A highly expressed intestinal cysteine protease of Ancylostoma ceylanicum protects vaccinated hamsters from hookworm infection.

BACKGROUND: Human hookworms (Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum) are intestinal blood-feeding parasites that infect ~500 million people worldwide and are among the leading causes of iron-deficiency anemia in the developing world. Drugs are useful against hookworm infections, but hookworms rapidly reinfect people, and the parasites can develop drug resistance. Therefore, having a hookworm vaccine would be of tremendous benefit. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the vaccine efficacy in outbred Syrian hamsters of three A. ceylanicum hookworm antigen candidates from two classes of proteins previously identified as promising vaccine candidates. These include two intestinally-enriched, putatively secreted cathepsin B cysteine proteases (AceyCP1, AceyCPL) and one small Kunitz-type protease inhibitor (AceySKPI3). Recombinant proteins were produced in Pichia pastoris, and adsorbed to Alhydrogel. Recombinant AceyCPL (rAceyCPL)/Alhydrogel and rAceySKPI3/Alhydrogel induced high serum immunoglobulin G (IgG) titers in 8/8 vaccinates, but were not protective. rAceyCP1/Alhydrogel induced intermediate serum IgG titers in ~60% of vaccinates in two different trials. rAceyCP1 serum IgG responders had highly significantly decreased hookworm burdens, fecal egg counts and clinical pathology compared to Alhydrogel controls and nonresponders. Protection was highly correlated with rAceyCP1 serum IgG titer. Antisera from rAceyCP1 serum IgG responders, but not nonresponders or rAceyCPL/Alhydrogel vaccinates, significantly reduced adult A. ceylanicum motility in vitro. Furthermore, rAceyCP1 serum IgG responders had canonical Th2-specific recall responses (IL4, IL5, IL13) in splenocytes stimulated ex vivo. CONCLUSIONS/SIGNIFICANCE: These findings indicate that rAceyCP1 is a promising vaccine candidate and validates a genomic/transcriptomic approach to human hookworm vaccine discovery.

Immune polarization by hookworms: taking cues from T helper type 2, type 2 innate lymphoid cells and alternatively activated macrophages.

Cellular and molecular investigation of parasitic helminth infections has greatly accelerated the understanding of type 2 immune responses. However, there remains considerable debate regarding the specific leucocytes that kill parasites and whether these mechanisms are distinct from those responsible for tissue repair. Herein, we chronicle discoveries over the past decade highlighting current paradigms in type 2 immunity with a particular emphasis upon how CD4(+) T helper type 2 cells, type 2 innate lymphoid cells and alternatively activated macrophages coordinately control helminth-induced parasitism. Primarily, this review will draw from studies of the murine nematode parasite Nippostrongylus brasiliensis, which bears important similarities to the human hookworms Ancylostoma duodenale and Necator americanus. Given that one or more hookworm species currently infect millions of individuals across the globe, we propose that vaccine and/or pharmaceutical-based cure strategies targeting these affected human populations should incorporate the conceptual advances outlined herein.

Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection.

Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection.

New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74.

Na-APR-1(M74) is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1(M74) and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1 wt in vitro, thereby providing proof of concept of Na-APR-1(M74) as a vaccine antigen. The mutated version, Na-APR-1(M74), was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1(M74) IgG. As little as 0.99 µg of recombinant Na-APR-1(M74) could induce anti Na-APR-1(M74) IgG in mice that were capable of inhibiting Na-APR-1w t-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1(M74) as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1 wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1(M74) which was assessed by fluorescence polarization, but with an ∼ 50-fold reduction in the dissociation constant. Taken together, these assays comprise a "toolbox" that could be useful for the analyses of Na-APR-1(M74) as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline.

Hookworm excretory/secretory products induce interleukin-4 (IL-4)+ IL-10+ CD4+ T cell responses and suppress pathology in a mouse model of colitis.

Evidence from human studies and mouse models shows that infection with parasitic helminths has a suppressive effect on the pathogenesis of some inflammatory diseases. Recently, we and others have shown that some of the suppressive effects of hookworms reside in their excretory/secretory (ES) products. Here, we demonstrate that ES products of the hookworm Ancylostoma caninum (AcES) suppress intestinal pathology in a model of chemically induced colitis. This suppression was associated with potent induction of a type 2 cytokine response characterized by coexpression of interleukin-4 (IL-4) and IL-10 by CD4(+) T cells, downregulation of proinflammatory cytokine expression in the draining lymph nodes and the colon, and recruitment of alternatively activated (M2) macrophages and eosinophils to the site of ES administration. Protease digestion and heat denaturation of AcES resulted in impaired induction of CD4(+) IL-4(+) IL-10(+) cell responses and diminished ability to suppress colitis, indicating that protein component(s) are responsible for some of the immunosuppressive effects of AcES. Identification of the specific parasite-derived molecules responsible for reducing pathology during chemically induced colitis could lead to the development of novel therapeutics for the treatment of human inflammatory bowel disease.

Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum.

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.

The mucosal response of hamsters exposed to weekly repeated infections with the hookworm Ancylostoma ceylanicum.

An experiment was carried out to assess mucosal changes in hamsters exposed to weekly repeated low-intensity infections with the hookworm Ancylostoma ceylanicum. The experiment included control groups of naïve, uninfected hamsters and groups that received a single-pulse primary infection. Changes in the intestinal architecture and in the density of inflammatory cells in the mucosa, including mast cells, goblet cells, Paneth cells and eosinophils were examined in relation to changes in hookworm burdens. As in the single-pulse primary infection, hamsters exposed to repeated infections responded with marked changes in the intestinal architecture and in mucosal populations of inflammatory cells. However, there were distinct differences in the kinetics of the responses to these two types of infection (primary single-pulse and repeated). The reduction in villous height and the increase in crypt depth in animals exposed to repeated infections were both initially slower but eventually equalled and exceeded the responses in hamsters given a chronic primary infection, despite the presence of fewer adult worms in the former. Similarly, changes in the mitotic figures of epithelial cells in the mucosa and the mast cell response were both initially slower and less intense in repeatedly infected hamsters, but eventually exceeded the response to primary infection. Furthermore, the eosinophil response was found to be initially greater in repeated infections and overall more persistent. In contrast, both goblet and Paneth cell responses were less marked in repeatedly infected animals compared to those carrying a primary infection. These results are discussed in the context of host protective resistance to infection with A. ceylanicum.

Ancylostoma ceylanicum excretory-secretory protein 2 adopts a netrin-like fold and defines a novel family of nematode proteins.

Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 Å resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-like fold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

The impact of concurrent and treated Ancylostoma ceylanicum hookworm infections on the immunogenicity of a recombinant hookworm vaccine in hamsters.

BACKGROUND: The effect of concurrent (active) and treated hookworm infections on the immunogenicity of vaccination with the recombinant fusion protein Ay-Ancylostoma-secreted protein 2 was analyzed in the Golden Syrian hamster. METHODS: Hamsters were infected with the hookworm Ancylostoma ceylanicum and vaccinated with the recombinant protein, with Quil A used as adjuvant. As controls, hookworm-infected hamsters were treated with the anthelmintic drug pyrantel pamoate before vaccination. Naive hamsters (i.e., those with neither previous hookworm infections nor treatment) were also vaccinated. RESULTS: The proliferation capacities of carboxyfluorescein diacetate succinimidyl ester-positive lymphocytes from the hookworm-infected vaccinated group were reduced by 50% relative to the capacities of lymphocytes from uninfected or treated vaccinated hamsters; capacities were comparable to the rates observed in lymphocytes from the hamsters vaccinated with the adjuvant alone. Immunoglobulin G1 antibody responses were also reduced in the actively infected, untreated hamsters, and interferon- gamma and interleukin-4 cytokine mRNAs were down-regulated. Conversely, interleukin-10 and tumor necrosis factor- alpha mRNAs were up-regulated in those hamsters. CONCLUSIONS: These results suggest that hookworm infections have an immunomodulatory effect by impairing the immune response to an exogenous antigen during infection. The hookworm-associated immunodepression may have important implications for design of clinical trials of human vaccines and vaccination strategies.

Protective immunity elicited by ultraviolet-irradiated third-stage infective hookworm (Necator americanus and Ancylostoma caninum) larvae in mice and hamsters.

The protective immunity elicited by ultraviolet-irradiated third-stage infective larvae of Necator americanus (UV-NaL3) and Ancylostoma caninum (UV-AcL3) was evaluated in laboratory mice (a non-permissive model) and hamsters (a permissive model). After optimizing the time of exposure to UV-irradiation, both oral and subcutaneous vaccination routes with UV-AcL3 in mice were explored. Oral vaccination was more effective at reducing the number of challenge AcL3 entering the lungs, whereas subcutaneous vaccination was more effective at blocking muscle entry. When UV-irradiated NaL3 and non-irradiated AcL3 were used as vaccines in hamsters, both of them were effective at reducing adult hookworm burdens. However, the length of protection afforded by UV-irradiated L3 was substantially greater than that resulting from immunization with non-irradiated L3. A single dose was less effective than multiple doses. The protective immunity elicited by UV-irradiated NaL3 given once every other week for a total of three immunizations was similar to that elicited by non-irradiated AcL3 given during the same schedule. Protection was not significantly affected by administering the L3 on a weekly basis for a total of three immunizations, even though the antibody titers were reduced using this schedule. These studies will facilitate the elucidation of the mechanisms underlying larval protection.

Computational analysis of Ancylostoma ceylanicum cysteine proteinase.

The potential tertiary structure of Ancylostoma ceylanicum cysteine proteinase was obtained by Automatic Program 3D-JIGSAW and used for finding homologues of known structure by VAST program. The results of computational analysis showed the presence of domains recognizing host immunoglobulins. Based on this analysis we suggest that this protein is involved in cleaving of host antibodies and therefore it may be promising vaccine candidate. In this paper we present the computational analysis of parasitic antigen which is very helpful in evaluation of the potential role of this protein.

Host cytokine production, lymphoproliferation, and antibody responses during the course of Ancylostoma ceylanicum infection in the Golden Syrian hamster.

The Syrian Golden hamster (Mesocricetus auratus) has been used to model infections with the hookworm Ancylostoma ceylanicum. New molecular immunological reagents to measure cellular immune responses in hamsters were developed and used to determine the impact of A. ceylanicum hookworm infection on host cytokine responses and lymphoproliferation. Initial larval infection with 100 third-stage A. ceylanicum larvae resulted in predominant Th1 responses (upregulation of proinflammatory cytokines) that lasted for the duration of larval migration and continued up to 14 days postinfection (prepatency). Subsequently, development of larvae into egg-laying adult hookworms (patency) coincided with a switch to Th2 predominant responses (interleukin-4 [IL-4]) as well as a marked increase in IL-10 production. This switch also concurred with reduced host lymphoproliferative responses to hookworm antigens. The findings demonstrate a similarity in immune responses between hamsters and humans infected with hookworms, suggesting that hamsters will be a useful animal model species for examining host immunity to human hookworm infections.

Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms.

We expressed a catalytically active cysteine protease, Ac-CP-2, from the blood-feeding stage of the canine hookworm Ancylostoma caninum and vaccinated dogs with the purified protease. Dogs acquired high-titer, antigen-specific antibody responses, and adult hookworms recovered from the intestines of vaccinated dogs were significantly smaller than hookworms from control dogs. There was also a marked decrease in fecal egg counts and the number of female hookworms in vaccinated dogs. Ac-CP-2 is expressed by the parasite in the brush-border membrane of its alimentary canal, and anti-Ac-CP-2 antibodies were bound to the gut of hookworms from vaccinated dogs, which suggests that these antibodies were ingested by the parasites with their blood meal. IgG from vaccinated dogs decreased proteolytic activity against a peptide substrate by 73%, which implies that neutralizing antibodies were induced by vaccination. These results indicate that cysteine proteases involved in parasite nutrition are promising candidates as vaccines against hookworm disease.

Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

Experimental approaches to the development of a recombinant hookworm vaccine.

Hookworm infection is a major parasitic cause of morbidity in the developing nations of the tropics. Development of a genetically engineered vaccine would be a useful tool in the control of this infection in highly endemic areas. Recombinant polypeptides belonging to the Ancylostoma secreted protein (ASP)-1 family have shown promise for reducing hookworm burdens after larval challenge infections in mice. Typically, these polypeptides are expressed in Escherichia coli and administered as an alum precipitate. Vaccine protection is antibody dependent. It is anticipated that a cocktail of different recombinant hookworm antigens may be required in order to effectively prevent heavy hookworm infections and disease. The progress of this work has been hampered by the absence of both a convenient laboratory animal with which to study hookworm infections resembling human infection, as well as the lack of easy availability of native hookworm antigens. In addition, useful human serologic correlates of antihookworm immunity are still poorly defined.

Expression of acquired immunity to the hookworm Ancylostoma ceylanicum in hamsters.

Four experiments are described in which hamsters, initially exposed to primary infection with Ancylostoma ceylanicum, were given a homologous challenge and components of the secondary response were quantified and compared to relevant control groups. The initial establishment of the L3 larvae was not prevented in immunized hamsters but their growth was slowed and virtually all larvae were lost within a week of challenge, when the majority were still at the L4 stage of development. The loss of worms was associated with an accelerated mucosal mastocytosis and increased systemic antibody. Thus acquired immunity to hookworm larvae in this system acted on L3 and L4 stages, thereby preventing larvae from maturing in immunized animals. In contrast to primary infections, immunized hamsters responding to a challenge infection did not lose weight nor did they experience significant anaemia, because of the lack of adult worms. The secondary immune responses therefore prevented manifestation of hookworm disease among immunized-challenged animals.

Secretion of cysteine proteinase activity by the zoonotic hookworm Ancylostoma caninum.

The zoonotic hookworm, Ancylostoma caninum, probably induces human eosinophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in other parasites, cysteine proteinases are involved in pathogenesis. We studied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activity using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity was observed against the cathepsins L and B-specific substrate Z-phe-arg-AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against gelatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in adult extracts was enhanced up to 15-fold by the reducing agent dithiothreitol (DTT), but was totally blocked by specific inhibitors of cysteine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-CHN2,E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gelatinolytic activity in ES antigens detected using substrate gels was enhanced by the addition of reducing agents and inhibited by Z-phe-ala-CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity in ES antigens was active over a wide pH range (pH 5-9). Similar cysteine proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the substrate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydrolysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cysteine proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of UV-irradiated larval vaccine of Ancylostoma ceylanicum (Looss, 1911) in golden hamsters (Mesocricetus auratus).

A vaccination trial in golden hamsters with UV-irradiated infective larvae of Ancylostoma ceylanicum was attempted. One oral vaccination of hamsters with 100 infective larvae irradiated by means of UV-tube (390 nm) at different time intervals induced the development of resistance. As the time exposure of irradiation was increased, there was a corresponding decrease in the subsequent worm establishment. A high level of protection afforded by larvae irradiated for 15 min UV-exposure was recorded giving 99.0% and 95.0% worm reduction against the challenge doses of 100 and 1000 normal larvae respectively. There was no marked difference in worm establishment in hamsters vaccinated either orally or subcutaneously, followed by oral challenge. In the vaccinated hamsters, the manifestations of resistance at 15 min UV-exposure were shown by marked reduction in worm establishment and highly reduced epg in pellets with significantly higher blood haemoglobin levels compared with those given normal larvae as vaccine and challenge controls.

Pattern of immune response in recipients after transfer of mesenteric lymph node cells sensitized with low and high doses of Ancylostoma caninum.

Cellular immune response was detected in female Swiss albino mice with mesenteric lymph node cells during Ancylostoma caninum infection. Sensitized lymphocytes responded vigorously to A. caninum antigens and it was found that the cells (singly or repeatedly) sensitized with either low or high doses could confer adoptive protective immunity in recipients later challenged with a single dose of 500 A. caninum larvae.

Efficacies of vaccines against canine hookworm disease.

Three types of vaccine (1) a sonicated extract of killed larvae, (2) 5-fluorouracil-treated larvae and (3) 40 krad. X-irradiated larvae have been evaluated. Pups aged 3-4 months, weighing 2 +/- 0.5 kg and free of natural hookworm infection were selected for experimental studies. The sonicated extract was given either in a single dose or as 2 doses at weekly intervals, whereas the 5-fluorouracil-treated and X-irradiated larvae were given in a single dose only. After completion of the vaccination schedule, the pups were challenged with filariform larvae and were sacrificed 3 weeks after challenge. Non-vaccinated pups and those vaccinated with a single dose of sonicated extract lost weight and had diarrhoea or dysentery after challenge. Furthermore, in non-vaccinated pups, haemoglobin levels fell and at autopsy the intestinal mucosa was found to be grossly congested and free blood was present in the lumen of the intestine. Such findings were not obtained in pups vaccinated with the other types of vaccines. The percentage of infective larvae which developed to the adult stage in non-vaccinated pups was 82.7 +/- 9.53 compared with 45.1 +/- 12.33, 30.1 +/- 10.05, 20.6 +/- 10.60 and 8.0 +/- 7.46%, respectively in pups vaccinated with a single dose of sonicated extract, double dose of sonicated extract, 5-fluorouracil-treated and 40 krad. X-irradiated larval vaccines. While the irradiated larval vaccine provided maximum protection against clinical ancylostomiasis, 2 doses of the sonicated larval vaccine could also afford almost equal protection to pups.