Antibodies against a truncated Staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in the rat

Vaccine 19 (2001) 3376– 3383 www.elsevier.com/locate/vaccine

Antibodies against a truncated Staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in the rat Anna Rennermalm a, Ying-Hua Li b, Linda Bohaufs a, Connie Jarstrand a, Annelie Brauner b, Frank R. Brennan c,1, Jan-Ingmar Flock a,* a

Department of Microbiology, Pathology and Immunology, Karolinska Institute, Huddinge, Sweden b Department of Clinical Microbiology, Karolinska Hospital, Sweden c Axis Genetics Ltd, Babraham, Cambridge, UK Received 6 September 2000; accepted 27 February 2001

Abstract Staphylococcus aureus bacteraemia (SAB) originating from local infections can lead to severe secondary infections such as endocarditis. The protective effect of antibodies against secondary infections was studied in a rat model, where a local joint infection leads to bacteraemia and endocarditis on damaged aortic valves. In this study, immunizations with a truncated D2-domain of the S. aureus fibronectin-binding protein displayed on a cow-pea mosaic virus (CPMV-D) carrier induced protection against endocarditis (PB0.05). Opsonization of S. aureus with antibodies raised against CPMV-D stimulated both neutrophil activity and macrophage phagocytosis in vitro. Furthermore, intravenous administration of these antibodies protected mice from weight loss due to SAB. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Arthritis/endocarditis model; Chimeric plant viruses; Fibronectin-binding protein; S. aureus

1. Introduction Staphylococcus aureus is a natural inhabitant of human skin and nasal mucosa. Also, it is an opportunistic pathogen, and a common cause of noscomial infections and bacteraemia. S. aureus bacteraemia causes a high rate of complications including osteomyelitis [1] and endocarditis [2–4]. The increasing resistance to antibiotics found in clinical isolates [5] calls for alternative therapeutic and prophylactic treatment, such as passive immunization or vaccination. A rat model to study the haematogenous spread of S. aureus to secondary infection sites is available. Briefly, a local infection is established in a temporomandibular joint (TMJ), resulting in bacteraemia. Subsequent dam* Corresponding author. Present address: Clinical Bacteriology, F82 Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel.: +46-8-58581169; fax: +46-8-7113918. E-mail address: [email protected] (J.-I. Flock). 1 Present address Microscience Ltd, 545 Eskdale Road, Winners Triangle, Wokingham, Berkshire RG41 5TU, UK.

age of aortic valves leads to the formation of vegetations composed of fibrinogen, other plasma proteins and platelets to which S. aureus adhere, causing infective endocarditis [6]. This model differs significantly from conventional endocarditis models previously described [7–9], where no primary focus of infection is first established. In the arthritis/endocarditis model used in this study, the bacteria propagate in vivo, and are continuously spread into the blood stream, thus mimicking a bacteraemia originating from a primary infection causing secondary infections. The bacteria will express a different set of potential virulence factors, as compared with a broth culture, when disseminating from the primary site to the secondary site of infection. The aim of this study was to decrease the number of bacteria spreading from the joint infection via the blood stream to the damaged aortic valves. S. aureus produces a number of secreted and cellbound proteins that specifically bind to host extracellular matrix (ECM). Most S. aureus strains encode two fibronectin binding proteins in tandem, FnBP-A and

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FnBP-B [10,11]. Immunization with a recombinant fibronectin binding protein resulted in partial protection in a conventional rat endocarditis model [7] and also protection in a mouse mastitis model [12]. However, the ability of S. aureus to bind to fibronectin does not seem to be crucial for the development of endocarditis, as demonstrated with isogenic strains, of which one lacked both fibronectin binding proteins [13]. Antibodies against FnBP have been shown to have opsonic activity [14,15], which might explain the protective effect by the immunization in the endocarditis model. The primary fibronectin binding sequences consist of three 37- or 38-amino acid repeats, designated D1– 3. The D-repeats are highly homologous between FnBP-A and FnBP-B, with binding amino acids located within the 20 C-terminal amino acids of each D-domain. An isolated D-domain has the capacity to bind to fibronectin, even though the binding is enhanced when placed in tandem [16,17]. The domains D1– D2 are identical in 33 of 37 amino acids, while the D3 domain is more divergent. In an effort to generate high titers of protective antibodies in the rat, we have examined the vaccine potential of a plant virus, cowpea mosaic virus (CPMV), genetically engineered to express and display foreign peptides on its surface. In this presentation system, termed EPICOAT®, peptides of up to  40 amino acids are incorporated into specific locations in either of the two virus coat proteins. This results in the presentation of 60 copies of the foreign peptide on the surface of each virus particle, circumventing the need to couple the peptides to carrier molecules. The resultant chimaeric virus particles (CVPs) are easily propagated in plants and large amounts of the CVPs (up to  1g/ kg fresh weight of leaves) can be readily extracted. They have the potential for cost– effective manufacture and are not known to infect mammalian cells, thereby circumventing the safety concerns associated with live attenuated bacteria or viruses. When expressed on CPMV, peptides derived from both viruses and bacteria can elicit high levels of antibody, which confers protection from challenge infections [18,19]. We have shown previously that CPMV expressing amino acids 1–30 of the D2 domain of FnBP-B, which does not bind Fn, elicited high titers of peptide-specific antibody that inhibited fibronectin binding [20,21]. However, it was not yet established whether antibodies against the recombinant CPMV-D particles could provide protection from S. aureus in a clinically relevant model of infection. In this study, we demonstrate that immunization of rats with CPMV-D conferred protection from endocarditis secondary to arthritis in the coupled arthritis/endocarditis model. The rate of infection and the amount of S. aureus recovered from the aortic valve vegetations were significantly lowered. S. aureus pre-opsonized with antibodies from the

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CPMV-D immunized rats were more readily ingested in vitro by rat macrophages and also stimulated neutrophil activity to a higher extent than S. aureus pre-opsonized with control antibodies from the wt-CPMV immunized rats. 2. Materials and methods

2.1. Bacterial strains and propagation The animals were infected with S. aureus strain Phillips [22]. The neutrophil stimulation and macrophage ingestion assays were performed with a protein A and clumping factor deficient strain Newman (Spa::Tcr, ClfA1::Tn9A) (Dr Tim Foster, Dublin). The bacteria were cultured overnight, re-inoculated with a 20-fold dilution and then grown for 2h at 37°C in LB-broth to achieve optimal expression of FnBPs. The bacteria were centrifuged and resuspended in phosphate buffered saline (PBS), frozen and stored at −70°C until use. Before use, aliquots were resuspended in PBS to appropriate concentrations.

2.2. Construction, propagation and purification of CPMV 6irions The construction, propagation and purification of CPMV-D have been described previously [20,21]. Briefly, the first 30 amino acids of D2 from FnBP (1GQNNGNQSFEEDTEKDKPKYEQGGNIIDID30) were inserted between amino acids 22 and 23 of the small coat protein (S-protein) of CPMV. The construct was inoculated onto young cowpea plants and propagated to generate CPMV-D. Each virion expresses 60 copies of the D2 peptide, and 1mg CPMV-D contains approximately 40 ng of the D2 peptide [20,21].

2.3. Immunization and challenge Female 5–6-week-old Wistar rats, weighing approximately 180 g were used. The animals were kept under standard laboratory conditions. Rats were immunized subcutaneously (s.c.) with either 250–500 mg CPMV-D or 150 –500 mg of the control virion, wt-CPMV, on days 0, 14 and 28. For all immunizations, the antigens were suspended to a total of 0.5 ml PBS together with 25 mg QS-21, a saponin purified from the bark of Quillaja saponaria [23]. Seven days after the last immunization (day 35), rats were sedated with chloralhydrate (Merck), 360 mg/kg bodyweight, intraperitionally (i.p). Following sedation, 0.1 ml of fentanyl citrate (0.315 mg/ml) and fluanisone (10 mg/ml) (Hypnorm®, Janssen) were injected intramuscularly (i.m.) for complete anesthesia. Blood samples were collected from the tail vein. Sera were kept at − 20°C.

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The coupled arthritis /endocarditis model in the rat has been described previously [6]. Briefly, under aseptic conditions, 0.1 ml triamcinolon acetonid 10 mg/ml (Kenakort®, Bristol-Myers Squibb) was injected in one of the (TMJ. As control, 0.1 ml PBS was injected in the contralateral joint. S. aureus (1 ml) strain Phillips 107 CFU/ml was administered intravenously. Seven days after injection of steroid and bacteria, animals were anaesthetized and vegetations induced on the aortic valve by introduction of a polyethylene catheter (0.7 mm outer diameter, Guerbet Biome´ dical, France) through the right carotid artery. The catheter was left in place until the animals were sacrificed 24 h later. Samples of sera were collected as well as both TMJs and aortic valve with vegetation. Kidneys from ten CPMV-D-immunized rats and from 10 wt-CPMVimmunized rats were collected. If a catheter was missing or out of place, the results from the animal were excluded. Rats rapidly loosing weight were killed and taken off the study. The number of excluded animals was the same in both CPMV-D and wt-CPMV immunized groups. Twenty one rats immunized with CPMV-D and 19 rats immunized with wt-CPMV were included in the study.

2.4. Sample analysis The blood samples were gently centrifuged to collect the sera, which were kept at − 20°C. Kidneys and aortic valve vegetations were placed in Brain– Heart infusion broth and homogenized with a Teflon homogenizer. TMJ tissue was homogenized by vortexing vigorously with glass beads. The homogenized samples were serially diluted in PBS and plated onto blood agar plates. Total CFU/tissue were determined with a detection level of 4 CFU/piece of tissue.

2.5. ELISA for detection of serum antibodies All ELISAs were performed in 96-well, flat-bottomed polystyrene plates (Costar).To determine the total IgG content in sera, plates were coated overnight at room temperature with serial dilutions of serum samples, blocked for 1 h at 37°C with 2% (w/v) bovine serum albumin (BSA, Sigma) in PBS and washed with 0.05% (v/v) Tween 20 (Merck) in PBS (PBST). Horseradish peroxidase (HRP) conjugated rabbit anti-rat IgG (Dako) diluted 1:1000 in PBST was added and incubated for 1 h at 37°C. Plates were washed and developed with OPD tablets according to the manufacturer’s instruction (Dako). The titers of antibodies directed against the D-domains of the fibronectin-binding protein were determined by coating plates with a fusion protein; glutathione-S-transferase fused with the D1, D2 and the major part of D3 of the FnBP A (GST-D). The

construction of the GST-D fusion protein has been described previously [20]. Wells were coated overnight at room temperature with 0.5 mg of GST-D. Wells were blocked for 1 h at 37°C with 2% (w/v) BSA in PBS, washed with PBST, and serial dilutions of sera in PBST were added. The plates were incubated for 1 h at 37°C, and were washed again with PBST. HRP conjugated rabbit anti-rat IgG (Dako) diluted 1:1000 in PBST was added and incubated for 1 h at 37°C. Plates were washed and developed with OPD tablets. Titres were defined as the dilution required to obtain an absorbance lower than the average absorbance obtained from a pool of wt-CPMV sera at a 1:100 dilution.

2.6. Separation of neutrophils Blood from six healthy volunteers was collected in vacutainer tubes containing heparin. The erythrocytes were sedimented for 30 min at 37°C with a 3% solution of dextrane in saline. The leukocyte-rich supernatant was removed to tubes containing Ficoll-paque (Pharmacia) and was centrifuged for 30 min at 110 g, washed twice in PBS and concentrated by centrifugation for 30 min at 110× g. The cells were counted and resuspended in Eagle’s medium to 5× 106 cells/ml.

2.7. Nitro-blue tetrazolium (NBT) test Neutrophil stimulation by, for example opsonized bacteria, results in the production of superoxide that can reduce colorless NBT to dark blue formazan [24,25]. In microtiter plastic plates (Costar), 20 ml of S. aureus, 109 CFU/ml, was mixed with 30 ml of pools of either CPMV-D-, wt-CPMV- or pre-immune sera diluted from 1:2 to 1: 32. The plates were incubated for 60 min at 37°C. One hundred microlitres of freshly prepared neutrophils, 5×106 cells/ml, and 50 ml 0.1% sterile-filtrated NBT solution were added to each well and mixed. The plates were incubated at 37°C for 1 h. The reaction was stopped by adding 50 ml of 0.5 M HCl. The formazan product was centrifuged at 1200 rpm for 10 min in a microplate centrifuge and extracted in 200 ml dimethyl-sulphoxide (DMSO). The optical density was determined at 540 nm.

2.8. Purification of antibodies Pooled sera from wt-CPMV- and CPMV-D- immunized rats were purified on a protein G sepharose column according to manufacturers instructions (Pharmacia Biotech, Uppsala, Sweden). The preparations were concentrated on Amicon filters (Beverly, MA) to 4.5 mg/ml. The protein levels were determined with a protein concentration determination kit according to manufacturer’s instructions (BioRad).

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2.9. Preparation of macrophages A cell line of rat alveolar macrophages from a normal male Sprague/Dawley rat was obtained from ATCC (Cat No. CRL-2192 or NR8383). The cells were maintained in a 75cm2 flask culture with 20 ml culture medium consisting of 85% Ham’s F12 nutrient media (Gibco) and 15% fetal bovine serum (Gibco). Fresh culture medium was supplied twice a week. Cells were harvested by scraping the bottom of the flask.

2.10. Macrophage ingestion test Cells were harvested, centrifuged at 125 × g for 10 min and resuspended in Ham’s F12 nutrient media to a concentration of 105 cells/ml. A 24-well cell culture plate (Costar) was filled with 1 ml of the cell suspension/well. While preparing the macrophages, S. aureus strain Newman (Spa::Tcr, ClfA1::Tn9A), 5× 108 CFU/ ml, was mixed with 10% (v/v) heat-inactivated (56°C 30 min), pooled sera from either CPMV-D immunized rats, wt-CPMV immunized rats or with PBS. After 45 min of incubating sera/bacteria, 25 ml of the suspension was added to the wells with macrophage suspension, giving 1.2×107 CFU opsonized bacteria/well. After incubating the plates for 20 min at 37°C, 55 ml of gentamicin, 2mg/ml (final concentration: 100 mg/ml/ well), was added to each well to kill extracellular bacteria. The plates were incubated for 2 h at 37°C, and centrifuged at 300× g in a plate centrifuge for 10 min. The supernatants were removed and replaced with 1 ml sterile water to lyse the macrophages. The wells were scraped and the contents were plated onto blood agar plates in serial dilutions. Plates were incubated at 37°C overnight and colony forming units were counted.

2.11. Infection with pre-opsonized S. aureus in mice Female NMRI mice (Mo¨ llega˚ rd), weighing approximately 30 g each, were used. The animals were kept under standard laboratory conditions. Sera from rats immunized with CPMV-D or wtCPMV were pooled. S. aureus strain Phillips was mixed with sera from the CPMV-D serum pool, the wt-CPMV serum pool, or PBS, to a final concentration of 5×107 CFU/ml in 10% (v/v) sera or PBS and was incubated for 1 h at 37°C with mild shaking. Two hundred microlitres of the samples were administered intravenously in the tails of 15 mice. The mice were weighed daily for a 2-week period.

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analyzed using the Mann–Whitney U-test. Neutrophil stimulation was compared using the Bonferoni multiple comparison test, and macrophage ingestion was analyzed with the Mann–Whitney U-test. Differences in mouse weight loss were analyzed with the Student t-test. 3. Results

3.1. CPMV-D elicits high titers of FnBP-D2 -specific antibody Rats were immunized with recombinant CPMV virions displaying a truncated fibronectin binding D-domain, containing only the first 30 amino acids. This truncated form is unable to bind to fibronectin (data not shown). A control group of animals was immunized with wt-CPMV virions. The serum antibody response against the D-domain was measured by using a GST-D fusion protein in an ELISA. The results in Fig. 1 show D2-specific antibodies in sera sampled at the time of challenge. Subcutaneous immunization of rats with CPMV-D elicits high titers of D2-specific antibody compared with the wt-CPMV-immunized rats. Specific antibody titers 1 week later, at the time of endocarditis, were generally two-fold lower (not shown).

3.2. Immunization with CPMV-D pro6ides protection from experimental endocarditis Animals immunized with either CPMV-D or wtCPMV were challenged with bacteria intravenously and a corticosteroid was injected into the right sided mandibular joint resulting in septic arthritis in the joint. One week later, the animals were catheterized to induce vegetations on the aortic valves. At this time, all animals had clinical signs of infection, such as weight loss. Samples were taken another 24 h later. The results in Fig. 2 show the number of bacteria recovered from various tissues.

2.12. Statistical analysis The difference between the number of colony forming units recovered on aortic valves, TMJs and kidneys from CPMV-D or CPMV-wt immunized animals was

Fig. 1. Elisa titration of antibodies directed against the D1-3 domain of FnBP A in sera from rats immunised with CPMV-D (circles; n =21) or wt-CPMV (triangles; n = 19). Standard deviations are indicated by bars.

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Fig. 2. Log10 number of viable S. aureus recovered from tissues. Symbols represent individual CFU values recovered from (A) aortic valves, (B) right sided temporomandibular joints (rTMJ) or (C) kidneys. Circles: rats immunized with CPMV-D (n =21 in A and B; n =10 in C). Triangles: rats immunized with wt-CPMV (n= 19 in A and B; n= 10 in C). Horizontal bars indicates median values in each group. The difference in CFU between the wt-CPMV and CPMV-D immunized groups are significant (PB 0.05) on aortic valves (A) and kidneys (C) but not rTMJ (P\ 0.05; Mann– Whitney U-test)

Left-sided mandibular joints, into which PBS alone had been injected as control, had significantly fewer bacteria than right-sided joints (PB 0.001, data not shown). This shows that injection of the steroid is required for arthritis to develop, as noted before [6]. Macroscopic examination clearly showed a different appearance of mandibular joints on the left and right sides; right sided joints generally showed tissue destruction and massive invasion of bacteria into bone tissue. The number of bacteria recovered from infected joints did not differ between the groups immunized with CPMV-D or wt-CPMV (median values 106.3 and106.8 respectively), showing that antibodies were unable to protect against the challenge dose of 107 CFU. On the other hand, analysis of CFU values obtained from aortic valves with vegetations, revealed that the animals immunized with CPMV-D had roughly 1000fold less median value of bacteria compared with animals immunized with CPMV-wt, 103.2 and 106.5, respectively (PB 0.05). Thus, immunization with CPMV-D can confer protection against dissemination of bacteria from the primary site of infection to the secondary site. Massive colonization of bacteria was found in all kidneys. Despite the high numbers, a difference in the amount of bacteria in kidneys were found between the groups; the median value was 107.2 for CPMV-D immunized animals and 108.2 for wtCPMV immunized animals (P B 0.05).

3.3. Opsonic acti6ity Bacteria were opsonized with pooled sera from animals immunized with either CPMV-D or wt-CPMV

and added to six independent samples of freshly prepared human neutrophils. The NBT reduction by the neutrophils was significantly different (mean absorbancy 0.99 and 0.64 at OD 492, respectively, PB 0.01) between the groups at a serum dilution of 1:2, as shown in Fig. 3A, but was true also at higher dilutions (data not shown). Opsonized bacteria were also used in a macrophage internalization test. The amount of bacteria ingested per macrophage was 3.2 times higher when opsonized with CPMV-D IgG than with wt-CPMV IgG (mean no. of ingested bacteria was 3.2 and 1.0 CFU/macrophage, respectively, PB0.05) (Fig. 3B), indicating a better recognition of S. aureus preopsonized with the specific antibodies against the D2-domain. In both opsonisation assays, a S. aureus strain Newman lacking both protein A and clumping factor (Clf A) was used since this strain is less resistant to opsonophagocytosis (Dr T. Foster, personal communication).

3.4. Infection with pre-opsonized S. aureus In order to examine the protective effect of passively administered antibodies on general systemic S. aureus infection, mice were challenged with S. aureus pre-opsonized with sera from rats immunized with CPMV-D or wt-CPMV. Mice were also challenged with non-opsonized bacteria. Weight loss was used to monitor the overall state of health, which is sensitive although not specific for any particular type of infection. In all the animal groups, a rapid decline of weight was observed. Weight loss was reduced in mice injected with S. aureus

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4. Discussion

Fig. 3. Opsonic activity of sera in (A) a neutrophil superoxide stimulation test or (B) a macrophage internalisation test. Bars indicate mean colorimetric reaction of metronidazol to superoxide release from neutrophils (A; n =12), or mean number of viable internalised bacteria per macrophage (B; n= 8), respectively. Maximum and minimum values are shown.

pre-opsonized with CPMV-D compared with those receiving bacteria pre-opsonized with wt-CPMV sera or PBS (PB 0.05). Regain of weight was also faster than in the other groups (Fig. 4).

Fig. 4. The graph illustrates the mean weight change in groups of 15 mice injected with S. aureus pre-opsonized with either a pool of sera from CPMV-D immunsed rats (circles) a pool of sera from wt-CPMV immunised rats (triangles) or PBS (crosses). The weight loss was significantly higher in the control groups (wt-CPMV and PBS) than in the CPMV-D group. For instance, at day 3 PB 0.001 and at day 11 PB 0.05 (Students t-test); SD 1.4 and 6.5 respectively.

S. aureus infections are often characterized by their propensity to spread from one site to another via the blood stream. This situation is mimicked in the animal model used here. In this animal model, not previously used in a vaccination study, bacteria are cultured in vivo in the rat temporomandibular joint (TMJ) prior to catheterization. This results in a continuous haematogenous spread of low doses of bacteria, thus mimicking a natural infection more than the conventional endocarditis model. Additionally, in vivo grown bacteria would be expected to have the advantage of being adapted to the host environment and express appropriate virulence functions, as is often the case in natural infections in humans. In previously published data it was demonstrated that the bacterial count in the blood during this coupled arthritis/endocarditis ranged from only 100.5 –103.5 CFU/ml, to be compared to an ID50 of 104 –105 in the conventional endocarditis model using broth–cultivated bacteria [6]. Prior to infection, rats were immunized with a recombinant cowpea mosaic virion (CPMV), expressing a fibronectin binding domain of the fibronectin binding protein (FnBP) from S. aureus, truncated in the C-terminal to lose its binding properties (CPMV-D). A significant decrease (PB 0.05) of bacteria attached to aortic valves was found in animals immunized with CPMV-D compared to wt-CPMV immunized animals. Median CFU values recovered from the aortic valves were 103.2 and 106.5, respectively. A significant difference in bacterial recovery was also found in the kidneys, but not in the TMJ in which the bacterial infection was established prior to catheterization. This strongly implies that the protective activity of the antibodies is at the bacteraemic stage, reducing dissemination of bacteria from the primary infection. The protective effects of immunizations with CPMV-D are significant in aortic valve vegetations and in kidneys, but do not completely protect the rats from endocarditis (Fig. 2). However, the overall reduction of bacterial load in secondary infection sites clearly demonstrates the possibility of reducing the dissemination of bacteria from the primary infection site. Protection against arthritis in the TMJ would be unexpected considering the high challenge dose required. We have previously shown that CPMV-D elicits high titers of FnBP-specific serum IgG in rats when Freund’s adjuvants are used [20]. Since Freund’s adjuvant has never been registered for use in humans, less toxic adjuvants were sought, that could also elicit high titers of FnBP-specific antibody. The saponin-based adjuvant QS-21, believed to be a less toxic but adjuvant-active fraction of QuilA, has been used in human clinical trials [23]. Thus QS-21 was used in this study. Sera from rats

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immunized subcutaneously with CPMV-D in QS-21 adjuvant contained high titers of FnBP D2-specific serum IgG, while wt-CPMV sera did not, showing that CPMV-D is immunogenic even when administered in a more clinically-relevant adjuvant. We have also studied the long-term immunity by boosting rats immunized with CPMV-D in Freund’s complete adjuvant more than 1 year earlier. In these rats, the D-specific titers had dropped from high levels to undetectable levels. High titers were obtained within 1 week of the booster with GST-D in Freund’s incomplete adjuvant (data not shown). The short response time and the high levels of specific IgG indicates a long-term memory induced by immunization with CPMV-D. Immunizations with FnBP have previously been demonstrated to protect animals against disease [7,12,14]. In this study it is implicated that immunization with only the 30 first amino acids of the D2-peptide is enough to protect to the same degree as in previous studies with all three D-domains. The truncated peptide does not bind to fibronectin in vitro, in contrast to peptides used in previous infection models. In theory, lack of binding to Fn is an advantage for a vaccine since it would decrease the risk for the host production of antibodies directed against ligand-induced binding sites (LIBS) that might increase S. aureus binding to fibronectin [26,27]. Furthermore, the binding of an immunogen to serum Fn may be expected to reduce its immunogenicity. We have previously found that antibodies directed to FnBP can block the interaction in vitro between Fn and FnBP [20,21,28,29]. In vivo, however, adherence of S. aureus to fibronectin is not essential for bacterial establishment on damaged heart valves [13]. The protective effect seen in this study is presumably due to factors other than blocking of the bacterial adhesion to host tissue. Instead, ingestion of opsonized bacteria by phagocytic cells is presumably more important here. However, for bacterial attachment to tissues other than aortic valvular vegetations, adherence blocking by specific antibodies might be of importance. Therefore, we looked at the stimulation in vitro of neutrophils incubated with opsonized S. aureus. We found that S. aureus incubated with sera from CPMVD immunized animals increased the release of superoxide anions from neutrophils to a level significantly higher (PB 0.01) than S. aureus incubated with pre-immune sera. The S. aureus incubated with serum from wt-CPMV immunized rats did not increase this stimulation. Additionally, in a macrophage internalization test, S. aureus opsonized with pooled IgG from CPMV-D immunized rats, was internalized to a three– four fold higher extent than when opsonized with wt-CPMV IgG from the control animals (P B0.05). In another study, antibodies raised against GST-D in rats were also

found to have opsonizing capacity (data not shown), indicating that this might be true for several types of anti-FnBP antibodies. Furthermore, weight loss due to S. aureus infection in non-immune mice was decreased when the bacteria were preopsonized with sera from CPMV-D immunized rats prior administration (PB 0.05). Opsonization with sera from wt-CPMV immunized rats did not protect from weight loss compared to non-opsonized bacteria. These results implicate that neutrophils and macrophages could be important for the host defense against S. aureus, and that the antibodies resulting from the immunization in this study enhance the efficiency of such a defense. In conclusion, we have shown that the FnBP D2 peptide is highly immunogenic when expressed on CPMV, eliciting high titers of specific antibody which induce a significant amelioration of S. aureus-mediated endocarditis resulting from primary infection in the TMJ. The antibodies raised against the D2 domain have an opsonizing ability that can stimulate neutrophil and macrophage activity in vitro, and decrease weight loss due to general infection in an in vivo model in the mouse. Since these plant virus-derived vaccines have the potential for cost–effective manufacture and are not known to infect mammalian cells, they have the potential for future development as a protective vaccine against human S. aureus infections.

Acknowledgements We thank Aquila Biopharmaceuticals, Inc., for supplying QS-21. This work was performed under MAFF licence No. PHL 91/2275 (08/1997). Financial support was obtained from the Swedish Medical Research Council, the Foundation for Strategic Research, Biostapro AB, KI-fonder and Magnus Bergvall foundations. We also thank Ingegerd Lo¨ fving-Arvholm and Karin Coster for excellent technical assistance.

References [1] Cunningham R, Cockayne A, Humphreys H. Clinical and molecular aspects of the pathogenesis of Staphylococcus aureus bone and joint infections. J Med Microbiol 1996;44:157 –64. [2] Arnow PM, Quimosing EM, Beach M. Consequences of intravascular catheter sepsis. Clin Infect Dis 1993;16:778 – 84. [3] Mylotte JM, McDermott C, Spooner JA. Prospective study of 114 consecutive episodes of Staphylococcus aureus bacteraemia. Rev Infect Dis 1987;9:891 – 907. [4] Raad II, Sabbagh MF. Optimal duration of therapy for catheterrelated Staphylococcus aureus bacteraemia: a study of 55 cases and review [see comments]. Clin Infect Dis 1992;14:75 –82. [5] John JF, Barg NL. Staphylococcus aureus. In: Mayhall CG (editor). Hospital epidemiology and infection control. Baltimore: Williams & Wilkins, 1996. p. 271 – 90.

A. Rennermalm et al. / Vaccine 19 (2001) 3376–3383 [6] Nozohoor S, Flock J-I, Heimdahl A. Experimental endocarditis in the rat secondary to septic arthritisinduced by Staphylococcus aureus. Clin Microbio Inf 1999;5:158 –63. [7] Schennings T, Heimdahl A, Coster K, Flock JI. Immunization with fibronectin binding protein from Staphylococcus aureus protects against experimental endocarditis in rats. Microb Pathog 1993;15:227 – 36. [8] Hienz SA, Schennings T, Heimdahl A, Flock JI. Collagen binding of Staphylococcus aureus is a virulence factor in experimental endocarditis. J Infect Dis 1996;174:83 –8. [9] Lee JC, Park JS, Shepherd SE, Carey V, Fattom A. Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats. Infect Immun 1997;65:4146 – 51. [10] Jo¨ nsson K, Signas C, Muller HP, Lindberg M. Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur J Biochem 1991;202:1041 – 8. [11] Signas C, Raucci G, Jonsson K, Lindgren PE, Anantharamaiah GM, Hook M, Lindberg M. Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc Natl Acad Sci USA 1989;96:699 –703. [12] Mamo W, Jonsson P, Flock JI, Lindberg M, Muller HP, Wadstrom T, Nelson L. Vaccination against Staphylococcus aureus mastitis: immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S. aureus. Vaccine 1994;12:988 –92. [13] Flock JI, Hienz SA, Heimdahl A, Schennings T. Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus. Infect Immun 1996;64:1876 – 8. [14] Mamo W, Jonsson P, Muller HP. Opsonization of Staphylococcus aureus with a fibronectin-binding protein antiserum induces protection in mice. Microb Pathog 1995;19:49 –55. [15] Rozalska B, Wadstrom T. Protective opsonic activity of antibodies against fibronectin-binding proteins (FnBPs) of Staphylococcus aureus. Scand J Immunol 1993;37:575 –80. [16] Huff S, Matsuka YV, McGavin MJ, Ingham KC. Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy. J Biol Chem 1994;269:15563– 70. [17] McGavin MJ, Raucci G, Gurusiddappa S, Hook M. Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor. J Biol Chem 1991;266:8343 –7.

3383

[18] Brennan FR, Gilleland LB, Staczek J, Bendig MM, Hamilton WD, Jr. Gilleland HE. A chimaeric plant virus vaccine protects mice against a bacterial infection. Microbiology 1999;145:2061 – 7. [19] Dalsgaard K, et al. Plant-derived vaccine protects target animals against a viral disease. Nat Biotechnol 1997;15:248 – 52. [20] Brennan FR, et al. Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses. Vaccine 1999;17:1846 – 57. [21] Brennan FR, Bellaby T, Helliwell SM, Jones TD, Kamstrup S, Dalsgaard K, Flock JI, Hamilton WD. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice. J Virol 1999;73:930 –8. [22] Patti JM, Bremell T, Krajewska-Pietrasik D, Abdelnour A, Tarkowski A, Ryden C, Hook M. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect Immun 1994;62:152 – 61. [23] Kensil CR, Soltysik S, Wheeler DA, Wu JY. Structure/function studies on QS-21, a unique immunological adjuvant from Quillaja saponaria. Adv Exp Med Biol 1996;404:165 – 72. [24] Urban T, Jarstrand C, Tunevall G. The influence of opsonization with specific antibody and complement on the nitroblue tetrazolium (NBT) reduction of neutrophil granulocytes A methodological study and a clinical application. J Clin Lab Immunol 1981;5:175 – 9. [25] Herting E, Jarstrand C, Rasool O, Curstedt T, Hakansson S, Robertson B. Effect of surfactant on nitroblue tetrazolium reduction of polymorphonuclear leucocytes stimulated with type Ia group B streptococci. Acta Paediatr 1995;84:922 – 6. [26] Casolini F, Visai L, Joh D, Conaldi PG, Toniolo A, Hook M, Speziale P. Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections. Infect Immun 1998;66:5433 – 42. [27] Speziale P, Joh D, Visai L, Bozzini S, House-Pompeo K, Lindberg M, Hook M. A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae. J Biol Chem 1996;271:1371 – 8. [28] Luk JM, Flock JI, Wadstrom T. Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay. FEMS Microbiol Immunol 1989;1:505 – 10. [29] Ciborowski P, Flock JI, Wadstrom T. Immunological response to a Staphylococcus aureus fibronectin-binding protein. J Med Microbiol 1992;37:376 – 81.