Analysis of peptide mimotopes of Burkholderia pseudomallei exopolysaccharide

Available online at www.sciencedirect.com

Vaccine 25 (2007) 7796–7805

Analysis of peptide mimotopes of Burkholderia pseudomallei exopolysaccharide夽,夽夽 Joseph B. Legutki a,b,∗ , Michelle Nelson c , Richard Titball d , Darrell R. Galloway a,e , Alfred Mateczun b , Leslie W. Baillie b,1 b

a Department of Microbiology, The Ohio State University, 484 W 12th Ave Rm 376, Columbus, OH 43210, United States Biological Defense Research Directorate, Naval Medical Research Center, 12300 Washington Ave, Rockville, MD 20852, United States c Defense Science and Technology Laboratory, Porton Down, Salisbury, WILTS SP4 OJQ, UK d School of Biosciences, Geoffry Pope Building, University of Exeter, EX4 4QD, UK e Joint Science & Technology Office, Chemical & Biological Defense Directorate, Defense Threat Reduction Agency, United States

Received 7 March 2007; received in revised form 8 August 2007; accepted 21 August 2007 Available online 14 September 2007

Abstract Previously two capsule-specific monoclonal antibodies (4VA5 and 3VIE5) were identified as protective against Burkholderia pseudomallei in passive transfer experiments. Panning these antibodies against evolutionary phage libraries identified reactive peptides capable of inhibiting its parent monoclonal from binding to B. pseudomallei. Mice immunized with peptide conjugated to thyroglobulin developed serum antibodies capable of recognizing the immunizing peptide of which a subset recognized exopolysaccharide in the context of whole B. pseudomallei cells. These serum antibodies recognized protease treated B. pseudomallei but not B. thailandensis suggesting that these peptides are mimotopes of the B. pseudomallei capsular exopolysaccharide. In a murine model of acute melioidosis, immunization with the mimotope of the 4VA5 binding site extended the mean time to death to 8.00 days over the 2.18 days afforded by immunization with thyroglobulin alone. This mimotope may be of use in developing an antibody response against B. pseudomallei exopolysaccharide. © 2007 Published by Elsevier Ltd. Keywords: Melioidosis; Mimotope; Vaccine

1. Introduction

夽

The views expressed in this article are those of the author and do not necessarily reflect the official policy or position or the Department of the Navy, Department of Defense, nor the U.S. Government. 夽夽 I am a military service member (or employee of the U.S. Government). This work was prepared as part of my official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. ∗ Corresponding author at: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, 1001 S. McClintock Road, P.O. Box 875901, Tempe, AZ 85287, United States. E-mail address: [email protected] (J.B. Legutki). 1 Present address: Cardiff University, Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, Wales, UK. 0264-410X/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2007.08.045

Burkholderia pseudomallei is the etiological agent of melioidosis, a glanders-like disease endemic in Southeast Asia and Northern Australia which accounts for 25% of community acquired sepsis in Thailand and 32% in tropical Australia [1,2]. Primarily a soil saprophyte, the bacterium usually infects individuals via the cutaneous and pulmonary routes following exposure to contaminated soil. However, the bacteria may also be water borne and cases of melioidosis resulting from aspiration of B. pseudomallei containing flood water were recently reported during the Asian tsunami of 2004 [3–5]. The low infectious dose by the aerosol route has resulted in B. pseudomallei being classified by the CDC as a Category B biological threat agent [6].

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

Following infection, the disease can take many forms ranging from an acute self-limiting episode lasting less than 2 months to a chronic disease. In some cases, the bacterium can persist in the host in a latent form where the organism remains dormant for up to 62 years [7]. Currently treatment with antibiotics is the only available therapeutic option. However, the effectiveness of treatment is complicated by the organisms inherent resistance to many classes of antibiotics in addition to being able to become dormant only to reemerge later in life. Currently there is no human vaccine that protects against B. pseudomallei infection. Animal studies have identified a number of potential vaccine targets including flagellin protein, lipopolysaccaride (LPS) and capsule [8]. Signature tagged mutagenesis studies have identified the capsule of B. peudomallei to be a key virulence factor which protects the bacterium from complement deposition [9–11]. The major component of this capsule is an exopolysaccharide (EPS) which consists of an unbranched repeating unit -3)2-O-acetyl-6-deoxy-␤-d-manno-heptopyranose-(1- which is conserved across strains making it an ideal vaccine candidate [10]. Serological analysis of human convalescent patients has identified the presence of antibodies specific for the EPS, which prior to its correct identification as the capsule had been described in the literature as type I O-PS [12]. Purified monoclonal antibodies reactive to EPS can protect mice against B. pseudomallei when administered prior to challenge [13]. Immunization with purified EPS raises low B. pseudomallei specific titers, but only extends the mean time to death in an acute model of murine melioidosis [14]. It is well known that immunization with purified carbohydrate can stimulate short-term immune responses but is unable to confer lasting memory. This is due to the fact that long polymeric carbohydrate antigens are T cell independent in that they directly stimulate B cells in the absence of T cell help. Thus, while infection or active immunization with capsular material results in the generation of exopolysaccharide specific antibodies, affinity maturation does not occur and there is no clonal expansion of antigen specific T memory cells. Capsule based vaccines such as the typhoid vaccine, which comprises purified capsular material derived from Salmonella enterica serovar Typhi (S. typhi), have been licensed for human use. While effective, these vaccines require multiple dosing to maintain protective levels of immunity. While memory responses can be induced by physically conjugating the carbohydrate antigen to a carrier protein this approach suffers from the fact that this chemical process can adversely effect the immunogenicity of the antigen [15]. An alternative strategy is to derive a short peptide known as a mimotope, which either directly mimics the physical structure of the carbohydrate antigen or is physically unrelated but is capable of raising antibodies in vivo which also bind the carbohydrate. The ability of this approach to confer protection against extracellular pathogens has been demonstrated using peptide mimotopes derived from the antibodies spe-

7797

cific for the EPS of Streptococcus pneumoniae and Neisseria meningitidis [16]. There is preliminary data to suggest that this approach may also be effective against intracellular bacterial pathogens such as S. typhi and Shigella flexneri [17,18]. Researchers have been able to use a capsule-specific antibody to generate peptide mimotopes which mimic the Vi capsular polysaccharide of S. typhi and the O-antigen of S. flexneri. It is hypothesized that antibodies raised against these mimotopes will be effective against the initial extracellular phase of infection. While these results are encouraging, there has been as yet no demonstration of protective efficacy using this approach against a pathogen which can invade host cells. To address whether peptide based carbohydrate mimotopes are capable of stimulating a protective immune response against pathogen which has the capability to invade and survive in host cells, we have chosen the etiological agent of melioidosis, B. pseudomallei as a model pathogen. Antibodies raised against these mimotopes are hypothesized to be effective against the initial extracellular phase of infection. Murine monoclonal antibodies 4VA5 and 3VIE5 which recognize the EPS of B. pseudomallei and have been shown to confer 80% and 20% protection, respectively, in animal challenge studies were used to generate mimotopes [8]. Candidate peptides were identified by panning the antibodies against M13 random peptide phage display libraries. From the resulting sequences, synthetic peptides were designed and evaluated for reactivity with 4VA5 and 3VIE5. Mice immunized with peptide conjugates generated predominantly IgG1 sera that recognized carbohydrate from B. pseudomallei. The mimotope of the 4VA5 epitope extended the mean time to death in a murine model of acute melioidosis.

2. Materials and methods 2.1. Strains and antibodies B. pseudomallei ATCC 23343 or Burkholderia mallei ATCC 23344 were grown on Brain Heart Infusion agar and re-suspended in PBS for use as an ELISA antigen. B. pseudomallei NCTC 4845 was used as the live challenge strain. Growth and manipulation of B. pseudomallei and B. mallei were conducted in a BSL-3 containment laboratory in accordance with the appropriate security regulations. Prior to use under BSL-2 conditions, culture-derived material was exposed to 3–4 × 106 total rads of radiation in a cobalt irradiator. Inactivation of whole cells was confirmed by the absence of culture growth following 96 h of incubation. Escherichia coli ER2738 was the F+ strain used for the propagation of the M13 phage. The monoclonal antibodies were previously raised against heat killed B. pseudomallei NCTC 4845 [13]. Corresponding antigens were identified as a protein for 4VH7 and exopolysaccharide for 3VIE5 and 4VA5 [13]. Capsular polysaccharide was isolated from B. pseudomallei strain NCTC 4845 as described previously [14]. Briefly, after

7798

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

extraction the capsular polysaccharide was purified using a monoclonal antibody 3VIE5 affinity chromatography column. Capsular polysaccharide was eluted with 3 ml × 1 ml volumes of sodium phosphate (0.02 M, pH 12) containing 0.5% sodium deoxycholate. The eluted material was neutralized by the addition of NaH2 PO4 . 2.2. Characterization of the relatedness of the 4VA5 and 3VIE5 epitopes The ability of the monoclonal antibodies to interfere with each other’s ability to bind B. pseudomallei cells was measured by a competitive inhibition sandwich ELISA. An Immulon IV microtiter plate was coated overnight with 3VIE5 (100 ng/well) in 0.1 M carbonate/bicarbonate buffer. A second plate was blocked overnight with 5% nonfat milk in PBST. The pre-blocked plate was washed and used to serially dilute 4VA5 in 50 ␮l PBST starting at 20.0 ␮g/ml. Whole irradiated B. pseudomallei cells were added to diluted 4VA5 in 50 ␮l PBST at 0.5 ␮g/ml and allowed to bind for 1 h at 37 ◦ C. The plate coated with 3VIE5 was washed 4 times in PBST and the mixture of 4VA5 and the B. pseudomallei cells was transferred to it. Following 1 h incubation at 37 ◦ C and being washed 4 times in PBST, a mouse IgA specific for B. pseudomallei LPS was added at 125 ␮g/ml for 1 h at 37 ◦ C to detect captured B. pseudomallei cells. The plate was washed 4 times with PBST and an anti-mouse IgA conjugated to HRP (KPL, Gaithersburg, MD) was added at 1:1000 in PBST for 1 h at 37 ◦ C. Following final four washes in PBST, the plate was incubated for 30 min at 37 ◦ C and absorbance read at 405 nm. To assay the interference of 4VA5 binding by 3VIE5, the monoclonals were reversed from what was described above between being coated on the plate and in solution.

2.3. Selection of phage clones displaying the mimotopes Three M13 phage display libraries were used (New England Biolabs, Beverly, MA), the libraries contained either a linear 7 amino acid sequence, a linear 12 amino acid sequence or a 7 amino acid sequence constrained in a loop between the disulfide bond of two flanking cysteines. Libraries were screened using monoclonal antibodies per the manufacturer’s directions. Briefly, individual wells of an Immulon IV microtiter plate (Thermo Labsystems, Franklin, MA) were coated with 2.5 ␮g monoclonal antibody in 100 ␮l of 0.1 M carbonate/bicarbonate buffer overnight at 4 ◦ C. Plates were washed with phosphate buffered saline +0.05% Tween 20 (PBST) and blocked with 0.5% bovine serum albumin in 0.1 M NaHCO3 . Phage were biopanned by incubating 2 × 1011 pfu in each well per round, gently washing away unbound phage, and eluting in 0.2 M glycine–HCl pH 2.2 at room temperature. Eluted phage was neutralized using 1 M Tris–Cl pH 9.1. Phage was amplified per manufacturer’s instructions and subjected to total three rounds of biopanning.

2.4. Immunoblot of reactive phage After three rounds of biopanning, the phage were plated and plaques lifted onto nylon membranes. After blocking for 1 h in 5% nonfat milk in PBST, membranes were washed 3 times in PBST and probed for 2 h with either 3VIE5 or 4VA5 at 1 ␮g/ml. Membranes were then washed and incubated with anti-mouse IgG conjugated to HRP (KPL, Gaithersburg, MD) for 1 h. To develop the immunoblots, membranes were washed 3 times in PBST, 2 in PBS and incubated with TMB (KPL) until a color developed. All washes and incubations were at room temperature. Reactive phage were identified, cored and amplified for further analysis. 2.5. Preparation of phage DNA for sequencing The epitope encoded by reactive phage was determined by DNA sequencing. Reactive phage was amplified in 20 ml cultures for 5 h at 37 ◦ C. Phage were separated from E. coli cells by centrifugation at 10,000 × g and precipitated by mixing the culture supernatant with 1/6 volume of 20% polyethylene glycol 8000 and 2.5 M NaCl. Precipitated phage were recovered by centrifugation at 10,000 × g for 10 min and resuspended in 200 ␮l NaI buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA, 4 M NaI) for 10 min and DNA precipitated by the addition of 500 ␮l absolute ethanol. DNA was washed with 70% ethanol and resuspended in 50 ␮l distilled water. DNA was further purified over an anion exchange column, per the manufacturer’s instructions (QIAGEN, Valencia, CA). DNA was sequenced using Big Dye terminator chemistry on an ABI 3730 (Applied Biosystems, Foster City, CA) using the M13 −96 sequencing primer (NEB). 2.6. Peptide synthesis Core consensus sequences were determined by CLUSTAL-W alignment using the MegAlign Program (DNASTAR, Madison, WI). The peptides used in this study were derived based on the core sequences and flanking Table 1 Mean times to death for mimotope immunized micea,b Immunogenc

MTTDd

B1–TGe

2.62 8.00 3.00 2.18 35.17

G3–TGf B1–TG + G3–TG TG controlg Capsule (crude preparation)h a

Female Balb\c mice, 6 per group. Mice were immunized on days 1, 14 and 28 and challenged on day 63 with an i.p. challenge of 250 MLDs of B. pseudomallei NCTC 4845. c 50 ␮g of the immunogen in PBS in a 1:1 ratio of Alum were given i.p. d Mean time to death was calculated as the average last day a mouse was observed alive. e Peptide B1 conjugated to thyroglobulin by glutaraldehyde. f Peptide G3 conjugated to thyroglobulin by glutaraldehyde. g Glutaraldehyde treated thyroglobulin. h Crude capsule preparation also included LPS and protein. b

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

sequences based on the pIII sequence. Peptides were synthesized with an N-terminal acetyl group to improve recognition by the antibody. Lysine was added near the C-terminal of the peptide to facilitate conjugation to a carrier protein. Peptides were synthesized by EvoQuest Services at 90% purity (Invitrogen, Carlsbad, CA). 2.7. Inhibition assays The ability of the peptide to inhibit binding of a monoclonal antibody to antigen was measured using an inhibition ELISA assay. Duplicate wells of an Immulon IV (Thermo Labsystems) microtiter plate were coated overnight with 100 ng/well antigen in 0.1 M carbonate/bicarbonate buffer. Diluted monoclonal antibody was preincubated for 2 h at 37 ◦ C on a separate pre-blocked plate with serially diluted (two fold) peptide beginning at 0.5 mg/ml. The final concentration of monoclonal antibody used was determined as the most dilute concentration capable of giving a maximum absorbance when detected by an immunoglobulin specific conjugated antibody. After incubation, the monoclonal and peptide mixture was transferred to the antigen coated plate which was blocked with 5% nonfat milk in PBST for 2 h and washed 4× in PBST. Uninhibited monoclonal antibody was allowed to bind by incubating for 1 h at 37 ◦ C. B. pseudomallei bound antibody was detected after washed 4× in PBST and incubated 1 h at 37 ◦ C with anti-mouse IgG conjugated to HRP (KPL) at 500 ng/ml. Plates were washed 4× in PBST and developed with ABTS (KPL) for 30 min at 37 ◦ C and A405 read. Inhibition curves of the absorbance versus concentration of peptide were plotted. 2.8. Conjugation of peptides Synthetic peptides were conjugated to either thyroglobulin for immunization or BSA for analysis of peptide specific immune sera. Five milligrams of peptide were combined with 5 mg of carrier protein for 1 h at room temperature in the presence of 0.15% glutaraldehyde. Glycine was added to a final concentration of 200 mM to ensure complete reaction of remaining glutaraldehyde [17]. Conjugated peptides were dialyzed against four changes of PBS using a 10,000 molecular weight cut off slide-a-lyser cassette (Pierce, Rockford, IL). 2.9. Immunization of mice Female BALB\c mice from Jackson Laboratories (Bar Harbour, ME) were immunized in groups of ten with either 50 ␮g of peptide conjugated to thyroglobulin, glutaraldehyde treated thyroglobulin or 25 ␮g/ml of a crude capsule preparation in a total of 100 ␮l PBS/Alum (1:1 ratio) per mouse. Immunizations were on days 0 and 14 with venous blood collected from the tail on days 0, 13 and 28. The experiments reported herein were conducted in compliance with the Animal Welfare Act and according to the principles set forth in the “Guide for the Care and Use of Laboratory Ani-

7799

mals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 1996. 2.10. Quantitation of antigen specific antibodies Microtiter plates were coated with 100 ng/well antigen in 0.2 M carbonate buffer overnight at 4 ◦ C. Wells were blocked with 5% nonfat milk in PBST. Serially diluted sera in PBST, was incubated for 1 h at 37 ◦ C. Plates were washed 4× with PBST then incubated for 1 h at 37 ◦ C with horse radish peroxidase labeled anti-mouse IgG 1:3000 (KPL, Gaithersburg, MD). After washing, plates were incubated with ABTS substrate (KPL) for 30 min at 37 ◦ C. The substrate reaction was stopped with the addition of 50 ␮l of 1% SDS. Absorbances read at 405 nm on a microtiter plate reader. Absorbances were compared to a standard curve generated against known concentrations of Mouse IgG (Sigma, St. Louis, MO). Values are presented as the average of triplicate assays in ␮g/ml sera. IgG subclass titers were determined similarly using isotype matched reagents from Zymed (South San Francisco, CA). 2.11. Determination of antibody affinity in a chaotropic ELISA Antibody avidity was determined in an ELISA by comparing the binding of a primary antibody at a constant concentration in the presence of increasing concentrations of chaotropic salt. The ELISA was run as described above with the following modifications. Immune sera or monoclonal antibody was diluted to a concentration determined above to give an absorbance between 0.4 and 0.7 for 1 h at 37 ◦ C. Plates were washed 4× with PBST and incubated for 15 min with NaSCN at 0.0, 0.25, 0.5, 0.75, 1.0, 1.5 and 3.0 M. Average absorbances from triplicate wells were plotted against NaSCN concentration and linear regression used to determine the affinity constant which is defined as the concentration of NaSCN at which 50% of the antibody remains bound [19]. 2.12. Challenge of mice For challenge, groups of mice were given a 0.1 ml suspension i.p. containing 4.7 × 104 cfu ml−1 of B. pseudomallei NCTC 4845 and observed for up to 5 weeks post-challenge. Once challenged the animals were handled under containment level 3 conditions within a half-suit isolator compliant with British home office regulations.

3. Results 3.1. Determination of the monoclonal antibody site relatedness To determine if individual monoclonals recognized a similar site on the EPS, the monoclonals were evaluated for their ability to prevent each other’s ability to capture whole B.

7800

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

Fig. 1. Monoclonal antibodies inhibit each others capture of whole B. pseudomallei cells in a sandwich ELISA, (A) inhibition of 3VIE5 and (B) inhibition of 4VA5. Whole irradiated B. pseudomallei cells were preincubated with either 3VIE5, 4VA5 to demonstrate self-interference, purified mouse IgG from Sigma, or 4VH7 an unrelated antibody that binds a surface protein of B. pseudomallei prior to use as the captured antigen in a sandwich ELISA. Captured cells were detected using an anti-B. pseudomallei LPS IgA antibody, followed by an anti-mouse IgA conjugated to HRP Average absorbance at 405 nm is plotted from duplicate wells and is representative of duplicate experiments.

pseudomallei cells. When 3VIE5 was used as the capture monoclonal, the amount of captured B. pseudomallei cells was reduced as the amount of 4VA5 pre-bound to the cells increased (Fig. 1A). Similarly, when 4VA5 was used as the capture monoclonal, capture of B. pseudomallei was reduced as the amount of pre-bound 3VIE5 increased (Fig. 1B). The presence of 4VH7 bound to the surface of the B. pseudomallei cells did not inhibit capture of cells by either monoclonal nor did the inclusion of irrelevant purified mouse IgG. In a separate experiment, neither 4VA5 nor 3VIE5 interfered with the binding of the IgA used to detect the captured cells (data not shown). The mutual ability to prevent capture of B. pseudomallei cells indicates that the epitopes recognized by 4VA5 and 3VIE5 are at least partially overlapping.

as the immobilized targets. Three rounds of panning produced a total of 18 reactive phages against each monoclonal antibody as determined by immunoblot. Antibody-reactive phages were selected from the 7 mer library that was constrained by the disulfide bond. No reactive phage resulted from biopanning against the 12 mer or the unconstrained 7 mer libraries. To ensure that these phages were recognized by the variable region of the antibody, each phage was probed against an irrelevant isotype matched antibody and the cutoff absorbance was defined as twice that of the nonspecific antibody (data not shown). A total of that 12 phage were identified with specificity to 4VA5 while a further 8 phages were bound by 3VIE5. The DNA sequences of the binding region of each phage are shown in Fig. 2. Consensus sequences were determined for each monoclonal using the CLUSTAL-W algorithm [18] (Fig. 2). The consensus sequence for 3VIE5 was CYLPFQLSC while the sequence for 4VA5 was CHPLFDARC. The amino acid compositions of our mimotopes are similar to the compositions of carbohydrate mimics reported in the literature. Aromatic and hydrophobic amino acids are the main residues found in peptide mimics of carbohydrates. Crystallographic studies of a peptide mimic of the Lewis Y antigen indicate that the aromatic residues are involved in van der Waals interactions and the charged residues are involved in hydrogen bonding with the antibody variable region residues [20]. Each nine amino acid consensus mimotope sequence contains a phenylalanine at the fifth position as the only aromatic amino acid in the G3 mimotope while mimotope B1 also contains a tyrosine. Both contain hydrophobic amino acids. Peptide G3 contained two charged amino acids arginine and aspartate, which are prevalent charged amino acids found in carbohydrate mimics [21]. Each mimotope contained a proline residue which may add to the constrained structure imposed by the disulfide bond. Proline residues have been reported in cysteine restricted mimotopes of other carbohydrates [22]. 3.3. Design of synthetic peptide mimotopes To accommodate the potential for the involvement of phage flanking residues in mimicry of the carbohydrate, synthetic peptides were designed to include amino acids from the N-terminal end of the mature pIII phage protein. Using the template ACXXXXXXXCGGKS the following peptides were synthesized: peptide B1 (ACYLPFQLSCGGKS) which includes the 3VIE5 reactive sequence and peptide G3 (ACHPLFDARCGGKS) which includes the 4VA5 consensus sequence. The lysine at the C-terminal of each peptide was included to facilitate conjugation to the carrier protein.

3.2. Biopanning of a phage displayed peptide library using monoclonal antibodies 4VA5 and 3VIE5

3.4. Ability of mimotypes to inhibit the parent mAb from binding B. pseudomallei

Three different M13 random phage display libraries were biopanned using the monoclonal antibodies 4VA5 or 3VIE5

The ability of peptides B1 or G3 to inhibit the binding of monoclonal antibody to B. pseudomallei EPS was evaluated

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

7801

Fig. 2. Alignment from immunoreactive phage recognized by (a) 3VIE5 and (b) 4VA5. DNA from reactive phage was purified and sequenced by Big Dye Terminator Sequencing on an ABI 3730 using the NEB M13 −96 sequencing primer. There resulting sequences were aligned using the ClustalW algorithm in the DNAStar MegAlign program. The identical residues are boxed.

in an ELISA against whole irradiated B. pseudomallei cells. When pre-incubated with the monoclonal antibodies prior to the ELISA, both mimotopes specifically inhibited their parent monoclonal from binding (Fig. 3). Neither monoclonal was inhibited by the other mimotope nor the control peptide. The control peptide consensus sequence is unrelated but shares the motif ACXXXXXXXCGGGKS suggesting that the flanking amino acids are not directly involved in binding to the monclonals. In a chaotropic ELISA, the relative avidities of 3VIE5 for B1 was (0.39) and 4VA5 for G3 was (0.42). 3.5. Immunogenicity of peptide conjugates in mice To enhance the immunogenity of the synthetic peptides they were conjugated to thyroglobulin via the C-terminal lysine and assessed for their abilities to induce the production of antigen specific antibodies in BALB\c mice. In May et

al, it was demonstrated that carbohydrates on KLH, the preferred carrier, generated serum antibodies which interfered with the anti-capsule antibodies generated by the peptide mimotopes [23]. Thyroglobulin was chosen based on our laboratory’s prior use of thyroglobulin to generate peptide specific antibodies. Prior to conjugation, no reactivity to thyroglobulin was seen in an ELISA using either monoclonal or serum from naive BALB/c mice (data not shown). Following immunization, the ability of immune sera to recognize individual peptides and capsular material from B. pseudomallei and B. mallei was determined by ELISA. The IgG response from the immunized mice was specific for the immunizing peptide. Mice immunized with the G3 conjugate had a geometric mean titer of 43.11 ␮g/ml and mice immunized with the B1 conjugate had a geometric mean titer of 27.0 ␮g/ml. No cross-reactivity between groups or reactivity of serum from glutaraldehyde treated thyroglobulin immunized mice for the peptides was observed. Serum antibodies

Fig. 3. Inhibition of parent monoclonal antibody binding by peptide mimotopes. The monoclonal antibodies 4VA5 (a) and 3VIE5 (b) diluted to 32.5 ␮g/ml were preincubated with either G3, B1 or a control peptide prior to an ELISA vs. whole irradiated B. pseudomallei cells. The control peptide consensus sequence is unrelated but shares the motif ACXXXXXXXCGGGKS. Points plotted represent the average absorbance at 405 nm of duplicate wells. Results are representative of two independent experiments. Error bars represent the standard deviation. The point corresponding to 1.0 on the X axis does not contain inhibitor.

7802

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

were primarily IgG1 and recognized both B. pseudomallei and B. mallei whole cells but not B. thailandensis (data not shown). Any response to the thyroglobulin carrier was negated by coating the ELISA wells with peptide conjugated to BSA. No thyroglobulin specific antibodies were detected in pre-immune sera. After immunization anti-thyroglobulin antibodies were detected, and were comparable across groups (data not shown). 3.6. The role of mimotope secondary structure in antibody recognition A mimotope is a peptide which mimics either the physical structure or the immunogenicity of an antigen. To confirm that the antisera from peptide-conjugate immunized animals was able to specifically recognize correctly folded peptide we probed peptide–BSA conjugates that had been treated with ␤-mercaptoethanol which disrupts a disulfide bond essential for maintaining the three dimensional structure of the peptide. Breaking the intrapeptide disulfide bond significantly reduced the binding of the parent monoclonals to the respective mimotope. Binding of 3VIE5 to B1 was reduced by 91% (p = 2 × 10−5 ) and 4VA5 binding to G3 was reduced by 98% (p = 2.45 × 10−8 ). 3.7. Specificity of peptide-conjugate immune serum for B. pseudomallei The ability of soluble peptide to inhibit the binding of immune serum to B. pesudomallei cells was determined using the previously described inhibition assay. At a serum concentration of 1:500 and a peptide concentration of 500 ␮g/ml, peptide G3 completely inhibited binding of sera from G3 conjugate immunized mice to B. pseudomallei cells and peptide B1 gave 80% inhibition of B1 conjugate immunized mice from binding B. pseudomallei. Neither peptide had an effect on the other serum’s binding ability. Each peptide was able to inhibit the binding of the corresponding anti-sera to the bacteria confirming that the anti-B. pseudomallei effect of the sera was due to antibodies raised by the peptide conjugates. Neither thyroglobulin nor the control peptide was able to inhibit immune serum antibodies from either group from binding B. pseudomallei cells (data not shown). To determine the nature of the B. pseudomallei antigen recognized by immune sera the ability of sera from immunized mice to recognize untreated or pronase digested B. pseudomallei cells was determined. Digestion of cellular protein was confirmed by gel electrophoresis and staining (data not shown). As expected, digestion with pronase dramatically reduced the ability of the monoclonal antibody 4VH7 to bind to the treated bacteria suggesting that it recognized a protein based epitope. In contrast pronase treatment had no effect on the binding of the parent monoclonals 4VA5 and 3VIE5 or antisera from corresponding peptideconjugates suggesting that they recognized a non-protein based epitope (data not shown).

Fig. 4. Survival curves of mice mimotope immunized mice. Female BALB/c mice were immunized 6 mice per group with either 50 ␮g mimotope conjugated to thyroglobulin, both mimotope conjugated to thyroglobulin, glutaraldehyde treated thyroglobulin, or 2.5 ␮g crude capsule preparation. The group receiving B1 and G3 conjugates received 25 ␮g each conjugate. Immunizations were given in a 1:1 ratio with Alum (Pierce, Rockford, IL) on days 0, 14 and 28. Mice were challenged on day 63 with an i.p. injection of 250 MLD of B. pseudomallei 4845. Survival was monitored on a daily basis and the number of survivors plotted.

3.8. Evaluation of mimotopes in a mouse model of melioidosis The protective efficacy of the exopolysaccharide mimotopes was evaluated in a mouse model of acute melioidosis. Mice were immunized on three occasions at a 2 weeks interval and were challenged 35 days after the last immunization with 250 mean lethal doses of B. pseudomallei NCTC 4845. The challenge schedule and dose were chosen to allow the results to be directly comparable to previous immunization with purified capsule in an acute model of disease [14]. B. pseudomallei causes deaths at doses lower than 104 cfu, however at these lower doses, there is an increased likelihood that some animals will present with a chronic form of the disease, rather than a more acute infection. Challenge 35 days after the last immunization negates the potential of non-specific protective effects from the adjuvant. Animals which received crude outer membrane preparation as a positive control survived to the end of the study (Fig. 4). These animals also demonstrated the highest IgG specific antibody response immediately prior to challenge (Fig. 5). Negative control mice immunized with glutaraldehyde treated thyroglobulin did not survive. In contrast, of the animals which received the peptide conjugates, only those immunized with the G3-thyroglobulin showed any protection with an extension of mean time to death from 2.18 days (TG control) to 8.0 days (Table 1) p = 0.09. IgG isotype analysis of the day of challenge sera gave IgG1 :IgG2a ratios of 2.94 for B1 conjugate immunized mice and 2.43 for G3 immunized mice which are indicative of a Th2 biased response. The response in the mice immunized with both mimotopes was more balanced with a ratio of 1.34 (Fig. 6). B. pseudomallei specific IgM and IgG3 mainly seen in the group immunized with the outer membrane preparation (Fig. 6) which is indicative of EPS being processed in an T independent manor. No IgG3 was raised in the

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

Fig. 5. Day of challenge anti-B. pseudomallei IgG geometric mean titers. Wells were coated with irradiated B. pseudomallei ATCC 23343. Day 63 sera from mice immunized with three immunizations on days 0, 14 and 28 were assayed in an ELISA. Values plotted the group geometric means and error bars represent the variation of individual titers within the group (B1 vs. TG; p = 0.006) (G3 vs. TG; p = 0.0075) and (B1 and G3 vs. TG; p = 5.11 × 10−11 ).

Fig. 6. Day of challenge B. pseudomallei specific antibody subclasses (A) IgM and (B) IgG isotype. Wells were coated with irradiated B. pseudomallei ATCC 23343. Day 63 sera from mice immunized with three immunizations on days 0, 14 and 28 were assayed in an ELISA. Values plotted are the values from pooled samples and error bars represent the standard deviation of triplicate samples.

7803

G3 immunized mice and a small amount (400 ng/ml) was raised in the B1 immunized mice (Fig. 6). IgM in the peptide immunized groups was equivalent to the thyroglobulin controls. Analysis of the B. pseudomallei specific antibody responses prior to challenge revealed that while the animals immunized with the B1 conjugate had twice the level of specific IgG of the animals given the G3 conjugate, there was no evidence of protection. Affinity analysis of the day of challenge sera indicated that mice immunized with the G3 conjugate had affinities for B. pseudomallei nearly twice that of mice immunized with the B1 conjugate (0.82 vs. 0.47). Comparison of the affinities of the monoclonal antibodies for B. pseudomallei, indicated that 4VA5, the parent monoclonal of G3, had a higher affinity than 3VIE5 (2.15 vs. 0.87).

4. Discussion Previous vaccine studies using peptide mimotopes of carbohydrate antigens have focused on viruses and extracellular bacteria. In this preliminary report we describe the efficacy of a peptide based carbohydrate mimotope against an intracellular bacteria. To be considered an immunological mimotope of a polysaccharide, a peptide must meet two criteria. First it should be able to inhibit the raising monoclonal from binding the target carbohydrate and second, antibodies raised by immunization with the peptide should recognize the carbohydrate [15]. In this experiment, synthetic peptides were developed and found capable of inhibiting the monoclonals 4VA5 and 3VIE5 from binding B. pseudomallei. Mice immunized with peptide–thyroglobulin conjugates generated sera capable of recognizing B. pseudomallei. Recognition of pronase treated cells implies that the antibody response generated by the mimotopes is to a carbohydrate component of B. pseudomallei. Inhibition of immune serum from binding B. pseudomallei by the immunizing mimotope indicates that the antibody response to B. pseudomallei was a product of immunization with the mimotopes. Taken together the data demonstrates peptides B1 and G3 are mimotopes of B. pseudomallei exopolysaccharide. The immunization of mice with purified capsular polysaccharide has previously been shown to extend the time to death after challenge with B. pseudomallei [14]. In this study the immunization of mice with peptide G3 also provided protection against experimental melioidosis, evidenced as an extended mean time to death. However, the degree of protection afforded after immunization with the peptide G3 was lower than that afforded after immunization with the capsular polysaccharide. Protection afforded after immunization with the crude capsule preparation may have been enhanced by other protein and LPS antigens present in the preparation. The relative efficacy of each peptide is related to the passive protection level of the generating monoclonal. Mimotope G3 was generated by 4VA5, the 80% protective antibody, and B1 was raised by 3VIE5 which was 20% passive protective.

7804

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

Despite binding an overlapping site on the capsular EPS, the monoclonal antibodies generated distinctive mimotopes, which did not generate cross-reactive serum antibodies. Previous studies using monoclonals to the same epitope have developed distinct non-cross-reactive mimotopes. This is most likely due to the spatial orientation of the bound monoclonals and the preference of the variable regions for one amino acid sequence over another [24]. Overlap in the EPS epitopes mimicked, may have contributed to the lack of protection in mice immunized with both mimotopes. Also this group contained half the amount of each mimotope conjugate as the individual groups resulting in lower titers of antibodies independently raised by both B1 and G3. Reduction of the number of mimotopes present, combined with competition between the 3VIE5-like and 4VA5-like antibodies for the same binding site would have decreased the expected benefit of the higher IgG titer seen in the co-immunized group. This suggests that as means are sought to improve the anti-EPS titer, only the more effective G3 mimotope should be used. Of the two mimotopes developed, peptide G3 was more effective than peptide B1 at inducing the production of antibodies with qualities similar to the parent monoclonal. Following challenge with B. pseudomallei, mice immunized with peptide G3 also had an extended mean time to death compared to the B1 immunized mice. The G3 mimotope was more effective than B1 due to the relative effectiveness of the parent monoclonal antibodies, the isotype of serum antibodies raised, and the affinity of serum antibodies raised. Antisera raised against peptide G3 was mostly IgG1 , the same subclass as its corresponding monoclonal antibody. Sera raised against peptide B1 was also predominately IgG1 , but differed in subclass from its corresponding IgG2a monoclonal 3VIE5. The ability of the mimotope to raise antibodies of the same isotype as the monoclonal is important for protection as other antibodies of different isotypes which recognize identical epitopes may not be protective [25]. This requirement for identical isotype may be related to the ability of the constant domain to influence the fine specificity of the variable region [26]. Cooper et al. have shown that the cooperative binding ability of IgG3 has the greatest effect on affinity however there is a slight difference between IgG1 and IgG2b [27]. In terms of the influence of isotype on the effectiveness of mimotopes, the difference may be in the functional role of the constant domain. Functionally, these antibodies may contribute to protection against melioidosis through the ability of IgG1 antibodies to facilitate opsonization [14]. Antibodies specific for EPS, then described as OPS-I, were demonstrated to facilitate phagocytic killing of B. pseudomallei by polymorphonuclear cells [12]. In the study by Nelson et al., the EPS immunized mice generated primarily IgG2b antibodies whereas our peptide immunized mice generated primarily IgG1 [14]. Immunization with the G3 peptide extended the MTTD of mice challenge with B. pseudomallei more than immunization with B1 peptide because of the higher avidity of the sera

for B. pseudomallei. This may reflect the higher avidity of 4VA5 than 3VIE5 for B. pseudomallei. The day of challenge, the B1 immunized mice had an anti-B. pseudomallei IgG titer twice that of G3 immunized mice. While more B. pseudomallei reactive antibodies were available in mice immunized with B1, the higher avidity of the antibodies in G3 immunized mice appears to have contributed to the extension of the MTTD. Olszewska et al. demonstrated the effect of affinity on the ability of mimotope raised sera to passively protect in a challenge. In that study, high affinity sera gave 100% protection against a viral challenge while equal titered low affinity sera gave only 50% protection [28]. This correlates with the differences in affinity between the two parent monoclonals and their relative passive protection at an equal dose [13]. Despite the mimotope G3 extending the MTTD, it did not provide the level of survival the original monoclonal did in the passive protection studies [13]. This might indicate that the level of antibody was insufficient to prevent the invasion of host cells. In the original passive protection studies, mice were given 40 ␮g of 4VA5 per mouse [13]. Mice immunized in this study with the G3 conjugates had B. pseudomallei specific titers that were 15 times less than the calculated serum dose in monoclonal protected mice on the day of challenge. Future studies involving this mimotope will need to involve an immunization scheme capable of raising the capsule-specific titer. Low carbohydrate specific serum antibody titers in the presence of high titers to the peptide mimotope have also been observed by others [29]. The peptide is a three dimensional structure which can interact with the immune system from multiple orientations. Only one orientation is expected to fit into the binding region of an antibody capable of also recognizing EPS and lead to expansion of that B cell clone. This orientation corresponds to how the peptide fits within the binding pocket of the original antibody. Based on the inhibition curves, it would be expected that the more effective inhibitor, B1 would also be the more effective mimotope, however G3 was more effective in extending the MTTD. The most likely explanation is that the G3 parent monoclonal 4VA5 is the more effective passive protective monoclonal. The apparent counterintuitive nature of the abilities of B1 and G3 to inhibit their respective parent mAb can be explained when the avidities of the mAbs for B. pseudomallei are considered. 4VA5 has a binding avidity 2.5 times that of 3VIE5 for B. pseudomallei cells while avidities of the parent monoclonal for the peptides were equivalent (4VA5 for G3: 0.39 and 3VIE5 for B1: 0.42). In this case 4VA5 had an affinity 5.5 times for the B. pseudomallei cells than the peptide (2.15 vs. 0.39) and 3VIE5 had an affinity twice that of the peptide for B. pseudomallei (0.87 vs. 0.42). Therefore, it is logical that the peptide mimotopes would be more effective at inhibiting the parent monoclonal with the lower avidity for B. pseudomallei than the monoclonal with the higher avidity. Since the avidity of the parent monoclonals, are equivalent for the peptides, B1 is simply an inferior mimotope biolog-

J.B. Legutki et al. / Vaccine 25 (2007) 7796–7805

ically, which fits the lesser ability of 3VIE5 to extend the MTTD. It is likely that an effective vaccine against B. pseudomallei infection will need to raise a strong humoral response to interdict extracellular bacteria and a cytotoxic T cell response to eliminate bacteria which have invaded host cells. Alternative means of presenting the G3 mimotope may be capable of raising the titer of 4VA5-like antibodies in immune sera. An improved titer of these G3 raised antibodies would be available to bind EPS upon entry of the bacterium to the body and reduced the number of bacteria required to be cleared by CTLs. Whether or not co-immunization with G3 improves protection seen with a CTL antigen remains to be tested. With further development, the exopolysaccharide mimotopes described in this work could provide the humoral component of a vaccine based defense against melioidosis.

[12]

[13]

[14]

[15] [16] [17] [18]

Acknowledgements

[19]

This work was supported by funded by work unit number 80000.000.000.A0031.

[20]

References [21] [1] Douglas MW, Lum G, Roy J, Fisher DA, Anstey NM, Currie BJ. Epidemiology of community-acquired and nosocomial bloodstream infections in tropical Australia: a 12-month prospective study. Trop Med Int Health 2004;9(7):795–804. [2] Chaowagul W, White NJ, Dance DA, Wattanagoon Y, Naigowit P, Davis TM, et al. Melioidosis: a major cause of communityacquired septicemia in northeastern Thailand. J Infect Dis 1989;159(5): 890–9. [3] Chierakul W, Winothai W, Wattanawaitunechai C, Wuthiekanun V, Rugtaengan T, Rattanalertnavee J, et al. Melioidosis in 6 Tsunami survivors in southern Thailand. Clin Infect Dis 2005;41(7):982–90. [4] Kongsaengdao S, Bunnag S, Siriwiwattnakul N. Treatment of survivors after the tsunami. N Engl J Med 2005;352(25):2654–5. [5] Allworth AM. Tsunami lung: a necrotising pneumonia in survivors of the Asian tsunami. Med J Aust 2005;182(7):364. [6] Currie BJ, Jacups SP, Cheng AC, Fisher DA, Anstey NM, Huffaman SE, et al. Melioidosis epidemiology and risk factors from a prospective whole-population study in northern Australia. Trop Med Int Health 2004;9(11):1167–74. [7] Ngauy V, Lemeshev Y, Sadkowski L, Crawford G. Cutaneous melioidosis in a man who was taken as a prisoner of war by the Japanese during World War II. J Clin Microbiol 2005;43(2):970–2. [8] Warawa J, Woods DE. Melioidosis vaccines. Expert Rev Vacc 2002;1(4):477–82. [9] Atkins T, Prior R, Mack K, Russell P, Nelson M, Prior J, et al. Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol 2002;51(7): 539–47. [10] Reckseidler SL, DeShazer D, Sokol PA, Woods DE. Detection of bacterial virulence genes by subtractive hybridization: Identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Infect Immun 2001;69(1):34–44. [11] Reckseidler-Zenteno SL, DeVinney R, Woods DE. The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

7805

serum by reducing complement factor C3b deposition. Infect Immun 2005;73(2):1106–15. Ho M, Schollaardt T, Smith MD, Perry MB, Brett PJ, Chaowagul W, et al. Specificity and functional activity of anti-Burkholderia pseudomallei polysaccharide antibodies. Infect Immun 1997;65(9):3648–53. Jones SM, Ellis JF, Russell P, Griffin KF, Oyston PC. Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins. J Med Microbiol 2002;51(12):1055–62. Nelson M, Prior JL, Lever MS, Jones HE, Atkins TP, Titball RW, et al. Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis. J Med Microbiol 2004;53(12):1177–82. Pirofski LA. Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens. Trends Microbiol 2001;9(9):445–51. Lindberg AA. Glycoprotein conjugate vaccines. Vaccine 1999;17(Suppl. 2):S28–36. Harlow E, Lane D. Antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1988, 726. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 1994;22(22):4673–80. Pullen JR, Fitzgerald MG, Hosking CS. Antibody avidity determination by ELISA using thiocyanate elution. J Immunol Methods 1986;86(1):83–7. Young AC, Valadon P, Casadevall A, Scharff MD, Sacchettini JC. The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes. J Mol Biol 1997;274(4):622–34. Luo P, Adagjanyan M, Qiu J, Westerink J, Stephlewski Z, Kieber-Emmons T. Antigenic and immunological mimicry of peptide mimotopes of Lewis carbohydrate antigens. Mol Immunol 1998;35(13):865–79. De Bolle X, Laurent T, Tibor A, Godfroid F, Weynants V, Letesson JJ, et al. Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella. J Mol Biol 1999;294(1):181–91. May RJ, Beenhouwer DO, Scharff MD. Antibodies to keyhole limpet hemocyanin cross-react with an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development. J Immunol 2003;171(9):4905–12. Xu Y, Ramsland PA, Davies JM, Scealy M, Nandakumar KS, Holmadahl R, et al. Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry. Mol Immunol 2004;41(4):411–9. Buchwald UK, Lees A, Steinitz M, Pirofski LA. A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice. Infect Immun 2005;73(1):325–33. Cooper JN, Shikhman AR, Glass DD, Kangisser D, Cunningham MW, Greenspan NS. Role of heavy chain constant domains in antibody-antigen interaction: Apparent specificity differences among Streptococcal IgG antibodies expressing identical variable region domains. J Immunol 1993;150(6):2231–42. Cooper JN, Robertson D, Granzow R, Greenspan NS. Variable domainidentical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance. Mol Immunol 1994;31(8):577–84. Olszewska W, Obeid OE, Steward MW. Protection against measles virus-induced encephalitis by anti-mimotope antibodies: the role of antibody affinity. Virology 2000;272(1):98–105. Beenhouwer DO, May RJ, Valadon P, Sharff MD. High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library. J Immunol 2002;169(12):6992–9.