Oral immunization using HgbA in a recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261 in the temperature-dependent rabbit model

Journal of Immunological Methods 375 (2012) 232–242

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Research paper

Oral immunization using HgbA in a recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261 in the temperature-dependent rabbit model Cathy Breau a, D. William Cameron a, b, Marc Desjardins c, B. Craig Lee a, b,⁎ a b c

Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 Department of Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 Department of Laboratory Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5

a r t i c l e

i n f o

Article history: Received 30 August 2011 Received in revised form 3 November 2011 Accepted 3 November 2011 Available online 10 November 2011 Keywords: Chancroid Haemophilus ducreyi HgbA Vaccine strain

a b s t r a c t Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of HIV. An effective vaccine against chancroid has not been developed. In this preliminary study, the gene encoding the H. ducreyi outer membrane hemoglobin receptor HgbA was cloned into the plasmid pTETnir15. The recombinant construct was introduced into the attenuated Salmonella typhimurium SL3261 strain and stable expression was induced in vitro under anaerobic conditions. The vaccine strain was delivered into the temperature-dependent rabbit model of chancroid by intragastric immunization as a single dose, or as three doses administered at two-weekly intervals. No specific antibody to HgbA was elicited after either dose schedule. Although the plasmid vector survived in vivo passage for up to 15 days following single oral challenge, HgbA expression was restricted to plasmid isolates recovered one day after immunization. Rabbits inoculated with the 3-dose booster regimen achieved no protective immunity from homologous challenge. These results emphasize that refinements in plasmid design to enhance a durable heterologous protein expression are necessary for the development of a live oral vaccine against chancroid. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chancroid (Trees and Morse, 1995; Schmid, 1998; Ronald and Albriton, 1999; Bong et al., 2002) is a sexually transmitted genital ulcer disease (GUD) caused by the Gram-negative bacterium Haemophilus ducreyi. The infection is common in many African, Asian and the Caribbean countries, where the organism causes 13–62% of GUD in sexually transmitted disease (STD) clinic populations (Behets et al., 1999; Risbud et al., 1999; Chen et al., 2000; Totten et al., 2000; Hoyo et al., 2005; Al-Mutairi et al., 2007). The outspoken biological importance of H. ducreyi infection has been shown in several studies ⁎ Corresponding author. Tel.: + 1 613 737 8899x74765; fax: + 1 613 737 8124. E-mail address: [email protected] (B.C. Lee). 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.11.002

in which chancroid facilitates the transmission of HIV (Cameron et al., 1989; Plummer et al., 1991; Wasserheit, 1992; Korenromp et al., 2001). H. ducreyi exhibits an absolute requirement for hemin that can be fulfilled by supplying the organism with bovine hemoglobin, human hemoglobin, and bovine catalase (Lee, 1991). The initial step in the acquisition of heme from hemoglobin involves the specific interaction of hemoglobin with a cognate outer membrane receptor, termed HgbA (Elkins, 1995; Elkins et al., 1995) or HupA (Stevens et al., 1996). The protein is an important H. ducreyi virulence determinant as an isogenic hgbA mutant displays an attenuated ability to cause disease in both the human challenge (Leduc et al., 2008) and temperature dependent rabbit models of chancroid (Al-Tawfiq et al., 2000). These properties coupled with the structural and functional conservation of HgbA between

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the 2 classes of H. ducreyi strains (Elkins, 1995; White et al., 2005), and with the protection afforded against chancroid following HgbA immunization in the experimental swine model of H. ducreyi infection (Afonina et al., 2006), supply the conceptual underpinnings for an HgbA-based vaccine. Such a vaccine targeted to groups of high-frequency STD transmitters, could both eliminate chancroid and decrease the incidence of HIV infection. The ideal vaccine would require no syringes, no cold chain distribution and single dose delivery. Live oral attenuated Salmonella vaccine vectors fulfill these requirements. By mimicking natural infections, they generate mucosal, humoral and cellular immune responses (Kotton and Hohmann, 2004). Furthermore, they act as intrinsic adjuvants because of the immunostimulatory molecules, such as lipopolysaccharide (LPS) and flagella, located on their cell surface (Kotton and Hohmann, 2004). Other advantages of using live attenuated bacteria as vaccine vectors include their simple and low cost of production, their stability without refrigeration (via lyophilization), their simple and safe oral administration, and their potential for increasing compliance through single dose vaccination (Burke et al., 1999; Kotton and Hohmann, 2004). Several attenuated Salmonella oral vaccines have been constructed to deliver heterologous antigens from other pathogens, such as HIV (Kotton et al., 2006), Eimeria tenella (Pogonka et al., 2003), Streptococcus mutans (Huang et al., 2000), Helicobacter pylori (Gomez-Duarte et al., 1998; Angelakopoulos and Hohmann, 2000) hepatitis B virus (Nardelli-Haefliger et al., 1996), Vibrio cholerae (Attridge et al., 1991), enterotoxigenic Escherichia coli (ETEC) (Ascon et al., 1998; Khan et al., 2007), Bacillus anthracis (Stokes et al., 2007), Clostridium tetani (Chatfield et al., 1992), Streptococcus pneumoniae (Nayak et al., 1998), Leishmania major (McSorley et al., 1997), and Shigella dysenteriae (Xu de et al., 2007), generating a pathogen specific protective immunity in relevant animal models. We have previously established an experimental framework that successfully uses Salmonella typhimurium SL3261 containing a mutation in the aromatic amino biosynthetic pathway (Hoiseth and Stocker, 1981) as a vector for the expression of a recombinant guest antigen in the temperaturedependent rabbit model of H. ducreyi infection (Ashby et al., 2005). In this study, we exploit this model to characterize the immune response to the hemoglobin receptor HgbA. 2. Materials and methods 2.1. Bacterial strains, plasmids, and growth conditions S. typhimurium SL3261 (his G46 (del) aroA 554) (Hoiseth and Stocker, 1981), S. typhimurium LB5010 (galE r− m +) (Bullas and Ryu, 1983), phage P22 (HT int-) (Schmieger, 1972; Schmieger and Backhaus, 1976), and phage P22.c2 (c2 mutation) (Zinder and Lederberg, 1952; Levine, 1972) were purchased from the Salmonella Genetic Stock Centre (SGSC/University of Calgary, Calgary, AB). Plasmid pTETnir15 was kindly supplied by Dr. D. Pickard (The Wellcome Trust Sanger Institute, Hinxton, UK). S. typhimurium and E. coli were grown on Luria Bertani (LB) agar plates or in LB broth at 37 °C for 16–20 h. For in vitro plasmid selection, ampicillin was added at 100 μg/ml (LB + amp). For anaerobic growth,

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an overnight LB broth culture was diluted 1/100 in fresh LB broth and then incubated at 37 °C with agitation by a magnetic stir bar in an anaerobic jar with AnaeroGen™ (Oxoid/Thermo Fisher Scientific, Nepean, ON) sachets to confirm that an anaerobic atmosphere was achieved. H. ducreyi 35000 was isolated from a 1975 chancroid outbreak in Winnipeg, Canada (Hammond et al., 1978) and was subcultured from stock cultures frozen at −70 °C onto chocolate agar (CA) plates [GC Medium Base with 1% (w/v) bovine hemoglobin supplemented with 1% (v/v) IsoVitalex and 5% (v/v) heat inactivated fetal bovine serum (FBS)] for 24 to 48 h at 35 °C in an atmosphere of 5% CO2 and saturated humidity prior to inoculation of GC broth [1.5% (w/v) proteose peptone 3, 0.4% (w/v) K2HPO4, 0.1% (w/v) KH2PO4, 0.5% (w/v) NaCl, 0.1% (w/v) starch, supplemented with 5% (v/v) FBS and 1% (v/v) IsoVitalex]. Broth cultures were grown at 35 °C in an atmosphere of 5% CO2 for 16–20 h with shaking at 225 rpm. 2.2. Construction of the recombinant Salmonella strains Plasmid pTETnir15 contains the tetC gene under the control of the anaerobically-induced nirB promoter from the nitrate reductase gene of E. coli (Oxer et al., 1991), and an ampicillin resistance gene cassette. To facilitate the cloning and expression of the H. ducreyi hgbA gene, the plasmid was modified using the approach described by McSorley et al. (1997) in order to provide a suitable restriction site for the insertion of hgbA (Fig. 1). Using restriction enzymes BglII and ClaI, all but 392 bp at the 3′-end of tetC was excised from pTETnir15. A 34-bp double stranded DNA linker [5′-GATCTTAATCATCCACAGGAGACTTTCATATGAT-3′ and 3′-AATTAGTAGGTGTCCTCTGAAAGTATACTACG-5′ (synthesized by Invitrogen, Burlington, ON)] incorporating BglII and ClaI restriction enzyme sites at the 5′ and 3′-end, respectively and containing a Shine– Dalgarno (SD) sequence (underlined); and a unique restriction enzyme site NdeI, which encloses a start codon, downstream of the nirB promoter region, was subsequently ligated to the 2.7 kb plasmid fragment. To remove possible DNA linker concatamers, the plasmid was subjected to ClaI digestion. The 2.7 kb gel extracted fragment was self-ligated before introducing it into OneShot-TOP10 chemically competent E. coli cells (Invitrogen, Burlington, ON). An appropriate sized 2.764 kb plasmid, designated pnirL, was recovered from an ampicillin resistant E. coli transformant. Nucleotide sequencing verified the expected location of the DNA linker in the vector (data not shown). The hgbA gene was PCR amplified from H. ducreyi 35000 genomic DNA using the primers 5′-GGAATTCCATATGGAAAGCAATATGCAAACAG-3′ and 5′-CGGGATCCTTAGAAAGTGATCT CTGCATTCAC-3′ incorporating NdeI and BamHI restriction sites (underlined) that permitted cloning of the hgbA gene into pnirL, which had been restricted with NdeI and BamHI in order to remove the remaining 392 bp 3′-end of the tetC gene. The PCR reaction conditions were as follows: 3 min of initial denaturation at 94 °C, 35 cycles of denaturation at 94 °C for 15 s, annealing at 68 °C for 30 s, extension at 68 °C for 3 min, followed by 5 min of final extension at 68 °C. As HgbA is toxic when heterologous expression of the protein includes the leader peptide (Elkins et al., 2000), this sequence was excluded in the primer design. A 5.225 kb plasmid isolated

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Fig. 1. Schematic representation of the construction of pnirL.

from an ampicillin-resistant E. coli transformant generated the expected fragment size following restriction enzyme digest (data not shown). The replicon was named pnirBhgbA. The 2.372 kb vector control pnirB was constructed by digesting pnirL with NdeI and BamHI to remove the remaining 392 bp 3′-end of the tetC gene, followed by repair of the 5′ ends and blunt end self-ligation (data not shown). Plasmids pTETnir15, pnirB or pnirBhgbA were introduced into Salmonella as described by Bowe et al. (2003). Briefly, the plasmids were first electroporated into intermediate strain S. typhimurium (galE r− m +) (Bullas and Ryu, 1983). Transductants bearing either pnirBhgbA or pnirB were confirmed by PCR amplification using primers annealing to vector sequences immediately upstream and downstream of the hgbA gene that produced two amplicons of 3169 bp and 295 bp, respectively (Fig. 2A and B).

S. typhimurium LB5010 transformants were infected with phage P22 (HT int-) and the resultant P22 lysate was used to introduce the plasmids into the S. typhimurium SL3261 vaccine strain by transduction. To preliminarily identify and isolate S. typhimurium SL3261 strains bearing phage introduced plasmid, colonies grown on LB + amp agar plates containing 5 mM ethylene glycol-bis (2-amino-ethylether)-N,N, N′,N′-tetra-acetic acid (EGTA) were inoculated onto green indicator plates [0.8% (w/v) tryptone, 0.1% (w/v) yeast extract, 1.5% (w/v) NaCl, 1.5% (w/v) Bacto-agar]. Each liter of autoclaved green indicator agar solution was supplemented with 34 ml of filter sterilized 40% (w/v) glucose, 25 ml of 2.5% (w/v) Alizarin yellow G and 6.6 ml of 2% (w/v) aniline blue. The plates were incubated at 37 °C for 24 h to obtain phage free colonies. Lightcolored colonies, indicating the presence of either a nonlysogen or a lysogen (Maloy, 1989), were streaked onto

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Fig. 2. Genotypic and phenotypic analysis of the recombinant S. typhimurium SL3261 strains. Plasmid DNA from pnirBhgbA and pnirB recovered from transduced strain S. typhimurium SL3261 served as template for PCR amplification reactions using primers that flanked the insertion site of the hgbA gene. Amplicons from 3 different clones of pnirBhgbA (Panel A) and of pnirB (Panel B) were applied to a 1% (w/v) agarose gel and the gel was stained with ethidium bromide. Lane + denotes PCR products amplified from plasmid pnirBhgbA or pnirB recovered from the E. coli and lane − is a sample containing no DNA template. In Panel C, Western immunoblots of whole cell lysates of S. typhimurium SL3261, carrying either pnirBhgbA or the control vector pnirB derived from aerobic or anaerobic cultures, were probed with polyclonal antibody against recombinant H. ducreyi HgbA. Lane + denotes H. ducreyi cell lysates prepared from cultures grown under heme restricted conditions and lane − represents S. typhimurium SL3261 (pnirB) lysates prepared from anaerobically grown cultures. In Panel D, Western blots of cell lysates of anaerobically grown S. typhimurium SL3261 (pnirBhgbA) (lanes 1–3) and S. typhimurium SL3261 (pnirB) (lanes 4–6) were probed with anti-rHgbA. Lane + denotes H. ducreyi cell lysates prepared from cultures grown under heme restricted conditions. In Panel E, anaerobically grown cell lysates of S. typhimurium SL3261 carrying pTETnir15 or the control vector pnirB were probed in Western immunoblots with rabbit polyclonal antibody against tetanus toxin peptides (anti-TetC). Lane + denotes E. coli (pTETnir15) and lane − S. typhimurium SL3261 (pnirB) anaerobically grown cell lysates, respectively. Sizes of the molecular mass markers (in kDa) are shown on the left.

LB+ amp plates. Nucleotide sequencing of pnirBhgbA recovered from S. typhimurium SL3261 corroborated the presence of hgbA (data not shown). The presence of pTETnir15 was confirmed by NcoI restriction digestion of recovered plasmid producing a

3.727 kb fragment (data not shown). The LPS profile of all three S. typhimurium SL3261 strains was identical to that of the parental isolate indicating that the LPS structural integrity was not compromised by transduction (data not shown). Stocks

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were prepared in glycerol (0.15% v/v) and frozen at −70 °C for future testing of phage P22 sensitivity. 2.3. Characterization of the Salmonella vaccine strain 2.3.1. Sensitivity of the Salmonella recombinant strains to phage P22 To distinguish between nonlysogenic and lysogenic Salmonella SL3261 transductants, the transduced S. typhimurium strains were tested for phage sensitivity as previously described (Maloy, 1989) with modifications (Dr. K. Sanderson, University of Calgary, Calgary, AB). An LB +amp agar plate was flooded with 500 μl of an overnight culture of the transduced strain. After diffusion of the bacterial culture into the agar, 10 μl of undiluted phage P22.c2 solution was placed onto the plate. After incubation at 37 °C for 24 h, the plate was examined for plaque formation. This virus contains a c2 mutation, which results in clear plaque formation (Barry et al., 1996; Levine, 1972; Zinder and Lederberg, 1952). Bacteria that are nonlysogenic are expected to be P22 sensitive, whereas lysogenized strains are P22 resistant (Maloy, 1989). 2.3.2. Growth kinetics of the recombinant Salmonella strains Following overnight growth of the Salmonella recombinant strains on agar plates, a single colony was incubated in 5 ml of LB broth until mid-log phase was reached (OD600 of 0.2). A 0.5–1 ml aliquot was inoculated into 50 ml of fresh LB broth. Cultures were continuously agitated with a magnetic stir bar. Growth at 37 °C was monitored by removing aliquots at 0, 2, 4, 6, 8, 10, 14, and 24 h for measurement of OD600. For cultures grown under anaerobic conditions, the AnaeroGen™ sachet was replaced after each aliquot was removed. 2.4. Characterization of the humoral immune response to guest antigens 2.4.1. Enzyme immunoassay (EIA) antigen preparation Despite the lack of its native leader sequence (Elkins et al., 2000), recombinant H. ducreyi HgbA (rHgbA) (provided by I. Leduc) is recognized in Western immunoblots probed with antibodies derived from individuals naturally infected with H. ducreyi (Elkins et al., 2000). Salmonella crude soluble antigens were prepared as previously described (Ashby et al., 2005). Tetanus toxin C fragment from C. tetani (TetC) was purchased from Sigma-Aldrich. Protein concentration for rHgbA, Salmonella and E. coli crude soluble antigens was determined using the BCA protein assay kit (Pierce, Rockford, IL). 2.4.2. EIA procedure The enzyme immunoassay was performed as described previously (Ashby et al., 2005). Test antigens were diluted in 0.1 M of carbonate buffer (30 mM Na2CO3, 70 mM NaHCO3, pH 9.6). Salmonella crude soluble antigens were diluted to a concentration of 50 ng/μl, TetC was diluted to 10 ng/μl and rHgbA to 2 ng/μl. 2.4.3. Western immunoblot To detect expression of HgbA and TetC, whole cell lysates were prepared from aerobic and anaerobic cultures of S. typhimurium SL3261 strains. Samples from broth cultures

grown as described above were removed at 1, 2, 3, 6, 12, and 24 h for S. typhimurium SL3261 (pnirBhgbA) and at 2, 4, 6, 12, and 24 h for S. typhimurium SL3261 (pTETnir15) and diluted to an OD600 of 0.2. After centrifuging a 1 ml aliquot, cells were resuspended in sample buffer and the cell lysates were fractionated by one-dimensional denaturing SDS-PAGE with the discontinuous buffer system (Laemmli, 1970). Western immunoblot analysis was performed as previously described (Negari et al., 2008). Membranes were probed with rabbit polyclonal antiserum raised against either recombinant H. ducreyi HgbA (anti-rHgbA) (kindly provided by I. Leduc, University of North Carolina, Chapel Hill, NC) (White et al., 2005) in a dilution of 1/20,000, or against tetanus toxin peptides 1300–1314 conjugated to KHL (anti-TetC; Biogenesis Ltd, Raleigh, NC) in a dilution of 1/16,000 dilution, followed by the addition of either a 1/10,000 dilution (for antirHgbA) or a 1/5,000 dilution (for anti-TetC) of goat anti-rabbit immunoglobulin horse radish peroxidase conjugated secondary antibody (BioSource, Camarillo, CA) solution. Membranes were developed with the addition of 3,3′,5,5′-tetramethylbenzidine (TMB peroxidase substrate; KPL Inc., Gaithersburg, MD) for 1–5 min. The reaction was stopped by rinsing the blot with ddH2O. 2.5. Colonization and in vivo stability of recombinant plasmids Rabbit feces were collected from the pan underneath the cages on the day that the pans were changed by the Animal Care Staff. Collection was done on 1, 3, 6, 8, 10, 13, 21, and 27 days post-gavage for the first vaccination trial or on 1, 6, and 10 days after each oral gavage for the second vaccination trial. The collection days were minimized from every 2–3 days in the first trial to every 4–5 days in the second trial for convenience purposes. Fecal pellets were collected and placed in a 1.5 ml microcentrifuge tube with forceps sterilized in 70% ethanol. After resuspension in 1.2 ml of PBS, 100 μl of serial tenfold dilutions were inoculated onto MacConkey (MAC) agar and MAC supplemented with ampicillin. After incubation at 37 °C in ambient air for 16–20 h, plates were examined for the presence of nonlactose fermenting (NLF) organisms. To determine the identity of the ampicillin resistant NLF organisms, selected colonies underwent further phenotypic analysis. For the first vaccination trial, single colonies were subjected to urea hydrolysis (Oxoid/Thermo Fisher Scientific), triple sugar iron (TSI) agar (PML Microbiologicals, Mississauga, ON), and motility medium (Oxoid/Thermo Fisher Scientific) tests (Imperatrice and Nachamkin, 1993). Organisms exhibiting reactions characteristic of Salmonella were re-streaked onto LB+ amp agar and grown overnight at 37 °C. Colonies were suspended in freezing media [2.4% (w/v) trypticase soybroth, 20% (v/v) glycerol] and stored at −70 °C. Nonlactose fermenting bacteria isolated from the second vaccination trial were re-streaked onto LB +amp agar and grown overnight at 37 °C. Colonies were identified as Salmonella using the VITEK-2 system (BioMérieux Canada Inc. St. Laurent, PQ). The QIAprep Spin Miniprep kit was used to retrieve plasmid from Salmonella recovered from rabbit fecal pellets. The isolated plasmid DNA underwent restriction digest analysis. For the second vaccination trial, PCR amplification to identify

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the plasmid construct bearing the hgbA gene was also performed using recovered plasmid DNA as template. Whole cell lysates were examined for HgbA and TetC expression by Western immunoblotting.

positivity, cumulative lesion size, peak lesion score, and duration of ulcer was performed with the Student's t test.

2.6. Pulse field gel electrophoresis (PFGE)

3.1. Phage sensitivity of the recombinant S. typhimurium SL3261 strains

Clonality of the Salmonella isolates was distinguished by DNA fingerprinting with SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) (Birren and Lai, 1993). 2.7. Intragastric immunization and in vivo protection assay Animal experiments were approved by the Animal Care Committee at the University of Ottawa. Twelve age-matched male New Zealand white (NZW) rabbits, between 2.2 and 2.5 kg in weight (Charles River Co., St. Constant, PQ), were used for this study. Intragastric immunization of the rabbits was performed as previously described (Ashby et al., 2005). For the booster-vaccination regimen, the oral gavage was performed three times at two week intervals. 2.8. Temperature-dependent rabbit model (TDRM) of H. ducreyi infection The temperature dependent rabbit model of H. ducreyi infection has been described in detail (Purcell et al., 1991; Desjardins et al., 1996). Briefly, rabbits were housed at an ambient temperature of 14 ± 1 °C (Thermo Air Plus air conditioning unit) and their backs shaved daily. Inocula at 3 titers (106, 105, or 104 CFU) were injected in triplicate in 100 μl volumes intraepithelially in a 3 × 4 grid drawn on each rabbit's back. To estimate the delivered inoculum size, each suspension was plated onto CA to determine viable colony counts. An operator blinded to the treatment observed lesions every other day for 20 days. Two lesions at each inoculum titer were measured for lateral diameter of induration using electronic calipers, and assigned a clinical score (1= redness, 2 = induration, 3 = suppuration, 4 = ulceration). The third lesion was cultured by injection of 100 μl PBS into the lesion followed by back-aspiration. This aspirate was plated on CA, and colonies were counted 2 days after plating. Culture positivity was determined by the presence of one or more colonies characteristic of H. ducreyi. Rabbits from the second vaccination trial were inoculated with H. ducreyi 10 days after the third oral gavage. Rabbits from the first trial were not challenged with H. ducreyi, as the first trial was designed to determine the immunization protocol that would generate a humoral response to HgbA. 2.9. Statistical analysis The size and score of the lesions at each inoculum titer were obtained by averaging the data of two non-manipulated lesions for each rabbit on each observation. Group means were calculated for each day of observation. Comparative statistical analysis of these data over 21 days of observation was performed using the one-way repeated measures analysis of variance (ANOVA), with Bonferroni t test for pairwise comparisons. Comparative evaluation of inoculum size, duration of culture

3. Results

Plasmids introduced into the S. typhimurium SL3261 by transduction used a phage that contained an int mutation, which prevents the formation of stable lysogens (Bong et al., 2002). To confirm that the transductants were nonlysogenic, the bacteria were tested for phage sensitivity to phage P22.c2. Plaque formation was seen when S. typhimurium SL3261 (pnirBhgbA), S. typhimurium SL3261 (pnirB), and S. typhimurium SL3261 (pTETnir15) was infected with phage P22.c2, indicating that all these strains were nonlysogens (data not shown). 3.2. Expression profiles of S. typhimurium SL3261 (pnirBhgbA) and S. typhimurium SL3261 (pTETnir15) To detect the expression of HgbA, a Western blot of whole cell lysates prepared from a S. typhimurium SL3261 (pnirBhgbA) grown under aerobic and anaerobic conditions was probed with anti-rHgbA (Fig. 2C). An immunoreactive band of 108.6 kDa was seen in cultures at 1 h and 2 h after exposure to an anaerobic environment (Fig. 2C, lane 2; Fig. 2D lanes 1 and 2). No expression was seen in whole cell lysates derived from cells grown anaerobically for 6 h and 9 h (data not shown). In contrast, no such immunoreactive band corresponding to HgbA was present in aerobically grown cell lysates (Fig. 2C, lanes 4–6) nor in lysates prepared from S. typhimurium SL3261 containing the empty vector pnirB (Fig. 2C, lane −; Fig. 2D, lanes 4–6). These results indicated that S. typhimurium SL3261 (pnirBhgbA) expressed HgbA and that the production of this protein was activated by anaerobic conditions. TetC expression was detected in cell lysates derived from both anaerobic and aerobic grown cultures (Fig. 2E). An increase in the expression of TetC was witnessed only during anaerobic growth. In contrast, the 50 kDa band corresponding to TetC was not seen in Western blots of cell lysates arising from anaerobic cultures of S. typhimurium SL3261 (pnirB) (Fig. 2E, lane −) that contained the empty vector. These results indicated that TetC is stably expressed in S. typhimurium SL3261 (pTETnir15) for 24 h, and that the anaerobic atmosphere does not tightly regulate expression. 3.3. In vitro growth of S. typhimurium SL3261 (pnirBhgbA) To ensure the absence of a growth defect in S. typhimurium SL3261 (pnirBhgbA), growth assays were conducted under both aerobic and anaerobic conditions. No major differences in growth between S. typhimurium SL3261 and S. typhimurium SL3261 (pnirBhgbA) were seen. The slower growth of both strains observed under anaerobic conditions compared to that seen under aerobic conditions was expected, as anaerobic metabolism reduces the rate of bacterial replication (Angelakopoulos and Hohmann, 2000). These results indicated that a metabolic burden is not incurred by the presence of

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the organism. The ability of rabbits fed S. typhimurium SL3261 (pTETnir15) to raise antibody to TetC (Fig. 3D) confirmed that a humoral response to a guest antigen delivered by the attenuated Salmonella vector can be detected in this system.

the recombinant plasmid pnirBhgbA in S. typhimurium SL3261 (data not shown). 3.4. Rabbit immune responses following oral vaccination No significant antibody response to HgbA was elicited in rabbits receiving a single oral dose of S. typhimurium SL3261 (pnirBhgbA) (data not shown). The observation that clearance of H. ducreyi required repeated infection in the swine model of chancroid (Cole et al., 2003) prompted the adoption of a multi-dose strategy. However, rabbits subjected to the booster protocol of three oral vaccinations also failed to produce an antibody against this antigen (Fig. 3A). However, these same animals generated a vigorous antibody response towards HgbA following experimental challenge with 10 5 CFU of H. ducreyi (Fig. 3B). As expected, no antibody to HgbA was detected in the mock challenged rabbit (Fig. 3B). Rabbits readily recognized the S. typhimurium SL3261 vector (Fig. 3C), mounting a brisk antibody response towards

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3.5. In vivo stability of plasmid derivatives Fecal shedding of the Salmonella strains was observed in all the orally challenged rabbits. The mean duration of shedding of the vaccine strain was similar in rabbits who received either one or three doses of this isolate (15.5 days versus 13.7 days, respectively, p ≤ 0.4). The two other recombinant Salmonella strains persisted longer following the three dose regimen [10 days versus 24 days for S. typhimurium SL3261 (pnirB), and 6 days versus 10 days for S. typhimurium SL3261 (pTETnir15), respectively]. No ampicillin resistant Salmonella species were isolated from stools collected 1 and 5 days prior to oral immunization (data not shown).

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Fig. 3. Serum rabbit antibody responses measured by enzyme immunoassay. In Panel A, sera from 3 rabbits fed the S. typhimurium SL3261 (pnirBhgbA) (open circle) vaccine strain, one rabbit fed S. typhimurium SL3261 (pnirB) (inverted closed triangle), and a PBS sham-immunized control (closed circle) were tested against rHgbA. In Panel B, sera from rabbits immunized with recombinant S. typhimurium SL3261 (pnirBhgbA) (open circle), S. typhimurium SL3261 (pnirB) (inverted closed triangle), S. typhimurium SL3261 (pTETnir15) (open triangle), and PBS sham-immunized control (closed circle) were tested against rHgbA. The arrow indicates when the rabbits were experimentally challenged with 105 CFU of H. ducreyi. In Panel C, sera from 4 rabbits receiving the S. typhimurium SL3261 (pnirBhgbA) vaccine strain were tested against crude soluble antigens of Salmonella SL3261. In Panel D, serum from one rabbit immunized either with S. typhimurium SL3261 (pnirB) (open circle) or with S. typhimurium SL3261 (pTETnir15) (closed circle), was tested against TetC. Each value represents the mean of three triplicate experiments. Standard deviations of the mean are shown by error bars.

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The macrodigestion profiles of the last Salmonella isolates recovered from the stool of all six immunized rabbits that participated in the first vaccine trial were identical to the strain used for immunization, indicating preservation of the original genotype. Although strains retrieved from the second multi-dose trial were not subjected to PFGE analysis, a similar result would be anticipated (data not shown). The genotype of plasmid derivatives extracted from Salmonella strains retrieved from the last day of documented stool shedding in rabbits fed a single dose of the vaccine corresponded to the original recombinant plasmids present in the inoculating Salmonella strains, as reflected by restriction digest analysis (data not shown), indicating that the constructs were stably maintained throughout the recorded days of in vivo passage. However, in the multi-dose trial, the recombinant plasmid in the vaccine strain exhibited a more limited in vivo stability as no product corresponding to hgbA was PCR amplified from organisms after 10 days. In contrast, the tetC gene was present in plasmids recovered on the last documented day of shedding (data not shown). In both trials, HgbA expression was limited to S. typhimurium SL3261 (pnirBhgbA) isolates recovered 1 day after immunization, except in one rabbit where the expression was present 6 days after immunization. Despite the presence of the recombinant plasmid, no protein was produced in strains shed beyond 24 h for two of three rabbits. In contrast, the ability to express TetC, was correlated with the presence of its plasmid construct as TetC production was detected on both the first and last days of fecal shedding (data not shown).

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no protective immune response was provided in rabbits inoculated with the recombinant Salmonella strain expressing HgbA. Rabbits challenged with 10 4 CFU were not analyzed, as this inoculum did not produce ulcerative disease in the PBS sham-immunized control rabbit (data not shown). This result is consistent with those of prior studies (Purcell et al., 1991; Desjardins et al., 1996) whereby the full clinical expression of chancroid in the TDRM is a dose dependent event. Intradermal injections of viable H. ducreyi less than 10 5 CFU failed to consistently lead to ulcer development. 4. Discussion In this preliminary study, live oral inoculation with recombinant S. typhimurium SL3261 expressing HgbA was unable to produce protective immunity against H. ducreyi infection in the TDRM of chancroid. Our results indicate that the most likely explanation was the inability of the Salmonella

3.6. Protective efficacy of the oral recombinant HgbA vaccine Rabbits participating in the three dose booster protocol received titred doses of 10 4, 10 5 and 10 6 CFU of H. ducreyi intraepithelially. In animals administered 10 5 and 10 6 CFU of H. ducreyi, viable organisms were recovered from the ulcerative lesions in the one naïve rabbit sham immunized with PBS, and in the single rabbit fed the S. typhimurium SL3261 (pnirB) control strain. In contrast, rapid sterilization was achieved in the three rabbits administered the S. typhimurium SL3261 (pnirBhgbA) vaccine strain at a dose of 10 5 CFU (Table 1). At both inocula, the duration, tempo, and severity of infection due to H. ducreyi were similar in all three groups (Fig. 4 and data not shown), and recapitulated the natural history of infection in the TDRM, indicating that

Table 1 Last day that H. ducreyi was cultured from lesions of rabbits experimentally challenged with H. ducreyi. Rabbit number

106 inoculum

105 inoculum

1 2 3 4 5

18 12 16 18 20

20 0a 0a 0a 14

Rabbits were administered by oral gavage PBS (rabbit 1), S. typhimurium SL3261 (pnirB) (rabbit 2), and S. typhimurium SL3261 (pnirBhgbA) (rabbits 3–5). a No viable H. ducreyi was isolated throughout the entire experiment from days 2 to 20.

Fig. 4. Comparison of experimental H. ducreyi lesions in control and immunized rabbits inoculated with 105 CFU in the TDRM. The data were for (A) lesion size and (B) lesion score for an average of two lesions for rabbits previously administered PBS (closed circles, and black bars), S. typhimurium SL3261 (pnirB) (open circles, and light gray bars), and S. typhimurium SL3261 (pnirBhgbA) (inverted closed triangles, and dark gray bars). Each value represents the mean of three triplicate experiments. Standard deviations of the mean are shown by error bars. Absence of error bars indicated that the standard deviation of the three replicate cultures was too small to be shown on the graph.

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recombinant strain to generate detectable antibody against HgbA. This unresponsiveness was intrinsic to the vector system as animals receiving the oral vaccine remained capable of mounting a vigorous humoral response against HgbA when subsequently challenged with H. ducreyi (Fig. 3B). In the swine experimental model of chancroid, a specific humoral response to native HgbA has been shown to be a crucial immunologic property of an HgbA vaccine (Afonina et al., 2006; Fusco et al., 2010), as passive transfer of polyclonal antiserum from pigs immunized with native HgbA was sufficient to protect against H. ducreyi homologous challenge (Leduc et al., 2011). These results attest that the formulation of a broadly effective HgbA vaccine necessitates the inclusion of specific conserved structural and functional HgbA domains. Although the immunological correlates necessary for protection are not well-defined, recruitment of innate and adaptive immune cells in both natural (Bauer et al., 2006) and experimental H. ducreyi infection (Janowicz et al., 2010), and the lack of efficacy of standard antimicrobial regimens for treatment of chancroid in HIV co-infected individuals (Tyndall et al., 1993), have implicated a role for cell-mediated immunity in the resolution of H. ducreyi infection. These studies offer an intriguing clue to the results in animals receiving the multi-dose vaccinations and who were subsequently challenged with 10 5 CFU of H. ducreyi. Induction of cell-mediated immunity promoting enhanced phagocytosis may have driven bacterial clearance (Janowicz et al., 2009). Lesion progression despite this sterilization may be a manifestation of a delayed-type hypersensitivity response (Desjardins et al., 1996). The absence of a similar effect following challenge with 106 CFU of H. ducreyi is not surprising as eliciting protective immunity in the TDRM is associated with inoculum size (Hansen et al., 1994). Thus, although compounding evidence supports involvement of a vigorous lymphocyte and plasma cell infiltrate in conferring immune clearance in the TDRM (Hansen et al., 1994; Desjardins et al., 1995, 1996), the debate whether this vaccine effect is context dependent remains to be resolved. Several factors likely accounted for the failure of the attenuated Salmonella vaccine strain to elicit antibodies directed to HgbA. First, a robust and durable synthesis of HgbA was lacking, despite the restricted anaerobic expression regulated by the anaerobically-inducible nirB promoter. Such controlled in vivo antigen delivery using nirB constructs has been associated with enhanced production of heterologous proteins (Kotton and Hohmann, 2004). Optimization of codon usage (Makoff et al., 1989; Gustafsson et al., 2004) and construction of C-terminal fusions to the highly immunogenic fragment C of tetanus toxin (Lee et al., 2000) have also been shown to improve and stabilize protein expression in a heterologous host. Incorporation of these alternate strategies in future approaches is imperative as immunogenicity of oral Salmonella vaccines has been strongly correlated with the sustained and stable production of the guest protein (Kotton and Hohmann, 2004). Secondly, the poor retention of the original plasmid vector compromised HgbA expression. Although plasmids were retrieved throughout the period of bacterial colonization, PCR analysis disclosed deletion of the HgbA gene in those plasmid derivatives recovered on the last day of stool collection. The presence of other genetic alterations, such as

point mutations, and gene inversions that might be responsible for the deficient HgbA expression, cannot be excluded as nucleotide sequencing of the hgbA insert was not performed. The use of a high-copy-number replicon may also have exerted a deleterious effect on plasmid maintenance (Glick, 1995; Galen and Levine, 2001), but in vitro growth kinetics did not indicate a metabolic burden imposed by hgbA on the Salmonella vaccine strain (data not shown). Thirdly, the influence of prior exposure to the Salmonella carrier on the immune response to the vectored antigen in rabbits participating in the booster vaccination schedule in particular cannot be discounted. Although this “vector-priming” may have contributed to the diminished capacity to respond to HgbA, prior human and animal studies have reported conflicting results regarding the role this immunomodulatory effect upon the humoral response to a foreign antigen (Bao and Clements, 1991; Whittle and Verma, 1997; Saxena et al., 2009; Attridge et al., 1997; Roberts et al., 1999; Kohler et al., 2000). Lastly, HgbA likely localized to a subcellular compartment in the Salmonella strain as the plasmid construct bearing hgbA lacked the leader peptide, in order to abrogate the toxicity incurred during high level expression in a heterologous host (Elkins et al., 2000). Although surface display of the vaccine antigen is recognized to be immunologically advantageous (Hess et al., 1996; Russmann et al., 1998; Galen and Levine, 2001; Kotton and Hohmann, 2004), this requirement may be antigen and vector dependent as non-surface exported proteins are capable of eliciting protective immunity (Oxer et al., 1991; Chatfield et al., 1992). The pursuit of a live orally delivered bacterial vector vaccine against chancroid remains a feasible goal. The use of HgbA as a potential vaccinogen will require both deft technical refinements to improve heterologous protein expression and vector fitness, and mapping of conserved structural domains. For example, given the insistence that a prominent humoral response against HgbA is an immunologic correlate of protection, identification and subsequent incorporation of specific B-cell epitopes of HgbA in the recombinant vector warrants consideration. The subsequent coupling of the guest antigen to the Salmonella enterica autotransporter MisL (Ruiz-Olvera et al., 2003) encoded from a single copy chromosomally integrated construct using a native in vivo-inducible Salmonella promoter to drive expression within the phagosome (Galen et al., 2009; Matic et al., 2009) would be expected to favor a stable sustained antibody production against HgbA. Recently developed novel S. enterica serovar typhimurium vaccine strains can be used to further enhance the immune response (Curtiss et al., 2009; Li et al., 2009). These regulated delayed attenuated strains display the wild-type phenotype at immunization and become attenuated during colonization of host tissues. One such highly immunogenic delivery vector has provided significant protection against pneumococcal infection in a bacteremic mouse model (Li et al., 2009). The promise of an HgbA subunit vaccine will await the application of these new techniques. Acknowledgments We are grateful to K.E. Sanderson for helpful advice, and to D. Pickard for the kind provision of the plasmid vector.

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