The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus.

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus-Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30-46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development.

Molecular cloning and characterization of Ac-MTP-2, an astacin-like metalloprotease released by adult Ancylostoma caninum.

Ac-MTP-2 is an astacin-like metalloprotease secreted by adult Ancylostoma caninum hookworms. Ac-mtp-2 cDNA was cloned by immunoscreening a cDNA library with antisera prepared against adult A. caninum excretory/secretory (ES) products. The full-length Ac-mtp-2 contains 850 bp cDNA encoding a 233 amino acid open reading frame (ORF) with 32% amino acid identity to Ce-NSP-4, a pharyngeal cell-derived secreted metalloprotease of the nematode Caenorhabditis elegans. The predicted ORF contained a conserved Met-turn sequence (SXMHY), but only a partial zinc-binding signature sequence (GXXXEHXRXER instead of HEXXHXXGXXHEXXRXDR) found in other astacins. However, by both gelatin gel electrophoresis and azocasein digestion, the recombinant Ac-MTP-2 exhibited proteolytic activity that was inhibited by the zinc chelator 1,10-phenanthroline and Ac-TMP, a putative tissue inhibitor of metalloprotease that was previously shown to be a highly abundant component of adult A. caninum ES products. By RT-PCR, Western blot Ac-MTP-2 was found only expressed in adult hookworms and secreted in the adult ES products. Immunolocalization with antisera shows that Ac-MTP-2 is located to the esophageal glands (confirming its role as a secretory protein), as well as to the parasite uterus. It is hypothesized that Ac-MTP-2 functions in the extracorporeal digestion of the intestinal mucosal plug lodged in the buccal capsule of the adult parasite.

Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration.

Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm Ancylostoma caninum secretes an astacin-like metalloprotease, Ac-MTP-1, upon activation in vitro with host serum. Recombinant Ac-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant Ac-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within A. caninum L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that Ac-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration.

Vaccination with irradiated Ancylostoma caninum third stage larvae induces a Th2 protective response in dogs.

While X-irradiated live parasites are not an acceptable proposition for human vaccination, they offer a ready experimental system to explore mechanisms by which immunity against hookworm infection may be induced in humans. As such, we sought to further elucidate the details of this highly protective immune response induced by the irradiated vaccine in canids, with special emphasis on the cellular aspects of the response. Vaccination with irradiated L3 induced high production of antibodies and strong PBMCs proliferation to crude L3 antigen preparation. Elevated IL-4 production was also observed in vaccinated dogs, especially in relation to IFN-gamma production (IL-4/IFN-gamma ratio). Serum from vaccinated animals inhibited penetration of L3 through canine skin in vitro by 60%. Finally, vaccinated animals had a strong antibody response to ASP-2, a promising vaccine antigen that is an excretory-secretory product of L3. These results add further support the idea that the Th2 immune response is required to generate protective immunity against hookworm larvae and that ES molecules released during this developmental stage are likely targets of this response.

Biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm Ancylostoma caninum.

We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection.

Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals.

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.

Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms.

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme-inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.

Ac-SAA-1, an immunodominant 16 kDa surface-associated antigen of infective larvae and adults of Ancylostoma caninum.

A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L(3). Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen.

A secreted protein from the human hookworm necator americanus binds selectively to NK cells and induces IFN-gamma production.

Parasitic helminths induce chronic infections in their hosts although, with most human helminthiases, protective immunity gradually develops with age or exposure of the host. One exception is infection with the human hookworm, Necator americanus, where virtually no protection ensues over time. Such observations suggest these parasites have developed unique mechanisms to evade host immunity, leading us to investigate the role of the excretory/secretory (ES) products of adult N. americanus in manipulating host immune responses. Specifically, we found that a protein(s) from ES products of adult N. americanus bound selectively to mouse and human NK cells. Moreover, incubation of purified NK cells with N. americanus ES products stimulated the production of augmented (4- to 30-fold) levels of IFN-gamma. This augmentation was dependent on the presence of both IL-2 and IL-12 and was endotoxin-independent. This is the first report of a pathogen protein that binds exclusively to NK cells and the first report of a nematode-derived product that induces abundant levels of cytokines from NK cells. Such an interaction could provide a means of cross-regulating deleterious Th2 immune responses in the host, thereby contributing to the long-term survival of N. americanus.

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

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.

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative.

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

Digestive proteases of blood-feeding nematodes.

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major blood-feeding nematodes - hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus - but also compares and contrasts these proteases with recent findings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the fine specificity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host-parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets.

Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum.

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3-6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion.

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity.

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine.

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D-like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 +/- 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5-18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastroinntestinal tract.