disposal mechanism may have been sufficiently effective at eliminating the Stx2 challenge dose in the mice before the circulating holotoxin had time to exchange with Gb3 receptors on target cells. An alternative but more remote explanation is that the two injection immunization protocols may have simply primed the murine immune system for it to respond in a booster-like fashion to the challenge dose of Stx2 holotoxin and quickly enough to produce de novo antibodies with an affinity that was high enough to be protective. These de novo antibodies would not have been present in the postimmunization but prechallenge serum samples which were evaluated in the Ramos B-cell apoptosis neutralization assays.
Several reports have indicated that the Stx1 B subunit, which lacks the enzymatic activity of the A subunit, activates the apoptosis program in certain tissue culture cells. This B-subunit-specific apoptogenic activity was seen only at relatively high concentrations, much higher than that needed by the holotoxin to achieve the same end. Nonetheless, such activity would still raise safety concerns about any vaccine preparation containing the Stx2 B subunit. Although we previously reported (30) that the Stx2 B subunit caused apoptosis in Ramos B-cells, we subsequently discovered (31) that this activity was in fact due to minute amounts of Stx2 holotoxin in the B-subunit preparations used in those studies. As a consequence, the recombinant Stx2 B-subunit preparations used in the present immunization studies were found to be completely free of any cytotoxic activity at a concentration which was 2.6 x 106 to 5 x 107 times greater than a 50% cytolethal dose for Stx2 in Vero and Ramos B-cells, respectively.
In summary, by conjugating the Stx 2 B subunit to KLH, it appears that the hyporesponsiveness of the endotoxin-poor Stx2 B-subunit preparation can thereby be overcome following only a primary injection and a single booster injection, inducing a protective immune response to Stx 2 holotoxin in mice. Further, the Stx2 B-subunit preparation used in these studies was found to contain below-detectable levels of apoptogenic activity in Ramos B cells. These results support additional evaluation of an endotoxin-poor Stx2 B-subunit-based EHEC vaccine using adjuvants and carrier proteins which have been approved for clinical use in humans. The vaccine could be used alone or in combination with other virulence factors to provide protection from the devastating consequences of foodborne or waterborne EHEC infections in human subjects. The availability of such vaccine preparations would also serve as a deterrent to using EHEC to compromise the safety of food or water intended for human consumption.
ACKNOWLEDGMENTS
This work was supported by operating grants MWS 56081 from the Canadian Institutes for Health Research (CIHR) and VP 17 from the Canadian Bacterial Diseases Network to G.D.A. P.M. was supported by doctoral scholarships from the CIHR and Alberta Heritage Foundation for Medical Research.
We thank Stefanie Wee for her technical assistance.
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