Vaccine 32 (2014) 464–469

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A monoclonal antibody that recognizes the E domain of staphylococcal protein A Hwan Keun Kim, Carla Emolo, Dominique Missiakas, Olaf Schneewind ∗ Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA

a r t i c l e

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Article history: Received 13 August 2013 Received in revised form 1 November 2013 Accepted 15 November 2013 Available online 27 November 2013 Keywords: Staphylococcal protein A B Cell superantigen Opsonophagocytic clearance Therapeutic vaccine Disease protection Monoclonal antibody

a b s t r a c t Staphylococcal protein A (SpA) binds Fc␥ and VH 3 clan Fab domains of human and animal immunoglobulin (Ig) with each of its five Ig binding domains (IgBDs), thereby supporting Staphylococcus aureus escape from opsonophagocytic killing and suppressing adaptive B cell responses. The variant SpAKKAA cannot bind Ig yet retains antigenic properties that elicit SpA-neutralizing antibodies and disease protection in mice, whereas S. aureus infection or SpA-immunization cannot elicit neutralizing antibodies. As a test for this model, we analyzed here mAb 358A76, which was isolated from SpA-immunized mice. Unlike SpAKKAA -derived mAbs, mAb 358A76 binds only the first IgBD (E) but not any of the other four IgBDs (DA-B-C) and its binding can neutralize only the E domain of SpA, which does not provide disease protection in mice. These results are in agreement with a model whereby wild-type SpA-immunization generates a limited antibody response without neutralizing and/or disease protective attributes. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Almost all clinical Staphylococcus aureus isolates express protein A (SpA) [1,2]. SpA is synthesized as a precursor that is cleaved and anchored by sortase A in the bacterial cell wall envelope [3,4]. During staphylococcal growth, SpA molecules are also released into the extracellular medium [5]. SpA is comprised of four to five repeats of the 56–61 residue immunoglobulin binding domains (IgBDs) [6,7]. Each IgBD folds into a triple-helical bundle with discrete binding sites for the Fc␥ and VH 3 clan Fab domains of human and animal immunoglobulin (Ig) [8,9]. Glutamine (Q) residues 9 and 10 in ␣helix 1 of each IgBD capture Fc␥ domains [9], thereby inhibiting antibody-mediated opsonization [10,11]. Aspartic acid (D) residues 36 and 37 in the linker region between ␣-helix 2 and 3 of each IgBD capture the Fab portion of VH 3-type IgG and IgM [8]. SpA association with VH 3-type B cell receptors triggers B cell superantigen activity [11,12]. These interactions explain why S. aureus spa mutants, in comparison with wild-type, are more susceptible to opsonophagocytic killing and unable to block adaptive immune responses in a mouse model of infection [13].

∗ Corresponding author at: Department of Microbiology, University of Chicago, 920 East 58th Street, Cummings Life Sciences Center 607b, Chicago, IL 60637, USA. Tel.: +1 7738349060; fax: +1 7738348150. E-mail address: [email protected] (O. Schneewind). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.11.054

SpAKKAA , a variant with 20 amino acid substitutions replacing Q9 and Q10 with lysine (K) as well as D36 and D37 with alanine (A) in each of the five IgBDs, cannot bind Fc␥ or VH 3 Fab domains [14]. When used as vaccine antigen in mice, SpAKKAA , but not SpA, elicited protein A-specific immune responses and protection from disease [14]. SpAKKAA was also used to study protein A-specific immune responses in humans and mice. Healthy volunteers with a history of staphylococcal infection did not harbor serum SpAKKAA -specific antibodies [14]. Mice that had been injected with recombinant wild-type SpA or infected with wild-type S. aureus strains (expressing SpA) did not mount significant SpAKKAA -specific antibody responses [15]. These data were incorporated into a model, whereby S. aureus infection does not elicit protective immune responses because of the B cell superantigen activity of protein A [13]. Mouse monoclonal antibodies (mAb) were isolated after immunization of animals with SpAKKAA [16]. Protection against S. aureus disease by candidate SpAKKAA -mAbs correlated with the ability to bind multiple IgBDs and inhibit protein A binding to Fc␥ and Fab VH 3 domains of Ig [16]. United States patents US2008/0118937 A1 and US2010/0047252 A1 describe a murine hybridoma cell line derived from mice that had been immunized with wild-type SpA. The corresponding antibody, mAb 358A76, was reported to bind SpA, however the nature of this binding was heretofore not known. Here, we analyze the molecular attributes of SpA-mAb 358A76 in comparison with SpAKKAA -mAb 3F6 [16].

H.K. Kim et al. / Vaccine 32 (2014) 464–469

2. Materials and methods 2.1. Bacterial strains and growth conditions S. aureus strain USA300 LAC [17] was grown in tryptic soy broth (TSB) at 37 ◦ C. Escherichia coli strains DH5␣ and BL21 (DE3) were grown in Luria-Bertani (LB) broth with 100 ␮g ml−1 ampicillin at 37 ◦ C. 2.2. Monoclonal antibodies The generation and characterization of the mouse monoclonal antibody 3F6 have been described previously [16]. Hybridoma cell line 358A76 was purchased from American Type Culture Collection (ATCC accession number PTA-7938) and expanded at the Fitch Monoclonal Antibody Facility, University of Chicago. Antibodies were affinity purified from the spent culture supernatant of cell lines as described previously [16]. 2.3. Purification of recombinant proteins Recombinant SpA variants tagged with N-terminal 6 histidine residues (SpA, SpA-E, SpAKKAA , SpA-EKKAA , SpA-DKKAA , SpA-AKKAA , SpA-BKKAA , and SpA-CKKAA ) and synthetic peptide fragments (H1, H2, H3, H1 + 2, and H2 + 3), were produced from plasmids or purchased, respectively, as described previously [16]. Briefly, purification of recombinant SpA variants was performed by diluting overnight cultures of E. coli BL21 carrying specific plasmids, 1:100 into fresh media and grown at 37 ◦ C. When cultures reached an absorbance at 600 nm (A600 ) of 0.5, isopropyl ␤-d1-thiogalatopyranoside (IPTG) was added at a concentration of 1 mM and cultures were incubated at 37 ◦ C for an additional three hours. Bacterial cells were sedimented, suspended in column buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl] and disrupted with a French pressure cell at 14,000 psi. Lysates were subjected to ultracentrifugation at 100,000 × g for 30 min and proteins in the supernatant were subjected to nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Proteins were eluted in column buffer containing increasing concentrations of imidazole (upto 500 mM). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Scientific). 2.4. Enzyme linked immunosorbent assay To determine binding affinities of candidate mAbs, ELISA plates were coated with either full length SpAKKAA (20 nM), individual IgBDs (100 nM), or synthetic peptides (H1, H2, H3, H1 + 3, and H2 + 3, 100 nM) derived from IgBD domain E of SpAKKAA (SpAEKKAA ) in 0.1 M carbonate buffer (pH 9.5 at 4 ◦ C) overnight. Plates were blocked and incubated with variable concentrations of mAbs 358A76 and 3F6. For competition assay, plates were coated with 20 nM of SpAKKAA , or wild-type SpA, or 100 nM SpA-E in 0.1 M carbonate buffer (pH 9.5 at 4 ◦ C) overnight. The following day, plates were blocked and incubated with dilutions of competing monoclonal antibodies at a starting concentration of 30 or 6 ␮g ml−1 prior to the incubation with HRP-conjugated SpA-specific mAbs (Innova Biosciences) or human IgG at a final concentration of 200 ng ml−1 . 2.5. Mouse renal abscess model Affinity purified antibodies in PBS were injected at 5 mg kg−1 into the peritoneal cavity of BALB/c mice (6 week old, female, Charles River Laboratories) 24 h prior to challenge with S. aureus. Overnight cultures of S. aureus were diluted 1:100 into fresh TSB and grown for 2 h at 37 ◦ C. Staphylococci were sedimented,

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washed, and suspended in PBS to the desired bacterial concentration. Inocula were quantified by plating dilution aliquots on TSA and enumeration of colonies after incubation of plates at 37 ◦ C overnight. Animals were anesthetized via intraperitoneal injection with 65 mg ml−1 ketamine and 6 mg ml−1 xylazine per kilogram of body weight and infected by injection with 5 × 106 CFU S. aureus USA300 (LAC) into the periorbital venous sinus of the right eye. On day 4 following challenge, mice were killed by CO2 inhalation. Kidneys were removed, and staphylococcal loads were analyzed by homogenizing renal tissue in PBS containing 0.1% Triton X-100. Serial dilutions of homogenates were plated on TSA and incubated for colony formation and enumeration. 2.6. Staphylococcal survival in blood Whole blood was collected from BALB/c mice by cardiac puncture and coagulation was inhibited by adding 10 ␮g ml−1 lepirudin. Fifty microliters of a suspension containing 5 × 105 CFU S. aureus USA300 were mixed with 950 ␮l of mouse blood in the presence of 10 ␮g ml−1 of mAbs. Samples were incubated 30 min at 37 ◦ C with slow rotation, placed on ice with 1% saponin/PBS to lyse blood cells and serially diluted prior to plating on TSA and enumeration of colonies. 2.7. Ethics statement All mouse experiments were performed at least twice and conducted in accordance with institutional guidelines following experimental protocol review and approval by the Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago. 2.8. Statistical analysis Colony counts from infected animals were analyzed with the two-tailed Mann–Whitney test. Unpaired two-tailed Student’s ttests were used to assess the statistical significance of ELISA data and ex vivo blood survival data. All data were analyzed with PrismTM (GraphPad Software, Inc.) and P values less than 0.05 were deemed significant. 3. Results 3.1. Monoclonal antibody mAb 358A76 recognizes the E domain of SpA To determine the affinity constant (Ka = [mAb·Ag]/[mAb] × [Ag]) and protein A binding site for SpA-mAb 358A76, microtiter plates were coated with SpAKKAA , a protein A variant that cannot capture IgG by binding Fc␥ or VH 3 Fab [13]. As control, the SpAKKAA derived mouse monoclonal antibody mAb 3F6 [16] bound with high affinity to SpAKKAA [Ka 22.81(±2.84) × 109 M−1 ] (Fig. 1A and B). We determined an affinity constant Ka 1.95(±0.55) × 109 M−1 for SpA-mAb 358A76, approximately 10 fold less than SpAKKAA mAb 3F6 (P = 0.0004; Fig. 1A and B). To identify the binding site of SpA-mAb 358A76, the five individual IgBDs: EKKAA , DKKAA , AKKAA , BKKAA , and CKKAA were purified [16] and used to coat ELISA plates. Further, we used synthetic peptides representing one (H1, H2, and H3) or two adjacent ␣-helices (H1 + 2 and H2 + 3) of the IgBD-EKKAA triple helical bundle as antigens in the ELISA assay. SpAKKAA -mAb 3F6 bound each of the five IgBDs with similar affinity (Ka 12.41–27.46 × 109 M−1 ). In contrast, SpA-mAb 358A76 bound only to EKKAA (Ka 0.21 × 109 M−1 ) but not to any of the other four IgBDs (DKKAA , AKKAA , BKKAA , and CKKAA ) (Table 1). Further, SpA-mAb 358A76 did not recognize synthetic peptides encompassing one or two ␣-helices of IgBD-EKKAA (H1, H2, H3, H1 + 2,

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Fig. 1. SpA-mAb 358A76 specifically recognizes the E domain of protein A. ELISA examining the association of (A) mAbs 358A76 and (B) 3F6 with the immobilized nontoxigenic SpA variant (SpAKKAA ), each immunoglobulin-binding domain (IgBD-EKKAA , -DKKAA , -AKKAA , -BKKAA , and -CKKAA ), and the synthetic linear peptides derived from the three helices (H1, H2, H3, H1 + 2, and H2 + 3) of IgBD-EKKAA . (C) Alignment of amino acid sequences of the five IgBDs of SpA. Amino acid residues identical to that of IgBD-E (top sequence) are depicted with a dot whereas those unique to the E domain are highlighted in yellow boxes. The four amino acid residues substituted in each IgBD of the non-toxigenic SpAKKAA variant are highlighted in the magenta boxes. (D) Amino acid sequence homology level was compared using ClustalW and the numbers represent the percent of amino acid homology between IgBDs.

and H2 + 3). By comparison, SpAKKAA -mAb 3F6 bound to the helix 1 + 2 peptide, but not to single ␣-helix peptides (H1, H2, and H3) or to the H2 + 3 peptide (Table 1). As reported previously [7], alignment of the amino acid sequences of the five IgBDs of protein A shows that the E domain is most dissimilar to the remaining four IgBDs with both conservative and non-conservative amino acid substitutions in ␣-helix 1 and 3 of the triple-helical fold [6,9] (Fig. 1C and D). The unique amino acid sequences of ␣-helix 1 and 3 of the E domain may explain why SpA-mAb 358A76 selectively binds only the first IgBD. Thus, immunization of mice with wild-type SpA led to the generation of an IgG2a antibody, which recognizes the E domain of protein A with Ka of 1 × 109 M−1 .

3.2. SpAKKAA -mAB 3F6, but not SpA-mAb 358A76, prevents IgG binding to protein A Do the monoclonal antibodies studied here bind protein A with sufficiently high affinity to block its binding to human immunoglobulin, which associates with SpA via its Fc␥ and VH 3 Fab domains with affinities ranging from 0.1 to 1 × 108 M−1 [18,19]? To address this question, we developed a competitive ELISA assay with either SpAKKAA (control) (Fig. 2A) or SpA (Fig. 2B) or the SpA-E domain alone (Fig. 2C) and measured binding of HRP-conjugated antibodies. As a control, IgG2a antibodies did not interfere with the binding of HRP-conjugated SpA-mAb 358A76 or SpAKKAA mAb 3F6 to SpAKKAA (Fig. 2A). Increasing amounts of SpA-mAb

Table 1 Association constants for the binding of mAbs 358A76 and 3F6 to SpAKKAA and its fragments. mAba

Association constant (×109 M−1 ) for antigen or antigen fragment

IgG2a 358A76 3F6c

SpAKKAA 1.00 22.97

IgG binding domains of protein A EKKAA DKKAA AKKAA b b 0.21 17.69 12.41 20.15

BKKAA

CKKAA

Segments of the EKKAA triple-helical bundle H1 H2 H3 H1 + 2

H2 + 3

b

b

b

b

b

b

b

27.46

26.46