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Vaccine assembly from surface proteins of Staphylococcus aureus Yukiko K. Stranger-Jones, Taeok Bae, and Olaf Schneewind* Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL 60637 Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved September 25, 2006 (received for review August 9, 2006) Staphylococcus aureus is the most common cause of hospital- acquired infection. Because of the emergence of antibiotic-resis- tant strains, these infections represent a serious public health threat. To develop a broadly protective vaccine, we tested cell wall-anchored surface proteins of S. aureus as antigens in a murine model of abscess formation. Immunization with four antigens (IsdA, IsdB, SdrD, and SdrE) generated significant protective im- munity that correlated with the induction of opsonophagocytic antibodies. When assembled into a combined vaccine, the four surface proteins afforded high levels of protection against invasive disease or lethal challenge with human clinical S. aureus isolates. disease protection opsonophagocytosis reverse vaccinology S taphylococcus aureus, a Gram-positive bacterial commensal of human skin and nares, is the leading cause of bloodstream, lower respiratory tract, and skinsoft-tissue infections (1, 2). The broad spectrum of important staphylococcal diseases also in- cludes endocarditis, septic arthritis, toxic-shock syndrome, scalded-skin syndrome, and food poisoning (3). S. aureus strains exhibiting multiple antibiotic resistances are isolated in 60% of community and up to 80% of hospital infections (4). For example, S. aureus strains with intermediate or full resistance to vancomycin, which is considered the therapy of last resort for methicillin-resistant S. aureus, have recently emerged (5, 6). Generating protective immunity against invasive S. aureus disease has been a goal since the discovery of this microbe. Whole-cell live or killed vaccines largely fail to generate pro- tective immune responses (7). Purified capsular polysaccharide, types 5 and 8 (which represent 80% of all capsular types found in clinical isolates; ref. 8), showed promise when used as a conjugate vaccine in experimental animals or in patients with end-stage renal disease (9–11). Immunization with poly-N- acetylglucosamine, a S. aureus surface carbohydrate synthesized by icaABC products (12), has been shown to protect mice against staphylococcal disease (13, 14). Subunit vaccines composed of individual surface proteins, for example, clumping factor A (ClfA) (15), clumping factor B (ClfB) (16), iron-regulated surface determinant B (IsdB) (17), or fibronectin-binding pro- tein (FnBP) (18), generate immune responses that afford partial protection against S. aureus challenge of experimental animals. However, for S. aureus vaccines to be successful, genetic deter- minants for specific antigens must be essential for staphylococcal virulence, which has not been observed for mutants lacking the genetic determinants of capsular polysaccharide or of individual surface proteins (20, 21). Rappuoli and colleagues (22) exploited information encrypted in bacterial genome sequences to distinguish pangenomes, i.e., genes in all strains of a pathogen, from conserved genes found in all members of its species. Rational vaccine design was achieved by interrogating conserved antigens (secreted or surface-displayed) for protective immunity, which led to the identification of multiple surface proteins of group B streptococci as candidates (reverse vaccinology) (23). By combining multiple antigens into a single vaccine, broad-spectrum protective immunity against many differ- ent clinical isolates was achieved (23). Here we used reverse vaccinology, and we tested surface proteins of S. aureus for gen- erating protective immune responses against invasive S. aureus disease in mice. By combining four antigens with the highest level of protective immunity, we generated a vaccine that protects mice against lethal challenge with diverse strains isolated from human clinical infections. Results Selection of S. aureus Surface Antigens. We wondered whether a broad-spectrum S. aureus vaccine may be derived by testing staphylococcal surface proteins for protective immunity. S. aureus sortase A (srtA) mutants, which cannot display surface proteins, are unable to establish infections in experimental models, indicating that the sum of all 23 surface proteins is essential for pathogenesis (24). Sortase A cleaves sorting signals of surface proteins and anchors polypeptides to the cell wall envelope (20). Eight staphylococcal genome sequences (25–27) were examined for the presence of sortase substrate genes by using sorting signals as queries in BLAST searches, and 19 conserved surface protein genes were identified (Table 3, which is published as supporting information on the PNAS web site). These genes were expressed in Escherichia coli as soluble His- tagged or GST fusions and recombinant proteins purified by affinity chromatography. Groups of mice were immunized by intramuscular injection with 100 g of purified protein emulsi- fied in complete Freund’s adjuvant and 11 days later with protein emulsified in incomplete Freund’s adjuvant. Blood samples were drawn before, during, and after immunization; and specific serum IgG levels were determined by ELISA, demonstrating that surface proteins generated humoral immune responses to immunization (Table 1). Mice were challenged 21 days after the primary immunization with 3–5 10 6 colony-forming units (cfu) of S. aureus Newman (28) by retroorbital injection. Four days after infection, mice were killed, kidneys were removed, and the bacterial load in homogenized tissues was measured (19). Com- pared with mock-immunized animals, some recombinant surface proteins generated specific immune responses that afforded partial protection against staphylococcal disease. Bacterial load in kidneys of animals immunized with ClfA, SdrD, SdrE, IsdA, or IsdB was reduced by three to four logarithms, whereas immunization with Spa, ClfB, IsdC, SdrC, SasD, or SasF af- forded a two to three logarithmic reduction in staphylococcal burden. FnBPA, SasG, or IsdH immunization generated even smaller reductions in bacterial load; whereas immunization with FnBPB, SasA, SasB, SasC, or SasK did not result in significant protection (Table 1). Antisera Against IsdA, IsdB, SdrD, and SdrE Stimulate Opsonophago- cytosis. Four surface-protein vaccine candidates (IsdA, IsdB, SdrD, and SdrE) generated the highest levels of protection Author contributions: Y.K.S.-J. and O.S. designed research; Y.K.S.-J. and T.B. performed research; Y.K.S.-J. contributed new reagentsanalytic tools; Y.K.S.-J., T.B., and O.S. ana- lyzed data; and Y.K.S.-J. and O.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS direct submission. Abbreviation: PMN, polymorphonuclear leukocyte. *To whom correspondence should be addressed. E-mail: [email protected]. © 2006 by The National Academy of Sciences of the USA 16942–16947 PNAS November 7, 2006 vol. 103 no. 45 www.pnas.orgcgidoi10.1073pnas.0606863103 Downloaded by guest on December 11, 2020

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Page 1: Vaccine assembly from surface proteins of Staphylococcus ... · Staphylococcus aureus is the most common cause of hospital-acquired infection. Because of the emergence of antibiotic-resis-tant

Vaccine assembly from surface proteinsof Staphylococcus aureusYukiko K. Stranger-Jones, Taeok Bae, and Olaf Schneewind*

Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL 60637

Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved September 25, 2006 (received for review August 9, 2006)

Staphylococcus aureus is the most common cause of hospital-acquired infection. Because of the emergence of antibiotic-resis-tant strains, these infections represent a serious public healththreat. To develop a broadly protective vaccine, we tested cellwall-anchored surface proteins of S. aureus as antigens in a murinemodel of abscess formation. Immunization with four antigens(IsdA, IsdB, SdrD, and SdrE) generated significant protective im-munity that correlated with the induction of opsonophagocyticantibodies. When assembled into a combined vaccine, the foursurface proteins afforded high levels of protection against invasivedisease or lethal challenge with human clinical S. aureus isolates.

disease protection � opsonophagocytosis � reverse vaccinology

S taphylococcus aureus, a Gram-positive bacterial commensalof human skin and nares, is the leading cause of bloodstream,

lower respiratory tract, and skin�soft-tissue infections (1, 2). Thebroad spectrum of important staphylococcal diseases also in-cludes endocarditis, septic arthritis, toxic-shock syndrome,scalded-skin syndrome, and food poisoning (3). S. aureus strainsexhibiting multiple antibiotic resistances are isolated in �60% ofcommunity and up to 80% of hospital infections (4). Forexample, S. aureus strains with intermediate or full resistance tovancomycin, which is considered the therapy of last resort formethicillin-resistant S. aureus, have recently emerged (5, 6).

Generating protective immunity against invasive S. aureusdisease has been a goal since the discovery of this microbe.Whole-cell live or killed vaccines largely fail to generate pro-tective immune responses (7). Purified capsular polysaccharide,types 5 and 8 (which represent �80% of all capsular types foundin clinical isolates; ref. 8), showed promise when used as aconjugate vaccine in experimental animals or in patients withend-stage renal disease (9–11). Immunization with poly-N-acetylglucosamine, a S. aureus surface carbohydrate synthesizedby icaABC products (12), has been shown to protect mice againststaphylococcal disease (13, 14). Subunit vaccines composed ofindividual surface proteins, for example, clumping factor A(ClfA) (15), clumping factor B (ClfB) (16), iron-regulatedsurface determinant B (IsdB) (17), or fibronectin-binding pro-tein (FnBP) (18), generate immune responses that afford partialprotection against S. aureus challenge of experimental animals.However, for S. aureus vaccines to be successful, genetic deter-minants for specific antigens must be essential for staphylococcalvirulence, which has not been observed for mutants lacking thegenetic determinants of capsular polysaccharide or of individualsurface proteins (20, 21).

Rappuoli and colleagues (22) exploited information encrypted inbacterial genome sequences to distinguish pangenomes, i.e., genesin all strains of a pathogen, from conserved genes found in allmembers of its species. Rational vaccine design was achieved byinterrogating conserved antigens (secreted or surface-displayed)for protective immunity, which led to the identification of multiplesurface proteins of group B streptococci as candidates (reversevaccinology) (23). By combining multiple antigens into a singlevaccine, broad-spectrum protective immunity against many differ-ent clinical isolates was achieved (23). Here we used reversevaccinology, and we tested surface proteins of S. aureus for gen-

erating protective immune responses against invasive S. aureusdisease in mice. By combining four antigens with the highest levelof protective immunity, we generated a vaccine that protects miceagainst lethal challenge with diverse strains isolated from humanclinical infections.

ResultsSelection of S. aureus Surface Antigens. We wondered whether abroad-spectrum S. aureus vaccine may be derived by testingstaphylococcal surface proteins for protective immunity. S.aureus sortase A (srtA) mutants, which cannot display surfaceproteins, are unable to establish infections in experimentalmodels, indicating that the sum of all 23 surface proteins isessential for pathogenesis (24). Sortase A cleaves sorting signalsof surface proteins and anchors polypeptides to the cell wallenvelope (20). Eight staphylococcal genome sequences (25–27)were examined for the presence of sortase substrate genes byusing sorting signals as queries in BLAST searches, and 19conserved surface protein genes were identified (Table 3, whichis published as supporting information on the PNAS web site).These genes were expressed in Escherichia coli as soluble His-tagged or GST fusions and recombinant proteins purified byaffinity chromatography. Groups of mice were immunized byintramuscular injection with 100 �g of purified protein emulsi-fied in complete Freund’s adjuvant and 11 days later with proteinemulsified in incomplete Freund’s adjuvant. Blood samples weredrawn before, during, and after immunization; and specificserum IgG levels were determined by ELISA, demonstratingthat surface proteins generated humoral immune responses toimmunization (Table 1). Mice were challenged 21 days after theprimary immunization with 3–5 � 106 colony-forming units (cfu)of S. aureus Newman (28) by retroorbital injection. Four daysafter infection, mice were killed, kidneys were removed, and thebacterial load in homogenized tissues was measured (19). Com-pared with mock-immunized animals, some recombinant surfaceproteins generated specific immune responses that affordedpartial protection against staphylococcal disease. Bacterial loadin kidneys of animals immunized with ClfA, SdrD, SdrE, IsdA,or IsdB was reduced by three to four logarithms, whereasimmunization with Spa, ClfB, IsdC, SdrC, SasD, or SasF af-forded a two to three logarithmic reduction in staphylococcalburden. FnBPA, SasG, or IsdH immunization generated evensmaller reductions in bacterial load; whereas immunization withFnBPB, SasA, SasB, SasC, or SasK did not result in significantprotection (Table 1).

Antisera Against IsdA, IsdB, SdrD, and SdrE Stimulate Opsonophago-cytosis. Four surface-protein vaccine candidates (IsdA, IsdB,SdrD, and SdrE) generated the highest levels of protection

Author contributions: Y.K.S.-J. and O.S. designed research; Y.K.S.-J. and T.B. performedresearch; Y.K.S.-J. contributed new reagents�analytic tools; Y.K.S.-J., T.B., and O.S. ana-lyzed data; and Y.K.S.-J. and O.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviation: PMN, polymorphonuclear leukocyte.

*To whom correspondence should be addressed. E-mail: [email protected].

© 2006 by The National Academy of Sciences of the USA

16942–16947 � PNAS � November 7, 2006 � vol. 103 � no. 45 www.pnas.org�cgi�doi�10.1073�pnas.0606863103

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against staphylococcal renal infection. To determine the natureof protection, antibodies against IsdA, IsdB, SdrD, and SdrEwere raised in rabbits and analyzed for their ability to induceopsonophagocytic killing of staphylococci in the presence ofwhite blood cells, an important immunological correlate ofprotective immunity against S. aureus (11, 14). Freshly isolatedhuman polymorphonuclear leukocytes (PMNs) were incubatedwith staphylococci in the presence of complement and specificantibodies. For this experiment, we used strain SEJ2, a �(spa)variant of S. aureus Newman carrying a deletion of the proteinA gene, to avoid nonimmune precipitation of antibodies on thebacterial surface. After incubation, staphylococcal killing wasmonitored by spreading sample aliquots on agar medium fol-lowed by colony formation and enumeration (Fig. 1). Antibodiesagainst all four surface-protein antigens induced opsonophago-cytic killing of S. aureus. Addition of a mixture of antibodiesagainst all four surface proteins to human PMNs led to anincrease in opsonophagocytosis compared with that mediated byantibodies against individual surface proteins (P � 0.03).

Combining Surface Antigens to Develop an Effective S. aureus Vaccine.IsdA, IsdB, SdrD, and SdrE were assembled into a combinedvaccine. To determine whether antibodies are generated againsteach of the four antigens in a combined vaccine, serum IgG titersof immunized mice were analyzed (Table 2). Antibody levelsgenerated by the combined vaccine were similar to thoseachieved by immunization with individual surface protein anti-gens. Mice were challenged with 3–5 � 106 cfu of S. aureus strainNewman. Four days after infection, animals were killed, andkidneys were removed. In contrast to immunization with indi-vidual antigens, the combined vaccine afforded complete pro-tection against staphylococcal challenge. We measured a reduc-tion in bacterial load of 5.062 log10 cfu per ml (P � 0.00074), i.e.,a reduction to a level below detection. Histological analysis of

kidney tissues failed to detect staphylococcal abscesses in micethat had been immunized with the combined vaccine (Fig. 2). Incontrast, organs removed from mock-immunized animals har-

Table 1. Protection against S. aureus infection conferred by immunization of mice with surface protein

Surface protein IgG titer*S. aureus in kidneys,†

log10 (cfu) per ml

Reduction ofStaphylococci,‡

log10 (cfu) per ml Significance§

Spa ND¶ 3.911 � 0.978 2.951 P � 0.00541FnBPA 18,000 � 6,771 4.891 � 0.922 1.971 P � 0.02316FnBPB 24,300 � 3,600 6.172 � 0.437 0.690 P � 0.07434ClfA 64,800 � 9,920 3.521 � 0.922 3.341 P � 0.00361ClfB 36,600 � 12,247 4.308 � 0.797 2.554 P � 0.01012SdrC 12,125 � 3,770 4.026 � 0.979 2.836 P � 0.01012SdrD 30,000 � 12,920 3.477 � 1.039 3.385 P � 0.00613SdrE 29,000 � 6,878 2.565 � 0.913 4.297 P � 0.00068SasA 8,500 � 1,936 5.207 � 1.048 1.655 P � 0.06821SasB 20,250 � 6,037 5.709 � 0.780 1.153 P � 0.09094SasC ND 5.649 � 0.781 1.213 P � 0.09642SasD 24,500 � 1,342 4.675 � 1.082 2.187 P � 0.03782IsdA�SasE 45,900 � 9,156 2.518 � 0.897 4.344 P � 0.00057SasF 16,900 � 3,932 3.916 � 0.781 2.946 P � 0.00187SasG�Aap 21,875 � 2,900 5.384 � 0.715 1.478 P � 0.03597HarA�IsdH�SasI 30,375 � 15,093 4.918 � 0.761 1.944 P � 0.02239IsdB�SasJ 36,300 � 2,741 3.394 � 0.982 3.468 P � 0.00318SasK 32,800 � 12,659 5.898 � 0.746 0.964 P � 0.11032IsdC ND 3.779 � 1.084 3.083 P � 0.00996PBS mock — 6.862 � 0.098 — —

*IgG titers (mean serum titers � SEM) in response to immunization with surface proteins were determined by ELISA (n � 5 animals).†Immunized mice challenged with S. aureus Newman (n � 8–10 animals), and staphylococci in kidney tissues were enumerated�log10(cfu) per ml � SEM�.

‡Reduction of the number of staphylococci in kidney tissues [log10 (cfu) per ml] is a measure for protective immunity, which is comparedwith control mice immunized with PBS (n � 8–10 animals).

§Statistical significance of reduction in staphylococcal burden was assessed with the one-tailed Student’s t test, and P values were recorded.¶ND, not determined.

Fig. 1. Antibodies against IsdA, IsdB, SdrD, and SdrE mediate complement-dependent opsonophagocytosis. Phagocytic assays were performed to deter-mine the mechanism of protection afforded by surface-protein immunizations.Rabbit antibodies, rabbit complement, freshly isolated human PMNs, and S.aureus were incubated and plated on agar medium to measure bacterial survivalas cfu; then the percent of killing was calculated. Antibodies were added eitheras individualantisera raisedagainstonesurfaceprotein (�-IsdA, �-IsdB, �-SdrD,or�-SdrE) or as a mixture of antisera (�-IsdA �-IsdB �-SdrD �-SdrE) to PMNs.Opsonsonophagocytic killing of staphylococci in the presence of complementwas significantly increased for mixed antisera compared with individual surface-protein antisera (P � 0.03). Error bars represent the SEM.

Stranger-Jones et al. PNAS � November 7, 2006 � vol. 103 � no. 45 � 16943

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Page 3: Vaccine assembly from surface proteins of Staphylococcus ... · Staphylococcus aureus is the most common cause of hospital-acquired infection. Because of the emergence of antibiotic-resis-tant

bored bacterial abscesses with central concentrations of staph-ylococci that were surrounded by a large cuff of white blood cells(Fig. 2). Kidney tissue of mice immunized with the combinedvaccine revealed the physiological architecture of renal tubulesas well as small infiltrates of PMNs, likely the anatomicalsubstrate of opsonophagocytic clearance of staphylococci.

Combined Vaccine Protects Animals Against Lethal Challenge withDiverse S. aureus Isolates. To test whether the combined vaccineprotects against lethal-challenge infections, mice were immu-nized with the combined vaccine or with individual purifiedsurface antigens (IsdA, IsdB, SdrD, and SdrE). Challenges of

2 � 1010 cfu of S. aureus Newman were administered intraperi-toneally, and mice were monitored for 7 days. Immunizationwith individual surface proteins either had no effect on survivalor it afforded only very modest protection (Fig. 3), as alreadyreported for purified IsdB (17). In contrast to individual proteinantigens, immunization with the combined vaccine affordedcomplete protection against a lethal challenge with S. aureusNewman (P � 0.03) (Fig. 3). These results suggest that immu-nization with the combined vaccine can generate increasedprotection against lethal challenge with a strain expressing allfour surface proteins.

For staphylococcal vaccines to be effective, protection must beachieved against a wide variety of different strains. By firsteliminating surface proteins that are not conserved amongstaphylococcal strains, we aimed at developing a vaccine thatmay be effective against a wide range of S. aureus isolates. FiveS. aureus strains were selected to test this hypothesis. NRS252 isa methicillin-sensitive S. aureus strain associated with toxic-shock syndrome after burn injury; whereas N315, NRS248,USA100, and USA400 are methicillin-resistant S. aureus strains(25, 29, 30). NRS248 is the causative agent of necrotizingpneumonia. USA400 carries the Panton–Valentine leukocidingenes associated with lethal lung infections (31). USA100 is themost frequent cause of healthcare-associated infections in theUnited States, and protection against this strain is of crucialimportance for vaccine efforts (29). Mice were immunized withthe combined vaccine or mock-immunized and then challengedby intraperitoneal injection of S. aureus suspensions harboring

Table 2. IgG titers for individual surface proteins and thecombined vaccine (IsdA, IsdB, SdrD, and SdrE)

Surface proteinIndividual

immunization*Combinedvaccine* Significance†

IsdA�SasE 27,000 � 12,000 41,000 � 14,000 P � 0.26964IsdB�SasJ 11,000 � 2,449 28,000 � 12,104 P � 0.14330SdrD 7,000 � 2,000 9,000 � 2,449 P � 0.18695SdrE 15,000 � 3,162 17,000 � 3,742 P � 0.35200

*IgG titers (mean serum titers � SEM) in response to immunization of mice asdetermined by ELISA (n � 5 animals).

†Specific antibody titers raised against surface proteins were not significantlydifferent when immunizing antigens were administered individually or incombination.

Fig. 2. Immunization with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) generates protective immunity against S. aureus abscess formation in mice. BALB�cmice were mock-immunized (A and B) or immunized with the combined vaccine (C and D) and challenged by retroorbital infection with S. aureus Newman. Fourdays after infection, animals were killed, and the kidneys were removed. Organ tissue was fixed in formalin, thin-sectioned, and stained with hematoxylin�eosin.Microscopic images of whole kidneys (A and C) or organ tissue at higher magnification (B and D) revealed abscess formation only in mock-immunized animals.Similar results were obtained for six organ tissues in each group. (Scale bars: A and C, 1 mm; B and D, 0.1 mm.) (B) Staphylococcal abscess (black arrow) with acentral concentration of staphylococci (white arrow). (D) PMN infiltrate (black arrow).

16944 � www.pnas.org�cgi�doi�10.1073�pnas.0606863103 Stranger-Jones et al.

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Page 4: Vaccine assembly from surface proteins of Staphylococcus ... · Staphylococcus aureus is the most common cause of hospital-acquired infection. Because of the emergence of antibiotic-resis-tant

3–10 � 109 cfu. Survival analysis revealed that the combinedvaccine afforded significant protection against lethal challengewith any one of the five clinical S. aureus isolates (Fig. 4).

DiscussionFor many years, surface proteins of Gram-positive bacterialpathogens have been tested as antigens to generate immuneresponses for the protection of humans against infection (32).For example, antibodies against M protein precipitate op-sonophagocytic killing of group A streptococci and clearance ofinfection for the leading cause of human pharyngeal inflamma-tory disease (33). Rational vaccine design for the prevention ofgroup A streptococcal disease has been hindered by both antigenand strain diversity (34, 35). However, recent advances ingenome sequencing and systematic analysis of antigens encodedby diverse strains led to the development of vaccine candidatesthat may prevent human infectious diseases (36). This approachhas been used successfully for the identification of protectiveantigens and vaccine development in group A streptococci (37),group B streptococci (23), and group B meningococci (38). In allcases examined, the antigens that elicited the most effectiveprotective immune responses were characterized as surfaceproteins.

Surface proteins of Gram-positive bacteria require C-terminalsorting signals for proper recognition and cell wall anchoring bysortases (39–41). Genes encoding surface proteins with sortingsignals can be identified in translated genome sequences on thebasis of amino acid sequence conservation (20, 42). By testing asantigens 19 conserved surface proteins found in the genomesequences of eight S. aureus strains, we could identify fourpolypeptides that generated the highest amount of protective

immunity: IsdA, IsdB, SdrD, and SdrE. IsdB binds to hemoglo-bin on the staphylococcal surface, and this interaction is aprerequisite for bacterial iron scavenging from host-derivedheme compounds (43). IsdA, a heme-binding surface protein, isalso involved in heme iron uptake (43). SdrD and SdrE aremembers of the family of surface proteins with serine (S)–aspartate (D) repeats (R), which includes also clumping factors(ClfA and ClfB) as well as SdrC (44). A molecular function hasnot yet been revealed for SdrD and SdrE; however, theseproteins are proposed to bind host extracellular matrix, similarto other members of the MSCRAMM family (44, 45).

Our data suggest that a combined vaccine of S. aureus surfaceantigens derived from conserved genome sequences can elicitimmune responses that achieve greater protective immunity thanimmunization with its individual components. Immunizationwith four surface proteins, IsdA, IsdB, SdrD, and SdrE, gener-ated the highest level of protection in mice compared with 15other antigens. Three of the four antigens in the combinedvaccine are already known to be immunogenic in humansbecause antibodies against IsdB, SdrD, and SdrE can be found

Fig. 3. Immunization with the combined vaccine (IsdA, IsdB, SdrD, and SdrE)generates protective immunity against lethal S. aureus challenge. Mice (n �8–10) were immunized with individual surface-protein antigens (IsdA, IsdB,SdrD, or SdrE), with the combined vaccine (IsdA, IsdB, SdrD, and SdrE) or withPBS. Animals were challenged by intraperitoneal injection of S. aureus New-man (2 � 1010 cfu), and then they were monitored for 7 days. Compared withanimals receiving mock-immunization (PBS), the significance of protectiveimmunity generated by various antigens was measured with Fisher’s exacttest: IsdA, P � 0.34372; IsdB, P � 0.22049; SdrD, P � 0.24006; SdrE, P � 0.31508;and combined vaccine, P � 0.02941. Compared with animals receiving thecombined vaccine, the significance of protective immunity was measured withFisher’s exact test: PBS, P � 0.02941; IsdA, P � 0.02941; IsdB, P � 0.05294; SdrD,P � 0.00377; and SdrE, P � 0.01131.

Fig. 4. Immunization with the combined vaccine generates protective im-munity against lethal challenge with five different clinical S. aureus isolates.Mice (n � 10) were immunized with the combined vaccine (IsdA, IsdB, SdrD,and SdrE) or with PBS, challenged by intraperitoneal injection with clinical S.aureus isolates (3–10 � 109 cfu), and monitored for 7 days for survival.Compared with animals receiving mock-immunization (PBS), the significanceof protective immunity generated by the combined vaccine was measuredwith Fisher’s exact test: (A) N315, P � 0.06502; (B) NRS248, P � 0.00036; (C)NRS252, P � 0.03215; (D) USA100, P � 0.00542; and (E) USA400, P � 0.00542.

Stranger-Jones et al. PNAS � November 7, 2006 � vol. 103 � no. 45 � 16945

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in healthy individuals or in patients with S. aureus disease (46).Our data also suggest a functional correlation between antibodytiters for surface antigens, opsonophagocytic properties of an-tibodies, as well as protective immunity. Further testing of thishypothesis may yield a molecular appreciation of immunityagainst S. aureus and permit rational development of a vaccinethat can protect humans at high risk for invasive S. aureusinfection.

Materials and MethodsBioinformatics. Genome sequences of S. aureus strains wereanalyzed for surface proteins by using sorting signals as queriesin BLAST searches. Signal peptide cleavage sites were predictedwith the SignalP 3.0 algorithm (47).

Bacterial Strains and Growth. Staphylococci were cultured ontryptic soy agar or broth at 37°C. S. aureus NRS70 (N315),NRS123 (USA400�MW2), NRS248, NRS252, and NRS382(USA100) were obtained through the Network on AntimicrobialResistance in S. aureus (NARSA, NIAID Contract no. N01-AI-95359). S. aureus SEJ2 is a �(spa) variant of strain Newmancarrying a deletion of the protein A gene. E. coli strains DH5�and BL21(DE3) were cultured on Luria agar or broth at 37°C.Carbenicillin (50 �g�ml) and erythromycin (10 �g�ml) wereused for plasmid selection.

Cloning and Purification. Coding sequences for surface proteinsexcluding the signal peptide and sorting signal were PCR-amplified by using S. aureus N315 template DNA (for a listing ofprimer sequences, see Table 4, which is published as supportinginformation on the PNAS web site). PCR products were clonedinto either pET-15b or pGEX-2T to express recombinant pro-teins with an N-terminal His6 tag or GST fusion, respectively.After bacterial disintegration, proteins were affinity-purifiedfrom cleared lysates by using nickel–nitrilotriacetic acid orglutathione–Sepharose. Protein eluate was subjected to endo-toxin removal by Triton X-114 phase separation, surface proteinswere purified further by gel filtration, and protein concentrationwas determined.

Immunization. BALB�c mice (24-day-old female, 8–10 mice pergroup, Charles River Laboratories, Wilmington, MA) wereimmunized by intramuscular injection into the hind leg withpurified protein. Proteins (100 �g of each) were administered ondays 0 (emulsified 1:1 with complete Freund’s adjuvant) and 11(emulsified 1:1 with incomplete Freund’s adjuvant). Blood sam-ples were drawn by retroorbital bleeding on days 0, 11, and 20.Sera were examined by ELISA for IgG titers with specificantigen-binding activity. Animal experiments were performed inaccordance with institutional guidelines following experimentalprotocol review and approval by the Institutional Animal Careand Use Committee.

Renal Abscess. Immunized animals were challenged on day 21 byretroorbital injection of 100 �l of bacterial suspension (3–5 � 106

cfu). Overnight cultures of S. aureus Newman were diluted 1:100into fresh tryptic soy broth and grown for 3 h at 37°C. Staphy-lococci were centrifuged, washed twice, and diluted in PBS toyield an A600 of 0.4 (3–5 � 107 cfu per ml). Further dilutions wereneeded for the desired inoculum, which was verified experimen-tally by agar plating and colony formation. Mice were anesthe-tized by intraperitoneal injection of 80–120 mg of ketamine and3–6 mg of xylazine per kilogram of body weight. On day 25, micewere euthanized by compressed CO2 inhalation. Kidneys wereremoved and homogenized in 1% Triton X-100. Aliquots werediluted and plated on agar medium for triplicate determinationof cfu. For histology, kidney tissue was incubated at roomtemperature in 10% formalin for 24 h. Tissues were embeddedin paraffin, thin-sectioned, stained with hematoxylin�eosin, andexamined by microscopy.

Lethal Challenge. Immunized animals were challenged on day 21by intraperitoneal injection with 2 � 1010 cfu of S. aureusNewman or 3–10 � 109 cfu of clinical S. aureus isolates. Animalswere monitored for 7 days, and lethal disease was recorded.

Opsonophagocytic Killing. PMNs were isolated from healthy hu-man volunteers. Cells were counted, examined for viability bytrypan blue exclusion, and diluted to 2–5 � 106 PMNs per ml. Toremove antibodies that react with the bacterial target, infantrabbit serum was preadsorbed with S. aureus Newman suspen-sions by mixing at 4°C for 30 min. Serum was then centrifuged,filter-sterilized, and used as a source of complement. S. aureusSEJ2 was adjusted to 2–5 � 105 cfu per ml. Rabbit antibodies toIsdA, IsdB, SdrD, and SdrE were normalized to the same IgGtiter. For individual antibody experiments, the normalized an-tibodies were further diluted 1:4, and for the combinationantibody experiments these diluted antibodies were mixed1:1:1:1. Equal volumes (100 �l) of PMNs, complement, bacteria,and diluted antibodies were mixed and incubated at 37°C for 90min before dilution, agar plating, and bacterial enumeration.The percent amount of bacterial killing was calculated as [1 (no. of cfu recovered in the presence of PMNs�no. of cfurecovered in the absence of PMNs)] � 100.

Statistical Analysis. One-tailed Student’s t tests were performed toanalyze the statistical significance of renal abscess data. Fisher’sexact test was used to analyze the statistical significance of thelethal challenge data.

We thank Nancy Ciletti for assistance with animal experiments; KatieOverheim and Derek Elli for ELISAs; and Dominique Missiakas, JulieBubeck-Wardenburg, Andrea DeDent, and Angelika Grundling forcritical comments. This work was supported by U.S. Public HealthService Grants AI38897 and AI52474 from the National Institute ofAllergy and Infectious Diseases, Division of Microbiology and InfectiousDiseases, National Institutes of Health (to O.S.).

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