Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials

Vaccine 23 (2005) 4754–4764

Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials Gaddam Narsa Goud a,∗,1 , Maria Elena Bottazzi a,∗,1 , Bin Zhan a , Susana Mendez a , Vehid Deumic a , Jordan Plieskatt a , Sen Liu a , Yan Wang a , Lilian Bueno a , Ricardo Fujiwara a , Andre Samuel a , So Yeong Ahn a , Maneesha Solanki e , Oluwatoyin A. Asojo c , Jin Wang b , Jeffrey M. Bethony a , Alex Loukas a,d , Michael Roy e , Peter J. Hotez a,∗ a

b

Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Ross Hall 736, 2300 Eye Street NW, Washington, DC 20037, USA Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA c Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA d Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, Qld, Australia e Science Applications International Corporation, Frederick, MD, USA Received 3 March 2005; received in revised form 26 April 2005; accepted 29 April 2005 Available online 6 June 2005

Abstract The ASP-2 protein secreted by infective larvae of the human hookworm, Necator americanus, is under development as a recombinant vaccine. Recombinant Na-ASP-2 was expressed in Pichia pastoris, and the purified protein was characterized. At the 60 L scale, the 21.3 kDa recombinant protein was produced at a yield of 0.4 g/L. When formulated with Alhydrogel® and injected into rats to determine immunological potency, three 50 ␮g doses of the formulated recombinant protein elicited geometric mean antibody titers up to 1:234,881. Rat anti-Na-ASP-2 antibody recognized larval-derived ASP-2 and also inhibited larval migration through skin in vitro. The processes developed and tested for the high yield production of recombinant Na-ASP-2 provide a foundation for clinical vaccine development. © 2005 Elsevier Ltd. All rights reserved. Keywords: Hookworm; Vaccine; Necator americanus; Na-ASP-2

1. Introduction Human hookworm infection caused by Necator americanus or Ancylostoma duodenale is a major cause of iron deficiency anemia and protein malnutrition in the developing world and a leading cause of parasitic disease burden [1,2]. An estimated 740 million people are infected in areas of rural ∗

Corresponding author. Tel.: +1 202 994 3532; fax: +1 202 994 2913. E-mail addresses: [email protected] (G.N. Goud), [email protected] (M.E. Bottazzi), [email protected] (P.J. Hotez). 1 Both GNG and MEB contributed equally to the manuscript. 0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.04.040

poverty in the tropics and subtropics resulting in up to 65,000 deaths and 22 million disability-adjusted life years [3,4]. The major approach to hookworm control relies on reducing morbidity through the frequent and periodic use of benzimidazole anthelminthic (BZAs) drugs, which can temporarily remove adult parasites in the human gastrointestinal tract [4,5]. Such treatment for school-aged children leads to improvements in health and educational achievement [4]. However, there are reasons why exclusive reliance on BZAs for controlling hookworm infection may not be effective. Among them, posttreatment hookworm re-infection rates are high in areas of high transmission [6], and the efficacy of BZAs can diminish

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with increasing and frequent use [7]. It has been further suggested that the emergence of BZA drug resistance is possible [7]. School-based programs are also not expected to interrupt hookworm transmission [8]. In order to develop new tools for the control of hookworm infection, studies were undertaken to develop recombinant hookworm vaccines. Based on previous success in the development of canine hookworm vaccines from third-stage infective larvae (L3) that were attenuated by X-irradiation [9], our antigen discovery efforts focused on the identification and isolation of critical L3-secreted molecules that mediate protective immunity [10]. These included a novel class of cysteine-rich secretory proteins known as the Ancylostoma secreted proteins or ASPs [10–14]. The ASPs belong to the pathogenesis-related (PR) protein superfamily whose members are found in a wide variety of animals, plants and fungi where they are typically produced in response to stress or injury, including invasion by pathogens [15]. Although the function of the hookworm larval ASPs is not known, the observation that they are released by L3 upon receiving host-specific stimuli suggests that these proteins are released during host entry and function in the transition from the environment to a parasitic existence [13]. ASP-2 was selected for evaluation as a recombinant vaccine candidate based on human immunoepidemiologic studies and laboratory animal vaccine trials [16–19]. These data led to the selection of ASP-2 from the human hookworm N. americanus for further development. Here we describe the cloning of the Na-asp-2 gene and the scale-up expression of the purified recombinant Na-ASP-2 protein in the yeast, Pichia pastoris. We further describe the immunological potency of Na-ASP-2 and the cross-reactivity of antibody against the recombinant protein with the native, parasitederived protein. The studies here also confirm the importance of Na-ASP-2 in hookworm tissue migration. These data lay the foundation for the clinical development of the Na-ASP-2 Hookworm Vaccine.

2. Materials and methods 2.1. Molecular cloning of Na-asp-2 cDNA and transformation into Pichia pastoris A cDNA library was constructed with mRNA from L3 of a Chinese strain of N. americanus [20], and probed with a heterologous cDNA fragment of Ac-asp-2 cloned from the dog hookworm Ancylostoma caninum [12]. The Acasp-2 pBluescript plasmid was cut with XhoI and BamHI, releasing a 739 bp fragment (28–767 bp). The fragment was gel purified (Qiagen) and randomly labeled with ␣-32 [P]dCTP using a Rediprime labeling kit (Amersham). Approximately 5 × 105 plaques of N. americanus cDNA library were screened as previously described [20]. The positive clones were excised and subjected to secondary screening with the same reagents to isolate single positive colonies. The posi-

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tive clones from secondary screening were excised in vivo as pBluescript phagemids according to the manufacturer’s instruction (Stratagene). Phagemid DNA was extracted using the alkaline lysis method (Qiagen), and both strands were sequenced using vector primers, T3 and T7. The complete 5 end of Na-asp-2 cDNA was isolated from first strand cDNA of N. americanus L3 by a modified RNA ligase-mediated rapid amplification technique (GeneRacer; Invitrogen) as described previously [21]. The Na-asp-2 gene specific primers used for isolating the 5 end were NaASP2-R2 (TGT CTA GAT CAA GCA CTG CAG AGT CCC TTC TC) and Na-ASP2-R1 (TGT CTA GAG CAC TGC AGA GTC CCT TCT C). The PCR products were ligated into the T-ended vector pGEM-T (Promega) according to the manufacturer’s instructions. Recombinant plasmids containing the correct insert were extracted using a Qiaprep Spin Miniprep kit (Qiagen) and submitted for dideoxy terminator cycle sequencing. DNA and predicted protein sequences were analyzed using ESEE version 3.1 [22]. Amino acid sequences were analyzed for putative signal peptides using signalP (http://www.cbs.dtu.dk/services/SignalP-2.0). Subsequently, an additional round of screening of the N. americanus-L3 cDNA library was performed to confirm that there were no other Ac-asp-2 cDNA orthologues. The entire coding sequence minus the N-terminal signal peptide of the Na-asp-2 gene was amplified by PCR from the first strand cDNA of N. americanus L3 with Naasp-2 gene-specific primers and subcloned into the Pichia expression vector, pPICZ␣A (Invitrogen, CA) as described previously [15]. The recombinant plasmids were linearized with SacI digestion and transformed into P. pastoris X33 strain as described previously [15–17]. Seed stocks were prepared from a colony of the transformed P. pastoris. 2.2. Expression of Na-ASP-2 in Pichia pastoris Expression of Na-ASP-2 in P. pastoris was conducted at the 60 L scale and at the 10 L scale. For 60 L scale expression, four 2.5 L Tunair shake flasks (Shelton Scientific, CT), each containing 1 L of Buffered Minimal Glycerol (BMG) medium (1.34% Yeast Nitrogen Base, 0.00004% (w/v) d-biotin, 1% (w/v) glycerol and 100 mM potassium phosphate, pH 6.0) were inoculated with 2 mL of the P. pastoris seed stock. The flasks were incubated at 30 ± 2 ◦ C for 24–25 h to a final OD600 of 5–15. Approximately 3 L of this culture was used to inoculate 30 L of heatsterilized basal salt media (BSM) containing 3.5 mL/L of a filter-sterilized trace element (PTM4) solution [16,23]. Fermentation was conducted in a BioFlo5000 Mobile Pilot Plant (New Brunswick Scientific Co. Inc.). The growth of P. pastoris at the 60 L scale was divided into two phases: a fed-batch glycerol phase and a methanol induction phase. The pH of the BSM was adjusted to and maintained at 5.0 with 14% ammonium hydroxide feed. The agitation speed was set at 550 rpm and the dissolved oxygen maintained at 30% throughout the fermentation. At approximately 18 h into the glycerol phase

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and after a sharp increase in the percentage of dissolved oxygen, 50% (v/v) glycerol was introduced into the cell culture media at a set flow rate of 15 g/(L h), for 6 h. The pH of the cell culture media was then increased linearly from 5.0 to 6.0 by adding 14% ammonium hydroxide over a 2-h period before the completion of the fed-batch glycerol phase. The temperature was decreased linearly from 30 to 26 ◦ C also over a 2-h period before the completion of the fed-batch glycerol phase. Excessive foaming was controlled with a feed-on-demand of 10% (v/v) antifoam KFO673 in DI water (KABO Chemicals Inc.). The methanol induction phase was initiated when the wet cell weight (WCW) reached approximately 200 g/L. Methanol was added at an initial flow rate of 1.5 mL/(L h) increasing to 11.0 mL/(L h) over an 8-h period, and then subsequently maintained at a flow rate of 11.0 mL/(L h) for another 57 h. The fermentation cell culture was diluted from 54 to 60 L with 50 mM sodium acetate buffer, pH 4.8 and then concentrated to 36 L. The concentrated P. pastoris cells were washed four times with 36 L of 50 mM sodium acetate buffer, pH 4.8 using a 0.075 ␮m microfiltration hollow fiber cartridge (UFP-750-E-55, Amersham Biosciences) and a Masterflex pump (Cole-Parmer instrument Company). Approximately 174 L of supernatant was collected. This supernatant was concentrated to 12 L by ultrafiltration using a ProFlux M30 Filtration System (Millipore Corporation) and a 3 kDa hollow fiber cartridge (UFP-3C-55-Amersham Biosciences). The final concentrated supernatant was filtered through a 0.22 ␮m membrane and frozen at −70 ◦ C until purification. Fermentation at the 10 L scale was conducted in a 10 L Bioflo 3000 (New Brunswick Scientific Co. Inc.) using methods similar to those described above. The cell culture supernatant was harvested, processed by microfiltration using a Flex Stand Pilot Scale system and a 0.075 ␮m microfiltration (UFP-750-E-6A) cartridge (Amersham Biosciences), concentrated by ultrafiltration (UFP-5C-9G) cartridge, and stored in 1 L aliquots at −70 ◦ C until purification. 2.3. Protein purification For purification of the recombinant Na-ASP-2 protein, a chromatography instrumentation system was assembled with a Masterflex Digital standard drive, Easy-Load II pump head (Model# 77200-60-Cole-Parmer instrument Company), UV1 Control and Optical units (Amersham Biosciences), and EzStart Chromatography data system with SS420 data acquisition box and EZ Chrom Elite software (Scientific Software Inc.). 2.3.1. SP-Sepharose FF To capture the Na-ASP-2 recombinant protein with SP Sepharose FF, approximately 92.5 mL of SP Sepharose Fast Flow (Amersham Biosciences) resin was packed into a Millipore Vantage L column (VL 32 × 250) at a linear flow rate of 410-cm/h, following the manufacturer’s instructions. The column was equilibrated with 8.6 column volumes of 50 mM sodium acetate buffer, pH 4.8 at a linear flow rate of 300 cm/h.

One liter of the 12 L of total concentrated supernatant from the 60 L fermentation (one-twelfth scale) or 1 L of the concentrated supernatant from the 10 L fermentation (one-half scale) was thawed at 4 ◦ C for 24 h and then at room temperature for another 6 h. The pH and conductivity were adjusted to 4.8 and below 5.5 mS/cm, respectively, and the supernatant was applied to the SP Sepharose FF column at a linear flow rate of 300 cm/h. The column was washed with 6 column volumes of equilibrating buffer (50 mM sodium acetate buffer, pH 4.8) and 16 column volumes of wash buffer (50 mM sodium acetate buffer, pH 4.8 containing 50 mM NaCl). The Na-ASP2 recombinant protein was eluted with 7 column volumes of 50 mM sodium acetate, pH 4.8 containing 200 mM NaCl. The SP Sepharose FF column fractions containing Na-ASP-2 recombinant protein (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE]) were pooled and concentrated to 100 mL using a tangential flow filtration system consisting of masterflex pump, 5 L reservoir and 5 K cut-off Omega membrane (Pall corporation). The concentrated protein sample was buffer exchanged with 1 L of 20 mM ethanolamine buffer, pH 9.5 by tangential flow filtration prior to further concentration to a volume of 260 mL. The pH was adjusted to 9.5 with 5N NaOH and conductivity was measured. Buffer exchange continued until the conductivity reached approximately 0.8–1.4 mS/cm. 2.3.2. Q Sepharose FF Approximately 46.25 mL of Q Sepharose Fast Flow (Amersham Biosciences) gel was packed into a Millipore Vantage L column (VL 32 × 250) at linear flow rate of 410 cm/h, following the manufacturer’s instructions. The column was equilibrated with 6.5 column volumes of 20 mM ethanolamine buffer, pH 9.5 at a linear flow rate of 300 cm/h. The eluate was concentrated and adjusted for pH and conductivity and was then applied to the Q-Sepharose FF column at a linear flow rate of 300 cm/h. The column was washed with 11 column volumes of 20 mM ethanolamine buffer, pH 9.5, followed by 13 column volumes of 20 mM ethanolamine buffer, pH 9.5 with 50 mM NaCl. The Na-ASP2 recombinant protein was eluted with 10 column volumes of 20 mM ethanolamine buffer pH 9.5, containing 150 mM NaCl. The eluted fractions containing Na-ASP-2 recombinant protein (as determined by SDS-PAGE) were concentrated nine-fold to 50 mL using a tangential flow filtration system. The concentrate was exchanged with 1 L of phosphate buffered saline (PBS: 1.04 mM KH2 PO4 , 2.97 mM Na2 HPO4 ·7H2 O, 154 mM NaCl, pH 7.4) and then further concentrated to 175 mL. 2.3.3. Sephadex G-25 Fine Approximately 200 g of Sephadex G-25 Fine resin (Amersham Biosciences) was swollen overnight in 2 L of PBS and a Millipore Vantage column (VL 44 × 500) was packed with 745 mL of this medium at a linear flow rate of 315 cm/h. The column was equilibrated with 2 column volumes of PBS at the flow rate of 216 cm/h. The purified and concentrated

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Na-ASP-2 was applied to a Sephadex G-25 column at a linear flow rate of 216 cm/h and then eluted with 0.4 column volumes of PBS. The eluted peak fractions were analyzed by SDS-PAGE. The protein concentration of the pooled fractions from the Sephadex G-25 Fine column was determined by measuring the OD280 and by using the theoretical extinction coefficient calculated for the Na-ASP-2 protein [24]. 2.4. SDS-PAGE, N-terminal sequencing, mass spectrometry and monosaccharide analysis SDS-PAGE was performed on 4–20% gradient Trisglycine polyacrylamide gels using an Xcell mini cell apparatus (Invitrogen) under denaturing and reducing conditions. The gels were stained overnight with Commassie Brilliant Blue (CBB) R-250 (Bio-Rad). N-terminal sequencing was performed by Edman degradation as described previously [16]. A Matrix Assisted Laser Desorption Ionization (MALDI) spectrum of recombinant Na-ASP-2 was recorded on a MALDI-Time of Flight (TOF) Mass Spectrometer Model # AXIMA-CFR (Kratos Analytical) using a linear mode for the detection of positive ions at the Proteomics Core Facility of The George Washington University (GWU). The instrument was externally calibrated with horse myoglobin. The MALDI matrix was sinapinic acid (10 mg/mL in water/acetonitrile 1:1 containing 0.1% trifluoroacetic acid). Monosaccharide analysis was performed by anion exchange chromatography with pulsed amperometric detection [25] at the Biologics Testing Facility, BioReliance Corporation, Rockville, MD. 2.5. Size exclusion high pressure liquid chromatography (SE-HPLC) SE-HPLC was performed using a Waters Liquid chromatography system (600S Controller, 626 Pump, and 2487 Dual Absorbance Detector) in combination with Size Exclusion and Guard Columns—G2000SWXL and SWXL, respectively (TSK-GEL-HPLC Column, from TOSOH Bioscience). Twenty micrograms of purified Na-ASP-2 protein in 50 ␮L of 50 mM sodium acetate buffer, pH 4.8 was injected into the size exclusion column through the aperture of the Rheodine Injector of a Waters 616/626 Pump and eluted with the same buffer at a flow rate of 0.25 mL/min. Qualification studies determined the range of detection (elution times and chromatograms of the Absorbance at OD280) of molecular weight controls. The limit of detection was 0.2 ␮g/50 ␮L of sample, which corresponded to a chromatogram peak with amplitude of 2 mV peaks. 2.6. Formulation of purified recombinant Na-ASP-2 protein with Alhydrogel® The recombinant Na-ASP-2 protein was adsorbed to Alhydrogel® (Superfos Biosector, NY) by adding 1.5 mL of a 3 mg/mL solution of Alhydrogel® to 8.5 mL of a 0.4 mg/mL

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solution of the protein. Aliquots (0.5 mL) were collected at 0, 5, 10 and 30 min and 1, 2, 4, 7 and 24 h, and centrifuged for 10 s at 16,000 × g to sediment the adsorbed protein. The supernatants were retained. To confirm that the protein bound to Alhydrogel® the starting material and unbound supernatants were analyzed by SDS-PAGE and CBB R-250 staining with densitometry using a desktop digital imaging system (Beckman Coulter, CA) and an optical scanner. Desorption of the Na-ASP-2 recombinant protein from Alhydrogel® was also performed by mixing equal amounts of formulated material and 20% sodium citrate (w:v in water). Aliquots (0.5 mL) were taken at 0, 10, 30 and 60 min and centrifuged at 16,000 × g for 10 s to sediment the Alhydrogel® . Supernatants were separated by SDS-PAGE and the percent desorption was calculated following CBB R-250 staining and densitometry as described above. 2.7. Immunological potency Immunological potency studies were conducted under the approval of a GWU Institutional Animal Care and Use Committee (Protocol 24-8,3). Randomly bred Sprague Dawley Rats (Harlan Laboratories) were received, acclimated for 7 days and examined prior to study initiation. The rats (5 per group) were vaccinated by intramuscular injection on days 0, 10 and 20. The vaccines consisted of 50 ␮g of recombinant Na-ASP-2, either bound with Alhydrogel® (300 ␮g) or nonadjuvanted in a volume of 0.1 mL; an additional group was injected with Alhydrogel® alone. Adjuvant formulation was carried out as described above by mixing the Alhydrogel® and the protein and stirring the mixture for 10 min at room temperature. The vaccine was prepared in a laminar flow hood to assure sterility of the product. Blood was collected on the day prior to beginning the vaccination series (T1, day 1), on the day of the second vaccination (T2, day 10) and at 10 days following the final vaccination (T3, day 30), when the study was terminated. Rat anti-Na-ASP-2 IgG titers were measured by indirect ELISA. To coat 96-well microtiter plates (NUNC Maxisorb F96 Fisher Scientific) with antigen, 1 ␮g/mL solution of recombinant Na-ASP-2 in 50 mM carbonate bicarbonate, pH 9.6 was incubated overnight at 4 ◦ C. After washing, the plates were blocked for 2 h at room temperature with PBS-Tween 20 (0.5%) and washed. Experimental rat sera were added to the plates and serially diluted. The plates were incubated at 37 ◦ C for 3 h and washed before 100 ␮L of 1:1000 horseradish peroxidase (HRP)-conjugated anti-rat total IgG (Zymed Laboratories, CA) diluted in PBS/Tween 20 (0.05%) was added to the plates prior to incubation for 1.5 h at room temperature. After washing, 100 ␮L per well of o-phenylenediamine substrate was added to each well and incubated in the dark for 15 min, when 50 ␮L of 2N H2 SO4 was overlaid to stop the reaction. Colorimetric reaction was read at a wavelength of 492 nm on a SpectraMax 340 PC (Molecular Devices) using SOFTmax Pro for Windows for data capture. Rat anti-NaASP-2 antibody responses were also analyzed by Western

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blot using either recombinant Na-ASP-2 or N. americanus L3 lysates as antigen as described previously [14], but using pooled rat anti-Na-ASP-2 antiserum at a dilution of 1:4000 and goat anti-rat IgG-HRP (Zymed Laboratories) diluted to 1:2000. 2.8. N. americanus larval migration inhibition assay Larval migration inhibition assays were adapted from a previously described method [26]. Briefly, fresh hamster skin was shaved, and approximately 4 cm2 section of skin was stretched and sandwiched between 2 × 20 mL syringe barrels that were clamped together with bulldog clips. The lower syringe was filled to the top with PBS so that the buffer was in contact with the underside of the skin. N. americanus L3, obtained from infected hamsters, were incubated with pooled sera (neat) from either rats immunized with Na-ASP-2 formulated with Alhydrogel® or rats injected with Alhydrogel® . The sera were obtained from the rats used in the immunological potency study described above. For these studies, the L3 (300 L3/group) were then incubated in 0.05 mL of undiluted serum pooled from either the Na-ASP-2-immunized rats or Alhydrogel® -injected rats for 1 h at 37 ◦ C. The L3 together with the antiserum were then applied to hamster skin to observe whether serum antibodies interfered with the migration in vitro. Each group of L3 was then placed on the upper side of the skin and allowed to migrate for 30 min at room temperature. L3 that remained on the surface of the skin were collected and counted by removing the remaining liquid with a pipette and washing the skin three times with 0.5 mL of PBS. The number of L3 in three different aliquots of 0.05 mL was counted, and the resulting mean was multiplied by a dilution factor of 30. To express the percentage of larval inhibition by the antiserum, the total number of L3 recovered was divided by 300 and multiplied by 100. Each assay was performed in triplicate.

3. Results 3.1. Identification and cloning of Na-asp-2 Two positive clones were obtained by screening the N. americanus L3 cDNA library with the radiolabeled Ac-asp-2 cDNA (28–767 bp) heterologous probe. Each clone contained a 731 bp cDNA encoding an open reading frame (ORF) of 206 amino acids with 65% amino acid identity with Ac-ASP-2. The Na-asp-2 cDNA sequence contained a 3 poly(A) tail but no translation initiation codon (ATG) at the 5 -end. To identify additional Na-asp-2 cDNAs, further screening of 5 × 105 plaques was undertaken using a probe designed from the conserved regions of Ac-asp-2 (87–275 bp and 465–598 bp) compared with other asp-2 genes from different species of hookworm. This resulted in the identification of 14 additional clones, each of which was nearly identical (99% identity at the nucleotide level) with the Na-asp-2 cloned during the ini-

tial screening. Minor nucleotide mutations (eight changes) were found among the 14 Na-asp-2 sequences. Each was a silent substitution except for the nucleotides at position 119. Ten of the 14 different sequences exhibited a T at position 119, while the other four exhibited an A; this resulted in an amino acid substitution from Leu-36 to Met-36 in the predicted open reading frame. The 5 end of Na-asp-2 was isolated from N. americanus L3 cDNA by rapid amplification of cDNA ends (RACE) using the GeneRacer kit and 5 GeneRacer primer. The full-length Na-asp-2 cDNA consisted of 753 bp encoding an ORF of 209 amino acids and a poly-A tail at the 3 end. There was a hydrophobic signal peptide sequence with a signal peptidase cleavage site between amino acids 18 and 19. The predicated ORF of mature Na-asp-2 (without signal peptides) had a calculated molecular mass of 20,621 Da, and a predicted pI of 8.00. There were no putative N-linked glycosylation sites detected in the sequence. The nucleotide and deduced amino acid sequence of Na-asp-2 is available in GenBank under accession number AY288089. Amino acid sequence comparisons revealed that Na-ASP2 exhibited 58–65% identity to other ASP-2 molecules from other hookworms of the genus Ancylostoma [16]. Compared to ORFs of Ancylostoma spp. ASP-2 molecules, Na-ASP-2 contains fewer amino acids including an eight amino acids deletion at the C-terminus, one amino acid deletion at position 21 and two amino acids deletion at position 146–147. Therefore, the Na-ASP-2 is approximately 1.0 kDa less in predicted molecular mass relative to the Ancylostoma ASP2 molecules. In addition, Na-ASP-2 was noted to contain a DSG signature sequence that was found previously in a PRP protease from cone snails [27]. 3.2. Expression and purification in Pichia pastoris The Na-asp-2 cDNA was cloned into the expression vector and transformed into P. pastoris, which was used to generate seed stocks for expressing recombinant Na-ASP2 at the 10 L scale and the 60 L scale, respectively (data not shown). Fermentation of the seed stocks resulted in wet cell weights ranging from 408 to 466 g/L and purified protein yields of 0.24–0.40 g/L (data not shown). Only biochemical data resulting from the 60 L fermentation is reported here, whereas immunological potency data will be reported from the 60 L fermentation and a 10 L fermentation. After 65 h of methanol induction the wet cell weight of P. pastoris cells from the 60 L scale fermentation was 454 g/L and yielded 0.4 g of purified recombinant protein per liter of BSM. The recombinant Na-ASP-2 protein was expressed as a secreted recombinant protein in yeast supernatants, and was purified using three major steps comprised of two ionexchange chromatography steps followed by desalting on Sephadex G-25 (Fig. 1). Chromatography with the cationic exchange resin SP Sepharose FF was effective in the group separation of Na-ASP-2. Relative to the starting material, the recombinant protein eluted together with only minor

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Fig. 1. Column elution profiles during the purification of recombinant Na-ASP-2: (a) SP Sepharose FF; (b) Q Sepharose FF; (c) Sephadex G-25 Fine, during the purification of Na-ASP-2. (d) SDS-PAGE analysis of the column eluates during Na-ASP-2 purification: lane 1, SeeBlue® Plus2 Pre-Stained Standard (Invitrogen); lane 2, concentrated supernatant from the 60 L fermentation; lane 3, SP Sepharose FF elution with 50 mM sodium acetate, pH 4.8 with 200 mM NaCl; lane 4, Q-Sepharose FF elution with 20 mM ethanolamine buffer pH 9.5 with 150 mM NaCl; lane 5, Sephadex G-25 Fine elution with PBS, pH 7.4.

impurities from SP Sepharose using 200 mM NaCl in a stepwise gradient (Fig. 1(a) and (d)). Further purification was achieved with anionic chromatography on Q-Sepharose FF (Fig. 1(b) and (d)), which resulted in highly enriched recombinant protein (Fig. 1(c) and (d)). Following purification, NaASP-2 was the only protein detected by SDS-PAGE and CBB staining, migrating with an apparent molecular weight of approximately 21 kDa. In order to estimate its purity, the protein was subjected to SE-HPLC that was qualified previously to a limit of detection of 0.2 ␮g. This detection limit corresponds to the appearance of a chromatogram peak equivalent to 2 mV at OD 280. Based on the calculated limits of detection it was determined that the recombinant Na-ASP-2 was greater than 95% pure (Fig. 2(a) and (b)). Additional studies using SDS-PAGE followed by silver staining confirmed the absence of additional contaminants (data not shown).

vector (after cleavage of the ␣-mating factor signal peptide) and GCPD represents the start of the Na-ASP-2 mature protein. The purified recombinant protein with the 6 amino acid vector tag has a predicted molecular mass of 21.3 kDa (21,298 Da), a finding in agreement with its apparent migration on SDS-PAGE as well as mass spectrometry. The MALDI-TOF spectrum of the recombinant Na-ASP-2 protein obtained revealed a single major species that corresponded to a molecular mass of 21.3 kDa and a minor species with an additional 161 Da of mass (Fig. 3). Since there are no N-linked glycosylation sites on the Na-ASP-2 ORF, the second species could correspond to a protein with an O-linked monosaccharide. This was confirmed by carbohydrate analysis that revealed the presence of 0.483 moles of mannose per mole of recombinant Na-ASP-2 (Table 1).

3.3. Biochemical characterization

Table 1 Analysis of monosaccharides in purified recombinant Na-ASP-2

The recombinant protein was expressed with an additional 6 amino acid EAEAEF N-terminal sequence, which corresponds to pPICZ␣ vector sequence when the EcoR1 site is used for cloning. It was noted that the addition of the sequence improved the yield of the secreted recombinant protein (data not shown). N-terminal amino acid sequencing of the first 10 residues of Na-ASP-2 by Edman degradation revealed the sequence EAEAEFGCPD, confirming the predicted sequence where EAEAEF is derived from the plasmid

Sugar

Quantity (mole/mole)a

Fucose Galactosamine Glucosamine Galactose Glucose Mannose

0 0 0 0 0 0.483b

a Mole/mole = pmol of each monosaccharide observed/total pmol per sample injected. b 113.3 pmol/234.8 pmol.

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Fig. 2. HPLC profile after purification of recombinant Na-ASP-2. Panel A shows the HPLC chromatogram of 50 ␮g (total) of Gel Filtration Standards (Bio Rad cat# 151-1901) comprised of five proteins, run at 0.25 mL/min. The profile denotes six peaks of which molecular weights can be determined. From left to right, peak 1: protein aggregates, peak 2: thyroglobulin (670 kD), peak 3: gamma globulin (158 kD), peak 4: ovalbumin (44 kD), peak 5: myoglobin (17 kD), peak 6: Vitamin B12 (1.35 kD). Panel B shows 20 ␮g of Na-ASP-2 run on same size exclusion column at 0.25 mL/min for 55 min. The elution time was determined to be between 38 and 42 min. This corresponds as expected between peak 4 (44 kD) and peak 5(17 kD) of the gel filtration standards shown in panel A. This qualified assay was shown to have a sensitivity of 0.2 ␮g equivalent to a 2 mV peak (data not shown).

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Fig. 3. MALDI-TOF spectrum of Na-ASP-2. The single ion species exhibited a molecular mass of 21,311.3. Two double ion species of 10,653.8 and 10,734.2 Da were also noted which correspond to molecular mass of 21,307.6 and 21,468.4 Da, respectively, as well as a triple ion species of 7106.7, which corresponds to a molecular mass of 21,320.1 Da.

3.4. Rat immune responses to vaccination The adsorption of recombinant Na-ASP-2 to Alhydrogel® was greater than 75%. The protein remained adsorbed to Alhydrogel® for up to 24 h since no unbound Na-ASP-2 was detected in the supernatants of the formulation when analyzed by SDS-PAGE (data not shown). To determine immunological potency, Sprague Dawley rats (n = 5) were vaccinated three times at 10-day intervals with a dose of 50 ␮g NaASP-2, with or without adjuvant (Alhydrogel® , 300 ␮g). The control group was vaccinated with the same dose of adjuvant. As shown in Table 2, vaccination of rats with two different batches of the Alhydrogel-formulated recombinant protein induced a marked antibody production (total IgG) with geometric mean titers ranging from 1:9784 to 1:20,949 after two doses, and 1:97,533 to 1:234,881 after three doses, Table 2 Mean total IgG titers (determined by ELISA) from sera of rats (n = 5) vaccinated three times (T1, day 0; T2, day 10; T3, day 20) at 10-day intervals with three different lots of the Na-ASP-2 Hookworm Vaccine containing 50 ␮g Na-ASP-2 and 300 ␮g Alhydrogel® Fermentation (L)

T1 (day 0)

T2 (day 20)

T3 (day 30)

10 60

<100 <100

9,784 20,949

234,881 97,533

Fig. 4. Identification of native Na-ASP-2 by Western blot with pooled rat antiserum raised against recombinant Na-ASP-2. Twenty-five nanograms of recombinant Na-ASP-2 (lane 1) and approximately 10 N. americanus L3 in 4 ␮L (lane 2) were homogenized in SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to a PVDF membrane. The Western blot was probed with a 1:4000 dilution of pooled rat anti-Na-ASP-2 antiserum (A). The same rat antiserum absorbed with recombinant Na-ASP-2 was used as a control serum (B). Arrows point to the putative Na-ASP-2.

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Fig. 5. Inhibition of N. americanus L3 hamster skin migration by rat antiNa-ASP-2 antiserum. For these studies, 300 N. americanus L3 were applied in buffer and the number of non-penetrating L3 was counted. To express the percentage of larval inhibition by the antiserum, the total number of L3 recovered was divided by 300 and multiplied by 100. The percentage inhibition resulting from either rat-anti-Na-ASP-2 antiserum or antiserum obtained from Alhydrogel® -injected rats is expressed as the mean ± standard deviation of assays performed in triplicate.

respectively. In contrast, immunization with the recombinant protein alone (unadjuvanted protein) elicited IgG titers less than 1:100 (data not shown), suggesting that the presence of Alhydrogel® is necessary for the hookworm vaccine to be immunogenic. Antiserum from rats injected with Alhydrogel® alone also contained IgG titers less than 1:100 (data not shown). On western blots the rat anti-Na-ASP-2 antiserum recognized both the recombinant protein as well as a band of approximately 21 kDa in N. americanus L3 extracts, which corresponds to the native protein (Fig. 4). As shown in Fig. 5, the rat anti-Na-ASP-2 antiserum inhibited larval skin migration 90 ± 7% in three different experiments compared with 17 ± 7% inhibition by serum from rats injected with Alhydrogel® (P = 0.019).

4. Discussion Here we describe the expression, purification and characterization of Na-ASP-2, a lead candidate antigen under development as a human hookworm vaccine. We also show that anti-Na-ASP-2 antibody inhibits larval migration in vitro, providing evidence for the role of this protein in host tissue migration by N. americanus L3. Phylogenetic analysis of the four hookworm ASP-2 molecules studied to date (one from N. americanus and three from different members of the genus Ancylostoma) reveals that they constitute a unique cluster of the PR-1 family of the PRP superfamily [12,14,16]. The cluster also includes Hc24, a protective vaccine antigen from the ruminant trichonstrongyle nematode, Haemonchus contortus [14,28]. No function has yet been assigned to any hookworm or trichostrongyle ASP-2. With the exception of the cone-snail PRP [27], which was revealed to be a protease, and the P14

plant PRP, which has antifungal properties [29], no function has been assigned to any of the PR-1 family proteins. The cone snail protease contains a DTG signature sequence, which is believed to have a pivotal role in enzyme catalysis [27]. Na-ASP-2 contains a DSG sequence, in which a serine may possibly substitute for a catalytic threonine. Although no protease activity for Na-ASP-2 has been detected so far (data not shown), our finding that anti-Na-ASP-2 antibody inhibits larval skin migration, suggests that the molecule has an important role in larval host entry and in migration through the tissues and pulmonary capillaries before reaching the intestine. This could provide the basis by which anti-ASP-2 antibodies are protective against larval challenge infections [16,17]. Hookworm infection is a disease of the world’s poorest, occurring almost exclusively in areas with poor sanitation and high levels of poverty [1,19]. Such a small commercial market demands that the production of recombinant hookworm antigens formulated for vaccines must be made in high yield and at low cost [19]. Although employment of Escherichia coli for expression of hookworm antigens ordinarily would meet these criteria, we have so far been unable to refold or solubilize either ASP-1 or ASP-2 from E. coli inclusion bodies [10]. Other investigators have also reported failure to express soluble PR-1 proteins [30], most likely because the high cysteine content causes improper protein folding secondary to aberrant disulfide bond formation [10]. Na-ASP-2 contains 11 cysteines. Expression of Na-ASP-2 in P. pastoris resulted in a secreted product that was soluble and did not require refolding. Codon optimization was not required in order to express Na-ASP-2 in high yield (data not shown). It was also determined that the addition of an acidic N-terminal EAEAEF sequence increased the amount of ASP-2 protein secreted into the fermentation broth. This sequence represented six amino acids of pPICZ␣A vector sequence that was introduced by incorporating an EcoRI restriction site into the cloning. We do not know if the addition of this sequence would increase the yield of other P. pastoris-expressed proteins. Capture of the Na-ASP-2 recombinant protein was achieved by an efficient SP Sepharose FF purification step. Subsequent elution of the highly enriched protein required only an additional anion-exchange chromatography step using Q-Sepharose FF and a desalting step with Sephadex G-25 Fine in order to obtain highly pure protein of consistently high quality. Further studies revealed that the fermentation and purification process was robust and resulted in a highly uniform, consistent, pure and stable recombinant protein. Using qualified assays for pH, color and appearance, identity (SDS-Silver Stain and Western Blots), and purity (SE-HPLC and SDS-PAGE Silver Stain), immune recognition (Indirect ELISA and Western Blots) and immunological potency, no major differences were detected among different pilot lots. The process development technologies reported here are sufficiently simple so that they could be transferred to manufacturers in middle-income countries. These would

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include countries where hookworm infection is endemic such as Brazil, India, and China [19]. Technology transfer to a manufacturing facility will be followed by toxicity and stability studies, and then a dose-escalating Phase 1 human trial to evaluate safety, tolerability, and immunogenicity of the Na-ASP-2 Hookworm Vaccine. To our knowledge this will be the first clinical trial attempted for a vaccine against any human nematode. Ultimately, immunization with the Na-ASP-2 Hookworm Vaccine would be indicated for the prevention of hookworm disease caused by high intensity infection with N. americanus. Vaccination would be expected to reduce the number of infective hookworm L3 that enter into the human gastrointestinal tract and become adult worms. This would reduce both the number of hookworms and the fecal egg counts in individuals exposed to L3 living in the soil.

Acknowledgements We wish to thank Drs. Allan Saul and Louis Miller of the Malaria Vaccine Development Branch of the NIAID, NIH for allowing us to use their fermentation facilities. These studies were supported by the Human Hookworm Vaccine Initiative of the Bill and Melinda Gates Foundation and the Sabin Vaccine Institute. JB is supported by an International Research Scientist Development Award (1K01 TW00009) and AL is supported by an RD Wright Career Development Award from the National Health and Medical Research Council of Australia. We would like to acknowledge Andrew Murdin, Mike Fletcher and Annie Dookie from Aventis Pasteur, Toronto, CA for helping optimize the Na-ASP-2 vaccine formulation.

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