Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm...

©2005 FASEB

The FASEB Journal express article 10.1096/fj.05-3936fje. Published online July 21, 2005.

Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals Jeffrey Bethony,* Alex Loukas,† Michael Smout,† Simon Brooker,‡ Susana Mendez,* Jordan Plieskatt,* Gaddam Goud,* Maria Elena Bottazzi,* Bin Zhan,* Yan Wang,* Angela Williamson,* Sara Lustigman,§ Rodrigo Correa-Oliveira,║ Shuhua Xiao,¶ and Peter J. Hotez* *Department of Microbiology and Tropical Medicine, The George Washington University, Washington DC; †Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, and Australian Centre for International Tropical Health and Nutrition, The University of Queensland, Queensland, Australia; ‡ Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom; §Lindsley F. Kimball Research Institute, New York Blood Center; ║Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil; ¶Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China

Corresponding authors: Jeffrey Bethony or Peter Hotez, Department of Microbiology and Tropical Medicine, The George Washington University, Ross Hall, Room 736, 2300 Eye St. NW, Washington DC 20037. E-mail: [email protected] or [email protected]

ABSTRACT

The development of a vaccine would provide an important new tool for the control of human hookworm infection. On the basis of successful vaccination of laboratory animals with living irradiated, third-stage hookworm larvae (L3), we examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant L3 secreted antigens, the ancylostoma secreted proteins, ASP-1 and ASP-2. Logistic regression was used to investigate the effects of antibody isotype responses to ASPs on the risk of an individual harboring heavy hookworm infection. A significant protective association was observed between increasing anti-ASP-2 IgE levels and the risk of heavy hookworm infection. To confirm that ASP-2 is a protective antigen, laboratory dogs were immunized with recombinant ASP-2 formulated with the GlaxoSmithKline Adjuvant, AS03. Sera obtained from the immunized dogs exhibited high geometric mean antibody titers, immunoprecipitated native ASP-2 from L3 extracts and localized the site of ASP-2 expression to the glandular esophagus and body channels exiting to the cuticle. The sera also exhibited an increased ability to inhibit migration of L3 through tissue in vitro relative to sera from AS03-injected controls. Upon L3 challenge, the ASP-2 vaccinated dogs exhibited significant reductions in fecal egg counts and intestinal hookworm burden. These findings provide strong support for the development of an effective recombinant vaccine against hookworm infection in humans.

Key words: ancylostoma secreted proteins • morbidity reduction • vaccine

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ookworm infection occurs in 740 million people living in developing countries where it is a major cause of intestinal blood loss, iron-deficiency anemia, protein malnutrition, and disability-adjusted life years lost (1–3). Hookworm control in endemic regions is

based on the principle of morbidity reduction through periodic deworming with benzimidazole anthelminthic drugs, including mebendazole and albendazole (4, 5). However, the variable efficacy of benzimidazoles for hookworm infection (6), the diminishing efficacy of benzimidazoles with repeated use (6) and the rapid reinfection that occurs following anthelminthic treatment (7) have made the successful development of an antihookworm vaccine a public health priority (8–10).

During the 1960s and 1970s, laboratory animals were successfully vaccinated with irradiated third-stage infective hookworm larvae (L3) (11). On the basis of the importance of L3-secreted products in mediating the protection induced by the irradiated vaccine (12), we examined the antibody responses of naturally infected individuals against the most abundant molecules released by hookworm L3 upon entry into the host, the ancylostoma secreted proteins (ASPs): ASP-1 and ASP-2 (13, 14). Both molecules are cysteine-rich secretory proteins belonging to the pathogenesis-related protein (PRP) superfamily (15). Although the function of the ASPs is unknown, their secretion by the parasite during the early stages of host invasion suggests that they have a key role in the molecular pathogenesis of infection (16).

Here, we report that individuals from Brazil and China who produced IgE against recombinant ASP-2 had a decreased risk of heavy hookworm infection. We subsequently confirmed that ASP-2 is a protective antigen in vaccinated laboratory dogs as evidenced by reductions in egg counts, worm burden, and an increased ability of sera to inhibit migration of L3 through tissue in vitro relative to controls. These findings provide an experimental basis for selecting ASP-2 for development as a recombinant vaccine against human hookworm infection.

MATERIALS AND METHODS

Recombinant ASP antigens

Expression and purification

Recombinant ASP-1 from the hookworm, Necator americanus (17), was expressed in the yeast Pichia pastoris and purified as described previously (18). Recombinant ASP-2 from the hookworm Ancylostoma caninum (14) was expressed in Spodpotera frugiperda Sf9 insect cells using the pMIB-V5/His expression system (Invitrogen, Carlsbad, CA). The entire ASP-2 open reading frame (GenBank accession number AF 089728) minus the N-terminal signal peptide (from Asn-18 to the C-terminal Gly-218) was cloned into pMIB-V5/HisA using the SphI and XbaI restriction sites so that the recombinant ASP-2 was fused in-frame with the vector-derived N-terminal melittin signal peptide and C-terminal V5 and 6-His epitopes. Sf9 cells were grown in Excell 420 medium (JRH Bioscience, Brooklyn, Victoria, Australia) and transfected with ASP-2 plasmid midi-prep (Qiagen, Valencia, CA) and Genejammer transfection reagent (Stratagene) according to the manufacturer’s instructions. Transfected cells were selected using Blasticidin S (Cayla, Toulouse, France) at a final concentration of 25 μg/ml in six well plates and maintained in 10 μg/ml blasticidin after selection. Selected cells were transferred successively from adherent populations to shaker flasks, according to the manufacturer’s instructions (Invitrogen). Stably selected cells in log phase were then used to seed a total of 4 L of Excel 420 medium to a final

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cell density of 1.0 × 106 cells/ml in a Bioflo 110 bioreactor (New Brunswick Scientific, Edison, NJ) with a 7.5-L vessel. The cells were maintained at a temperature of 27°C and stirred at 70 rpm in the presence of 55–80% dissolved O2. The pH was not adjusted and remained between 6.1–6.4. Cells were grown until a cell density of 1.0 × 107 cells/ml was attained. Supernatant was harvested by centrifugation at 4,000 g and concentrated 10-fold by ultrafiltration using a 10-kDa cut-off ultrasette membrane (Pall Corporation, East Hills, NY) and peristaltic pump. Concentrated supernatant containing recombinant ASP-2 was buffer-exchanged into milliQ H2O followed by binding buffer (0.05 M NaH2PO4, 0.3 M NaCl, 10 mM imidazole, pH 8.0) before being applied to a nickel-NTA agarose column (Novagen, Madison, WI) with a settled bed volume of 2.0 ml. The column was washed with 10 volumes of binding buffer followed by 5 column volumes each of 20, 40, and 60 mM imidazole in binding buffer. Proteins were eluted in 5 column volumes of 250 mM imidazole in binding buffer. Fractions were assessed for recombinant protein and resulting purity by sodium dodecyl sulfate PAGE (SDS-PAGE) using precast 4–20% Tris-glycine gradient minigels (Invitrogen) stained with Coomassie Brilliant Blue. Fractions containing purified protein were pooled, concentrated and buffer-exchanged into PBS, pH 7.2 at 4°C. Protein concentration was determined using a micro BCA kit (Pierce, Rockford, IL).

Biochemical characterization of the ASP antigens

Recombinant ASP-1 was characterized as described previously (18). Recombinant ASP-2 was analyzed by SDS-PAGE (as described above) and by mass spectrometry. A matrix-assisted desorption ionization (MALDI) spectrum of recombinant ASP-2 was recorded on a MALDI-time of flight (TOF) mass spectrometer, model #AXIMA-CFR (Kratos Analytical, Chestnut Ridge, NY) using a linear mode for the detection of positive ions at the Proteomics Core Facility of The George Washington University. The instrument was externally calibrated with horse myoglobin. The MALDI matrix was sinapinic acid (10mg/ml in water/acetonitrile 1:1 containing 0.1% trifluoroacetic acid.

To produce a rabbit polyclonal antibody against recombinant ASP-2, 50 μg of the recombinant protein was emulsified in Freund’s complete adjuvant and injected intramuscularly into a white rabbit. The rabbit was subsequently boosted two times with 50 μg of ASP-2 in incomplete Freund’s advjuvant. The reactivity of the rabbit antiserum with either recombinant protein or N. americanus larval extracts was tested by Western blotting, as described previously (19). N. americanus L3 were obtained from infected hamsters (20).

Human investigations

Patient sample and parasitological methods

A cross-sectional parasitologic and serologic survey was conducted in two hookworm-endemic regions of Hainan Province, China and Minas Gerais State, Brazil, respectively. A description of each testing site was reported previously (21, 22). For human investigations in China, ethics committee approval was obtained from the Institutional Review Board (IRB) of Yale University (Protocol 10932) for the years 1999–2000, and subsequently by the IRB of The George Washington University (GWU) (Protocol 080004). Verbal consent was obtained. For human investigations in Brazil, ethics committee approval was obtained from the National Ethics


Committee of Brazil, FIOCRUZ (Protocol 003/2000) and the IRB (Protocol 090303) of GWU. Verbal or written consent was obtained from all participants.

Houses were assigned a unique household identification number, and each resident was given a unique personal identity number. Fecal samples and a blood sample were requested of all residents aged ≥ 2 years. The presence of soil-transmitted helminth eggs was determined by saline float. Blood was obtained by venipuncture. For persons whose fecal sample tested positive, three subsequent fecal samples were obtained over the course of 3 days. Two slides from each day’s fecal sample were prepared ≤24 h after receipt by use of the Kato-Katz thick smear technique (23), and quantitative egg counts were obtained and expressed in eggs per gram. Egg-positive (infected) individuals were designated as either “lightly” (1–499 epg) or “heavily” infected (≥500 epg). These categories were established to reflect differences in the severity of the loss of fecal hemoglobin due to the intensity of hookworm infection (24). Hookworm species (Ancylostoma duodenale or N. americanus) was determined by morphological identification of L3 reared from eggs by coproculture. After collection of fecal samples for egg counts and blood for serum, all infected subjects were treated with albendazole. Individuals who refused to participate in the study were offered a fecal examination and treatment for any diagnosed helminth infection.

Human indirect ELISA

Serum samples (10 ml) were obtained from whole blood collected into siliconized tubes. An indirect ELISA was then used to study isotype responses of participants to three crude A. caninum antigen preparations (L3 soluble extract, adult worm soluble extract, and adult worm excretory/secretory [ES] products) and the two hookworm L3 recombinant proteins, ASP-1 and ASP-2. Nunc Maxisorb Surface 96-well plates (Nunc) were coated with 5 μg/ml (100 μl/well) of crude antigen preparation in 20 mM sodium bicarbonate/27 mM sodium carbonate, pH 9.6, and stored overnight at 4°C. For IgG2 assays, 96-well plates were adsorbed overnight at room temperature with 100 μl/well of Poly-L-lysine (PLL) at 1 μg/ml in 50 mM sodium carbonate, pH 9.0. Plates were then washed with PBS, and crude antigen was added and incubated in the manner described above. Plates were washed with PBST and blocked with 1% fetal calf serum. Serum samples were diluted 1:100 in PBST, and 100 μl was added to each well in duplicate. The following dilutions of horseradish peroxidase-conjugated anti-human antibodies (Zymed Laboratories, Burlingame, CA) were added to each well: 1:5,000 of IgG1; 1:1,000 of IgG2, IgG3, and IgG4; and 1:800 of IgE. Plates were washed and developed for 30 min with o-phenylenediamine (Sigma, St. Louis, MO) containing 0.03% hydrogen peroxide. For the IgE assay, other isotypes from serum were not preadsorbed because it did not affect the detection of IgE in our ELISA (data not shown). Assays were standardized as described elsewhere (25).

Human data entry and statistical methods

All data involving human subjects were collected under Good Clinical Practices. Data from case report forms (CRFs) were double-entered, with conflicts in double entry of data resolved by referring to the CRFs. Data from both study areas were then combined, with area coded as 0 for Brazil and 1 for China and transferred to SPSS 10.0 for analyses. Because of the difficulty interpreting serological data from egg-negative individuals living in hookworm endemic areas, analyses were limited to egg-positive individuals only, who were assigned either a 0 (1–499 epg) or a 1 (≥500 epg). Logistic regression was then employed. Age was divided into tertiles and


numbered 1–3, from youngest to eldest. Sex was coded as 0 for females and 1 for males. Plots of lowess smoothed logit vs. IgE and IgG4 levels supported treating them as linear variables in the logistic regression. A separate analysis adjusted for age, sex, and area was undertaken for the ratio of IgG4/IgE against ASP-2. All computations were done using SPSS 10.00 software. Tests of the hypotheses that a regression coefficient was 0 were done with the Wald χ2 for logistic regression.

Canine studies

Animal husbandry

The test and control animals were purpose-bred, parasite naïve male beagles 56 ± 4 days of age (body weights equal to or exceeded 2.5 kg, hematocrit equal to or exceeded 28.0, WBC did not exceed 20×106) on arrival. All dogs in a trial were purchased from the same vendor (Marshall Farms), identified by ear tattoo, and maintained in the GWU Animal Research Facility. The experiments were conducted according to a protocol approved by the GWU Animal Care and Use Committee (Protocol 48-12,1). Dogs were housed in groups for approximately 1 month and 1–3 animals per cage thereafter; controls were housed identically to test animals. After larval challenge, all dogs were individually housed.

Vaccine study design and antigen-adjuvant formulation

The vaccine trial was designed to test ASP-2 formulated with Adjuvant System 03 (AS03), which was obtained as a gift from Drs. Joe Cohen and Sylvie Cayphas of GlaxoSmithKline Biologicals (Rixensart, Belgium). The 10 purpose-bred beagles were randomized by the study statistician and allocated either to a vaccine or placebo group. The intervention assignment of each canine was restricted only to the study veterinarian (for the monitoring of severe adverse events), but kept from the investigators for the duration of the study. A single canine was removed from the experimental (vaccine) group because it became severely anemic (hematocrit lower than 30%) and developed intercurrent giardiasis, with both cysts and trophozoites detected in the diarrheic feces. This animal then received a 10-day course of metronidazole. This dog was excluded from the study because of its debilitated state

To prepare six doses of ASP-2/AS03, 600 μg of recombinant protein (0.3 ml of ASP-2 at a concentration of 2 mg/ml) was mixed with 1.2 ml of 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9 and 1.5 ml of AS03; the contents of the tube were vortex-mixed for 30 s, then shaken at low speed for 10 min. Dogs were immunized intramuscularly 4 times with 100 μg of ASP-2/AS03 or AS03 alone in a final volume of 0.5 ml, on days 0, 21, 42, and 64. AS03-only control was prepared as described above, with PBS included instead of ASP-2. Test and control articles were prepared on the day of injection.

Canine antibody measurements by Indirect ELISA

Blood was drawn at least once every 21 days, and serum was separated from cells by centrifugation. Each animal's specific antibody response was measured by an indirect ELISA, as described previously (26, 27). Briefly, recombinant ASP-2 was coated onto microtiter plates at a concentration of 5 μg/ml. Dog sera were titrated between 1:100 and 1:2 × 106 to determine endpoint titers. Anticanine IgG1, IgG2, and IgE antibodies conjugated to horseradish peroxidase (Bethyl Laboratories) were used at a dilution of 1:1,000. For the IgE assay, we did not preadsorb


other isotypes from serum because it did not affect the detection of IgE in our ELISA (data not shown).

Immunoprecipitation of native ASP-2 from larval extracts

Sera obtained from the ASP-2/AS03-vaccinated dogs were tested for their ability to immunoprecipitate native ASP-2 from A. caninum larval extracts. A. caninum L3 were obtained by coproculture of feces from infected dogs (28). L3 were homogenized and sonicated in PBS to generate soluble protein extract, which was then biotinylated using NHS-LC-biotin (Pearce) following the manufacturer’s instructions. The extract was dialyzed against PBS to remove free biotin. Ten microliters of biotinylated A. caninum L3 extract (bio-L3X) was added to 485 μl of PBST and 5 μl of antiserum (neat) and rotated overnight at 4°C. One hundred microliters of Protein A-agarose (50% slurry) was washed with 1.0 ml of PBST for 5 min at room temperature, added to the antigen-antibody complex, and rotated at room temperature for 1.5 h. The samples were then briefly centrifuged, and the supernatant removed and retained as unbound protein. Pelleted agarose was washed 3 times for 5 min with 1 ml of PBST and resuspended in 100 μl of reducing SDS-PAGE loading dye, mixed, and boiled for 5 min before a final centrifugation for 30 s. The supernatant (in SDS-PAGE loading dye) was retained and referred to as the bound sample. Ten microliters of the unbound material and 20 μl of the bound material were electrophoresed on 4–20% gradient gels (Invitrogen) at 120 V. Protein in the gel was transferred to nitrocellulose for 1 h at 250 mA. The membrane was blocked for 1 h at room temperature in 5% skimmed milk in PBS, probed with horseradish peroxidase-conjugated streptavidin (Amersham Biosciences, Castle Hill, Australia) diluted 1:5,000 in PBST/2.5% skimmed milk for 1 h at room temperature, washed an additional 3 times with PBST, and developed with enhanced chemiluminescence (ECL, Amersham Biosciences).

Immunolocalization

A. caninum L3 were fixed for 60 min in 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 1% sucrose and processed for immunoelectron microscopy, as described previously (29). Thin sections of embedded worms were probed with 1:100 dog or rabbit antiserum raised against recombinant mature ASP-2, before interaction with 15 nm gold-labeled goat anti-rabbit IgG (H+L; Amersham Biosciences) diluted 1:20. Sections stained with the dog antibody were then incubated with gold-labeled rabbit anti-dog IgG (50 μg/ml). Preimmune serum was used as the control.

In vitro L3 migration assays

Migration assays were conducted by a modification of a previously described method (30). Briefly, live A. caninum L3 were incubated with sera (neat) from ASP-2 vaccinated dogs or control dogs for 1 h at 37°C. L3 were then applied to canine skin to observe whether serum antibodies interfered with the migration of L3 through tissue in vitro. The ear of a dog was removed after euthanasia, shaved, and ~4 cm2 section of skin was stretched and sandwiched between 2 × 20 ml syringe barrels 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. One milliliter of PBS was placed on the skin for 15 min to check integrity of the skin. L3 (300 L3/group) were then incubated in 50 μl of PBS, pH 7.2, or undiluted serum from different vaccinated or control dogs for 30 min at 37°C. Each group of L3 was then placed on the upper


side of the skin (added to the 1.0 ml of PBS already present) 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. Experiments were conducted in triplicate. Nonparametric statistics (Wilcoxon signed-rank test) were used to test the difference between the number of L3 remaining on the skin between ASP-2 vaccinated dogs and control dogs. The baseline was determined by counting the number of L3 remaining on the skin in the presence of control dog serum. The number of L3 that did not migrate in the presence of anti-ASP-2 sera was calculated by subtracting this baseline and then determining the percent reduction in L3 migration.

Hookworm challenge infections and parasite recovery

A. caninum larvae were cultured as described above. All hookworms in the infective challenge were approximately equal age (17±7 days post hatching). The species identities of the infective larvae were confirmed using a species-specific polymerase chain reaction (31). All dogs were infected by the footpad method with the same single dose of 500 L3 of A. caninum (27). Because of the large number of dogs in this study, larval challenge was staggered and occurred on one of three consecutive days (at age 120±9 days), 14–16 days after the final immunization. Dogs were anesthetized using a combination of ketamine and xylazine (20 mg/kg and 10 mg/kg, respectively), and 500 A. caninum L3 in a final volume of 50 μl were applied to the footpad.

Canine data entry and statistical methods

For the animal studies, data were entered directly into SPSS 10.00. Because of the small sample size (n=9), we could not determine if the values were normally distributed so nonparametric methods of statistical inference were used. A Wilcoxon rank sum test was used to compare control vs. vaccine groups, which evaluates the null hypothesis that the medians of the two samples are identical. All two-sided tests were conducted at the 0.05 level of significance. Spearman correlation coefficient was used as a nonparametric measure of correlation between ordinal variables. For all of the cases, the values of each of the variables were ranked from smallest to largest, and the Pearson correlation coefficient was computed on the ranks.

RESULTS

Biochemical characterization of the recombinant ASP antigens

ASP-1 and ASP-2 are the two major antigens secreted by hookworm L3 after host entry (16). ASP-1 from N. americanus is a 406-amino acid protein with a predicted molecular weight of 44 kDa (17, 18). The recombinant ASP-1 was expressed as a secreted protein from P. pastoris and isolated by ion-exchange and size exclusion chromatography (18). Purified recombinant ASP-2 from A. caninum was expressed in insect cells as a soluble protein containing C-terminal V5 epitopes and 6x His epitopes, with a predicted molecular weight of 24.2 kDa. On SDS-PAGE, the recombinant protein migrates as a doublet between the 21 kDa and 30 kDa size markers (Fig. 1A). Both bands were recognized by monoclonal antibodies against the V5 and 6x His tags (data not shown). On mass spectrometry the predominant species exhibited a molecular mass of 24.5 kDa, and the second most abundant species exhibited a mass of 24.6 kDa (Fig. 1B). Endoglycosidase digestion removed the higher molecular weight band seen on SDS-PAGE (data not shown) implying that the doublet reflects glycosylated and nonglycosylated forms of the


protein. ASP-2 contains a single putative N-linked glycosylation site (14). As shown in Fig. 1C, antibody produced against ASP-2 from A. caninum recognized both the recombinant protein and the native protein from N. americanus on Western blots.

Human anti-ASP-2 antibodies are associated with a significantly decreased risk of heavy hookworm infection

Details of the Brazilian (Minas Gerais State) and Chinese (Hainan Province) study samples can be found in Table 1. The mean age for these two populations was 36 and 33 years, respectively. The prevalence of hookworm infection was 54% in the Brazilian endemic area and 59% in the Chinese endemic area. The mean quantitative fecal egg counts were 1,645 epg in Brazil, and 1,723 epg in China. Because 20% of the study sample from each site had epg values over 3,999, both study sites can be considered high transmission sites with respect to hookworm infection by WHO criteria (4). N. americanus was identified as the predominant hookworm in both regions (data not shown).

Logistic regression was then used to investigate the effects of antibody levels on the risk of an individual harboring a heavy hookworm infection. After controlling for age, sex, and the study area (China or Brazil), increasing IgE anti-ASP-2 levels protected against heavy hookworm infection (Odds Ratio [OR]=0.38; 95% confidence interval [CI]=0.152, 0.959; P<0.001); that is, increasing IgE anti-ASP-2 levels reduced the risk of heavy hookworm infection by 62% (Table 2). In contrast, a significant positive association was observed on the risk of heavy hookworm infection and increasing IgG4 anti-ASP-2 levels (OR=4.97; 95% CI=1.87, 13.19; P<0.0001). In a separate age, sex, and area-adjusted analysis (data not shown), a significant positive association was observed for the ratio of IgG4/IgE anti-ASP-2 (OR=2.81; 95% CI=1.410, 5.598; P<0.0001). These findings suggest that heavy infection is influenced by a balance between the protective effects of IgE and the risk effects of IgG4 against ASP-2. Additional analysis did not show significant relationships between antibody responses to crude hookworm antigen extracts or recombinant ASP-1 (data not shown). These results suggested that host antibody against ASP-2 could influence worm burdens and led us to test ASP-2 as a vaccine antigen in an animal model of hookworm infection.

Vaccination of dogs with ASP-2 elicits antibodies that recognize native, hookworm-derived ASP-2

To confirm that ASP-2 was a protective antigen when used as a vaccine, a randomized, controlled vaccine trial was conducted using helminth-naïve, purpose-bred beagle pups, who received either an intramuscular injection with four doses of recombinant Ac-ASP-2/AS03 (intervention) or four doses of the adjuvant AS03 alone (control). Sera obtained from the immunized dogs before challenge exhibited high anti-ASP-2 IgG1 and IgG2 titers (geometric mean antibody titers [GMAT] of 13,500 and 63,850, respectively), but only moderate IgE titers (GMAT of 1,204) (Fig. 2A). IgG from canines vaccinated with ASP-2 immunoprecipitated the native protein from L3 extracts (Fig. 2B), indicating that IgG from dogs vaccinated with recombinant ASP-2 recognized the native ASP-2 protein in crude L3 extract.

ASP-2 immunolocalized both to the glandular esophagus and the esophageal lumen of A. caninum L3. In addition, the protein localized to the channels that connect the glandular esophagus to the L3 surface. ASP-2 was also detected on the L3 surface itself (Fig. 3). There


were some differences between the localization of the native protein using dog antibodies vs. rabbit antibodies; rabbit antibodies localized protein in both the glandular esophagus and cuticle, whereas the dog antibody labeled primarily the epicuticle and the cuticle of the L3 surface.

Anti-ASP-2 antibodies inhibit larval invasion through tissue in vitro

Three replicates from three different experiments were used to determine the effect of anti-ASP-2 sera on migration of L3 through tissue in vitro. A median of 60 out of 300 L3 that had been incubated in 50 μl of control sera remained on the skin. i.e., did not migrate through tissue. A median of 240 of our 300 L3 that had been incubated in 50 μl of anti-ASP-2 sera remained on the skin (P=<0.01), reflecting a 60% reduction in migration of L3 through tissue in the presence of anti-ASP-2 sera (Table 3).

Vaccination of dogs with ASP-2 results in reduced hookworm burden and fecal egg counts following hookworm larval challenge

Two weeks following the final immunization, vaccinated dogs and control dogs were infected with hookworm by percutaneous administration of 500 L3 of the canine hookworm A. caninum. Relative to controls, the vaccinated dogs exhibited reductions in fecal egg counts (69%, 10,613±5,306 vs. 3,336±1,681; P=0.05) (Fig. 4A). A 26% (204±12 vs. 151±21; P=0.03) reduction in worm burdens was also observed (Fig. 4B).

DISCUSSION

The studies here report on three independent lines of evidence indicating that ASP-2 is a lead vaccine candidate for human hookworm infection: 1) the presence of antibodies against ASP-2 protected against heavy infection in humans; 2) anti-ASP-2 antibodies immunoprecipitated native ASP-2 from larval extracts and inhibited hookworm larval invasion through tissue in vitro; and 3) vaccination with ASP-2 resulted in lower worm burdens and fecal egg counts in dogs. Our study also shows that it is important for an infected person to mount the appropriate antibody response (IgE and not IgG4) to ASP-2 in order to exhibit a reduction in the intensity of hookworm infection. Previously, we showed that ASP-2 vaccination reduces hookworm burden in hamsters challenged with the hookworm Ancylostoma ceylanicum (32, 33), and other investigators have found that ASP-2-like molecules are protective antigens for onchocerciasis in laboratory mice (34) and for ruminant haemonchosis caused by the trichostrongyle nematode, Haemonchus contortus (35). Interestingly, our canine vaccine trials show that vaccination with ASP-2 results in a strong and protective IgG response. It is possible that the protection from a vaccine is mediated by a different process than the protection that occurs during a chronic hookworm infection.

The mechanism by which the presence of anti-ASP-2 antibodies results in reduction in host hookworm burdens and fecal egg counts is not known. Evidence to date indicates that anti-ASP-2 antibodies interact primarily with the tissue-invading L3 stage. ASP-2 protein has not been detected in other hookworm life-history stages, and asp-2 mRNA was found only in L3 and not adult hookworms (36). We cannot rule out the possibility, however, that anti-ASP-2 antibodies cross-react with other ASP molecules found in adult hookworms, e.g., ASP-3, ASP-4, ASP-5 and ASP-6 (36), or other adult hookworm PRPs (37–39). This could account for the reduction in canine fecal egg counts seen in ASP-2 vaccinated dogs, which exceeded what would be expected


based solely on the reduction in host worm burdens. An explanation supported by observations with the irradiated hookworm L3 vaccine of canines (11) is that the ASP-2 vaccine induces an immune response that can attenuate L3. Hence, some of those parasites that reach adulthood in the host gut are sexually compromised and therefore less fecund.

From the immunolocalization studies reported here, L3 produce ASP-2 in their glandular esophagus before secretion. Secretion probably occurs by at least two different mechanisms. First, ASP-2 was found in the lumen of the L3 esophagus, suggesting that it could be secreted from its oral opening during a larval feeding process that resumes following entry into the host (16). Second, the protein was also detected in channels that connect the esophagus with the cuticle. The channels are believed to function as a network akin to vascular channels that are capable of efficiently transporting protein (40), including transport to the cuticle located on the parasite surface. In this study, ASP-2 was also detected on the parasite surface. Therefore, anti-ASP-2 antibody could interfere with larval invasion either by an interaction with its target antigen on the parasite surface or oral opening. This also includes the possibility that anti-ASP-2 interacts with the parasite surface through an association with effector leukocytes (antibody dependent cellular cytotoxicity or ADCC), an important effector arm in immune destruction of larval helminths (41, 42). Alternatively, anti-ASP-2 antibody may also neutralize some critical biochemical function of the target protein. It has been proposed that ASP-2 could function as an immunomodulator with chemokine-like properties (15). Our observation that anti-ASP-2 antibodies inhibit L3 invasion through tissue in vitro further supports the hypothesis that the immune response resulting from ASP-2 vaccination interferes with the early stages of parasite invasion in the host. Therefore, ASP-2 vaccination would decrease the number of L3 that reach the gastrointestinal tract, leading to reduced adult hookworm burdens and host blood loss (Fig. 5).

In public health terms, the impact of a hookworm vaccine using ASP-2 would be the reduction of moderate/heavy hookworm infection and the morbidity associated with hookworm disease, that is, the heavy blood loss and iron deficiency anemia that accompanies this level of infection. Similar reductions in worm burdens using anthelminthic drugs were proposed at the World Health Assembly 2001 Resolution 54.19, with the goal of attaining a minimum target of regular administration of chemotherapy (anthelminthic deworming) to at least 75% and up to 100% of all school-age children at risk of morbidity by 2010 (4). Because ASP-2 vaccination may target only L3 and not adult hookworms, it is likely that an ASP-2 vaccine used in childhood would likely be administered following a round of anthelminthic deworming (8). In this way, an ASP-2 hookworm vaccine could become an integral part of international deworming programs. To this end, we are also exploring additional vaccines from the adult worm (27). A significant advantage of vaccination over chemotherapy would be the possible requirement for only a single health intervention (completion of the vaccine series) in order to administer the vaccine, as opposed to the requirement for twice- or thrice-yearly administration of anthelminthic drugs in areas of high transmission (4). Studies, including phase I clinical trials, are in progress to develop and test human hookworm vaccines comprised of an ASP-2 antigen (10).

ACKNOWLEDGMENTS

These studies were supported by the Human Hookworm Vaccine Initiative of the Bill and Melinda Gates Foundation and the Sabin Vaccine Institute, and a Clinical Research Grant (FY00-791) from the March of Dimes Birth Defects Foundation. JB is supported by an


International Research Scientist Development Award (1K01 TW00009); AL is supported by an RD Wright Career Development Award from the National Health and Medical Research Council of Australia; SB is supported by a Wellcome Trust Advanced Research Fellowship (073656). We would like to acknowledge Drs. Joe Cohen and Sylvie Cayphas of GlaxoSmithKline Biologicals (Rixensart, Belgium) for providing AS03 and technical assistance with formulation, as well as Yelena Oksov MS of the New York Blood Center Electron Microscopy Laboratory for her work in preparing the electron micrographs.

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Received April 13, 2005; accepted June 13, 2005


Table 1 Descriptive statistics for cross-sectional serological surveys carried out in Brazil and China

The term “CI” refers to 95% confidence interval, and the term SD refers to standard deviation.

Age Sex Prevalence Eggs Per Gram of Feces (epg) Infection Level n Mean (Range) F:M % (CI) Mean (SD) Range 1-499 ≥500 Brazil 302 36 (3-84) 141:124 54% (47, 61) 1,645 (3,375) 2-19,488 101 61 China 244 33 (2-86) 118:147 59% (51, 67) 1,723 (6,001) 24-66,432 86 59


Table 2 Logistic regression associating the risk of heavy hookworm infection to antibodies against ASP-2 (n=307)

95% Confidence Interval Variable n Odds Ratio Lower Upper Area (Brazil:China) 162:145 1.254 0.772 2.035 Sex (female:male) 165:142 0.809 0.496 1.138 Age group:

Young (5–29) 72 1.000 – – Middle (30–49) 141 1.070 0.574 1.99 Old (50+) 94 1.798 0.920 3.516

IgG4 anti-ASP-2 307 4.967 1.871 13.188 IgE anti-ASP-2 307 0.382 0.152 0.959

Log likelihood = –192.81; χ2 = 25.2; P<0.0001. Plots of lowess smoothed logits vs. IgE and IgG4 levels to ASP-2 supported treating them as linear variables in the logistic regression. No other IgG subclass against ASP-2 gave significant results. In a separate analysis, also adjusted for age, sex, and area, the ratio IgG4/IgE against ASP-2 was shown to be a significant risk factor for heavy hookworm infection.


Table 3 Inhibition of A. caninum larval tissue penetration by pooled sera from dogs vaccinated with ASP-2 and dogs injected with AS03 alone

Group N Median Mean Min Max SD % Reduction* P†

ASP-2 9 60 37 0 60 29 AS03 9 240 223 60 240 34

60 < 0.01

Approximately 300 L3 were applied to the skin and the number of L3 that did not successfully penetrate was counted. *Percent reduction in migration ASP-2 vs. control. †P value from Wilcoxon sign rank test comparing median of inhibition of A. caninum larval tissue penetration by pooled sera from dogs vaccinated with ASP-2 and dogs injected with AS03 alone.


Fig. 1

Figure 1. Biochemical characterization of recombinant ASP-2. A) Profile of ASP-2 eluted from nickel affinity resin with a stepwise gradient of imidazole. Arrows refer to the recombinant ASP-2 doublet where the higher band represents the glycosylated product. B) MALDI-TOF spectrum of recombinant ASP-2. C) Western blot showing recognition of recombinant ASP-2 and a protein of the predicted molecular weight from N. americanus L3 extract (NaL3E) using rabbit antiserum raised to recombinant ASP-2 (rb α-ASP-2) or preimmune serum (NRS) (right).


Fig. 2

Figure 2. Anti-ASP-2 immune responses in vaccinated dogs. A) The geometric mean titers of the IgG1 (�), IgG2 (�), and IgE (�) antibody responses against recombinant ASP-2 in canines vaccinated with recombinant Ac-ASP-2. The control group was vaccinated with AS03 only and had no titers (data not shown). The term “primary” refers to prevaccination sera. The “C” inside the arrowhead refers to larval challenge; the “N” inside the arrowhead refers to necropsy. B) Individual dogs (A–E) vaccinated with recombinant Ac-ASP-2 generate antibodies at day 75 (before larval challenge) that immunoprecipitate the native ASP-2 protein from L3 extracts (L3E). Sera from canines vaccinated with adjuvant AS03 alone (control) did not precipitate any proteins. The letter “p” refers to biotinylated A. caninum L3E proteins that were precipitated by canine sera bound to protein A-agarose. The term “np” refers to L3E proteins that were not precipitated by sera and protein A agarose (i.e., proteins remaining in the supernatant after precipitation).


Fig. 3

Figure 3. Immunolocalization of ASP-2 in A. caninum L3. Antibodies raised against ASP-2 in rabbits or dogs were used to study the subcellular localization of ASP-2 in L3. A) Normal dog serum (similar results were obtained with normal rabbit serum [data not shown]). B) Dog anti-ASP-2 antibodies recognized proteins in channels and the cuticle and epicuticle. C) Rabbit anti-ASP-2 antibodies strongly labeled the granules of the glandular esophagus, the basal lamina of the body cavity or the channels connecting the glandular esophagus with the cuticle. The cuticle and the epicuticle were also labeled.


Fig. 4

Figure 4. A. caninum fecal egg counts and worm burdens (intestine and colon) from canines vaccinated with ASP-2/AS03 and AS03 alone (controls). A) Fecal egg counts (all egg counts were performed twice) were reduced in canines vaccinated with ASP-2 (n=4) compared with the adjuvant AS03 alone (control) (n=4; one of the dogs did not produce stool on the day of sampling). B) Fewer adult worms were retrieved during necropsy from the colon and small intestine of canines vaccinated with ASP-2/AS03 (n=4) compared with AS03 alone (control) controls (n=5).


Fig. 5

Figure 5. Schematic of the effects of anti-ASP-2 antibodies on host hookworm burdens. Anti-ASP-2 antibodies inhibit early host entry of hookworm L3 through the skin and other tissues. As a result, reduced numbers of L3 gain entry into the host gastrointestinal tract. This causes reduction in adult worm burden, which leads to reduced hookworm-associated blood loss and fecal egg counts. Reductions in blood loss prevent the development of anemia and malnutrition.


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