WO2003032917A2

WO2003032917A2 - Hookworm vaccine

Info

Publication number: WO2003032917A2
Application number: PCT/US2002/033106
Authority: WO
Inventors: Peter Hotez; James Ashcom; Mahnaz Bdamchian; Bin Zhan; Yan Wang; John Hawdon; Alexander Loukas; Angela Williamson; Brian Jones
Original assignee: The George Washington University
Priority date: 2001-10-17
Filing date: 2002-10-17
Publication date: 2003-04-24
Also published as: CN1571677A; WO2003032917A3; AU2002335061A1; BR0213656A

Abstract

Preparations which elicit an immune response to hookworm antigens and which may be uitilized as hookworm vaccines are provided. In addition, a method of increasing the effectiveness of vaccinations against infectious diseases in patients infected with hookworm is provided. The method involves chemically treating the hookworm infestation prior to administering the vaccine.

Description

HOOKWORM VACCINE

DESCRIPTION

BACKGROUND OF THE INVENTION

Field of the Invention The invention generally relates to a vaccine for hookworm. In particular, the invention provides vaccines based on parasite-derived antigens.

Background of the Invention

Hookworm infection is a significant public health concern in developing countries around the world, causing enteritis, intestinal blood loss, anemia, developmental delays, and malnutrition. It is estimated that there are more than one billion cases of human hookworm infection worldwide, with 194 million cases in China alone (Hotez et al. 1997). In some regions of China such as Hainan Province in the South China Sea more than 60 percent of the population harbor hookworms (Gandhi et al. 2001).

Most of the pathology caused by hookworm results from the adult stages of the parasite in the human intestine. The attachment of adult Ancylostoma hookworms to the mucosa and submucosa of the vertebrate small intestine is one of the best-defined examples of host-parasite relationships in all of parasitology. Comprised of several cubic millimeters of host mucosal and submucosal tissue lodged in the buccal capsule of the parasite, it is possible to actually touch the host-parasite relationship at necropsy or autopsy (Kalkofen, 1970; Kalkofen, 1974).

The dog hookworm Ancylostoma caninum is a major cause of morbidity and mortality in dogs throughout the world including subtropical regions of North America. Hookworm- associated blood loss leading to severe anemia and even death can occur in dogs between 2 and 3 weeks after a single primary infection (Soulsby, 1982; Jones and Hotez, 2002). Significantly, A. caninum has also been recently identified as an important human pathogen. Zoonotic infection with one adults, caninum parasite can result in eosinophilic enteritis syndrome, an inflammatory condition of the intestine in response to invasion by the parasite (Prociv and Croese, 1990). The pathogenesis of A. caninum infection is associated with the intestinal blood loss that occurs during adult worm attachment and feeding in the mammalian small intestine (Kalkofen, 1970; Kalkofen, 1974).

Current efforts for the treatment and control of hookworm infestations are limited to periodic removal of adult hookworms from patients with anthelmintics. This approach has several limitations, including rapid reinfection following treatment, requiring multiple visits, and the eventual development of anthelmintic resistant strains of hookworms following several years of heavy anthelmintic treatments (Savioli et al. 1997; Geerts and Gryseels, 2000). Thus, it would be of great benefit to have available additional methods for both treating and preventing hookworm infection in mammals. For example, it would be highly advantageous to have available vaccines to treat or prevent hookworm infection.

SUMMARY OF THE INVENTION

The present invention provides preparations for eliciting an immune response against hookworm. The preparations contain various hookworm antigens which have been identified as useful for eliciting an immune response. These preparations may be used as vaccines against hookworm in mammals, for example, in humans. As a result of the administration of the preparations, the vaccinated mammal may develop an immune response against hookworm which causes immunity to infection by the parasite, or may display a lower worm burden, decreased blood loss, or a decrease in size of parasitizing hookworms. To that end, the invention provides a composition comprising a recombinant or synthetic antigen or a fragment thereof derived from hookworm, and a pharmacologically acceptable carrier. The recombinant or synthetic antigen may display at least about 80% identity to an antigen such as Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac- APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, or Ay-TTR. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1, or Ac-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale. The invention also provides a method of eliciting an immune response to hookworm in a mammal. The method includes the step of administering to the mammal an effective amount of a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm, and a pharmacologically acceptable carrier. The recombinant or synthetic antigen may display at least about 80% identity to an antigen such as Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac- APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1, orAc-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The inventions further provides a method of vaccinating a mammal against hookworm. The method includes the step of administering to the mammal an effective amount of a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm and a pharmacologically acceptable carrier. The recombinant or synthetic antigen may display at least about 80% identity with an antigen such as Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac- APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1, or Ac-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The invention further provides a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm. The recombinant or synthetic antigen display at least about 80% identity with an antigen such as Na-ASP-1, Na-ACE, Na- CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac- ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1₅ Ay-API, and Ay-TTR. The composition further comprises a pharmacologically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1, or Ac-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The invention further provides a vaccine comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm. The recombinant or synthetic antigen displays at least about 80% identity with an antigen such as Na-ASP-1, Na-ACE, Na- CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac- ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR, The vaccine further comprises a pharmacologically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac- MEP-1, or Ac-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The present invention further provides a composition for eliciting an immune response comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm. The recombinant or synthetic antigen displays at least about 80% identity with an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na- APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac- MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay- ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR. The composition further comprises a pharmacologically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac- MEP-1, orAc-MTP-1. The antigens may be derived from a hookworm from species such as Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The invention further provides a method for enabling vaccination of a patient against infectious diseases. The method includes the steps of treating hookworm infection to a degree sufficient to increase lymphocyte proliferation, and vaccinating the patient against an infectious disease such as HIV, tuberculosis, malaria, measles, tetanus, diphtheria, pertussis, or polio.

The present invention also provides a method for enabling hookworm vaccination. The method includes the steps of chemically treating a hookworm infected patient to ameliorate hookworm infection, and vaccinating the patient with a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm after amelioration of hookworm infection. In the method, the hookworm infection may be completely eradicated by treatment, or may be lessened to such an extent that hookworm vaccination is effective. The recombinant or synthetic antigen may display at least about 80% identity with an antigen such as Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac- MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac- 103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR.

The present invention also provides a method for reducing blood loss in a patient infected with hookworm. The method includes the step of administering to the patient a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm, and a pharmacologically acceptable carrier.

The present invention also provides a method for reducing hookworm size in a patient infected with hookworm. The method includes the step of administering to the patient a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm, and a pharmacologically acceptable carrier.

The invention further provides a method of reducing hookworm burden in a patient infected with hookworm. The method comprises the step of administering to the patient a composition comprising a recombinant or synthetic antigen (or a fragment of the antigen) derived from hookworm, and a pharmacologically acceptable carrier.

The present invention also provides the following nucleic acid and amino acid sequences: SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63 and SEQ ID NO. 64.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and B. Na-ASP-1 : A, cDNA (SEQ ID NO. 1) and B, deduced amino acid sequence (SEQ ID NO. 2). GeneBank accession # AF079521.

Figure 2A and B. Na-ACE: A, cDNA (SEQ ID NO. 3) and B, deduced amino acid sequence

(SEQ ID NO. 4). GeneBank accession # AF536813.

Figure 3A and B. Na-CTL: A, cDNA (SEQ ID NO. 5) and B, deduced amino acid sequence

(SEQ ID NO. 6).

Figure 4A and B. Na-APR-1 : A, cDNA (SEQ ID NO. 7) and B, deduced amino acid sequence (SEQ ID NO. 8).

Figure 5A and B. Na-APR-2: A, cDNA (SEQ ID NO. 9) and B, deduced amino acid sequence (SEQ ID NO. 10).

Figure 6A and B. Ac-TMP: A, cDNA (SEQ ID NO. 11) and B, deduced amino acid sequence (SEQ ID NO.12).

Figure 7A and B. Ac-MEP-1: A, cDNA (SEQ ID NO. 13) and B, deduced amino acid sequence (SEQ ID NO.14). GeneBank accession # AF273084.

Figure 8A and B. Ac-MTP-1: A, cDNA (SEQ ID NO. 15) and B, deduced amino acid sequence (SEQ ID NO. 16). GeneBank accession # AY036056.

Figure 9A and B. Ac-ASP-1 : A, cDNA (SEQ ID NO. 17) and B, deduced amino acid sequence (SEQ ID NO. 18). GeneBank accession * AF 132291.

Figure 10A and B. Ac-ASP-2: A, cDNA (SEQ ID NO. 19) and B, deduced amino acid sequence (SEQ ID NO. 20). GeneBank accession # AF089728.

Figure 11A and B. Ac-ASP-3: A, cDNA (SEQ ID NO. 21) and B, deduced amino acid sequence (SEQ ID NO. 22).

Figure 12A and B. Ac-ASP-4: A, cDNA (SEQ ID NO. 23) and B, deduced amino acid sequence (SEQ ID NO. 24). Figure 13A and B. Ac-ASP-5: A, cDNA (SEQ ID NO. 25) and B, deduced amino acid sequence (SEQ ID NO. 26).

Figure 14A and B. Ac-ASP-6: A, cDNA (SEQ ID NO. 27) and B, deduced amino acid sequence (SEQ ID NO. 28).

Figure 15 A and B. Ac-TTR: A, cDNA (SEQ ID NO. 29) and B, amino acid sequence (SEQ

ID NO.30) deduced from nucleotides 25-531.

Figure 16A and B. Ac-103: A, cDNA (SEQ ID NO. 31) and B, amino acid sequence (SEQ

ID NO. 32).

Figure 17A and B. Ac-VWF: A, cDNA (SEQ ID NO. 33) and B, amino acid sequence (SEQ

ID NO. 34).

Figure 18A and B. Ac-CTL: A, cDNA (SEQ ID NO. 35) and B, amino acid sequence (SEQ

ID NO. 36).

Figure 19A and B. Ac-API-1 : A, cDNA (SEQ ID NO. 37) and B, amino acid sequence (SEQ

ID NO. 38) deduced from nucleotides 23-706.

Figure 20A and B. Ac-MTP-1 : A, cDNA (SEQ ID NO. 39) and B, amino acid sequence

(SEQ ID NO. 40).

Figure 21A and B. Ac-MTP-2: A, cDNA (SEQ ID NO. 41) and B, amino acid sequence

(SEQ ID NO. 42).

Figure 22A and B. Ac-MTP-3: A, cDNA (SEQ ID NO. 43) and B, amino acid sequence

(SEQ ID NO. 44).

Figure 23A and B. Ac-FAR-1 : A, cDNA (SEQ ID NO. 45) and B, amino acid sequence

(SEQ ID NO. 46). GeneBank Acession # AF529181

Figure 24A - C. Ac-KPI-1 : A and B, cDNA (SEQ ID NO. 47) and C, amino acid sequence

(SEQ ID NO. 48) deduced from nucleotides 12-2291.

Figure 25A and B. Ac-APR-1 : A, cDNA (SEQ ID NO. 49) and B, amino acid sequence

(SEQ ID NO. 50).

Figure 26A and B. Ac-APR-2: A, partial cDNA sequence (SEQ ID NO. 51) and B, partial amino acid sequence (SEQ ID NO. 52).

Figure 27A and B. Ac-AP: A, cDNA (SEQ ID NO. 53) and B, amino acid sequence (SEQ

ID NO. 54).

Figure 28A and B. Ay-ASP-1 : A, cDNA (SEQ ID NO. 55) and B, amino acid sequence (SEQ ID NO. 56).

Figure 29A and B. Ay-ASP-2: A, cDNA (SEQ ID NO. 57) and B, amino acid sequence (SEQ ID NO. 58).

Figure 30A and B. Ay-MTP-1: A, cDNA (SEQ ID NO. 59) and B, amino acid sequence (SEQ ID NO. 60).

Figure 31A and B. Ay-API-1 : A, cDNA (SEQ ID NO. 61) and B, amino acid sequence (SEQ ID NO. 62) deduced from nucleotides 23-703.

Figure 32A and B. Ay-TTR: A, partial cDNA (SEQ ID NO. 63) and B, partial amino acid sequence (SEQ ID NO. 64).

Figure 33A and B. Spearman rank order correlations between hookworm burden and anti-MTP-1 antibody titer. A) total worms; B) median EPG.

Figure 34A-C. Antigen-specific geometric mean IgGl antibody titers in dogs vaccinated with A. caninum recombinant fusion proteins as a function of time. Geometric means were calculated for a total of 6 dogs in each group, except for Ac-AP in which only a single dog developed an antigen-specific antibody response. The arrows denote timed vaccinations. (A) Anti-Ac-APR-1 responses (n=6). (B) Anti-Ac-TMP responses (n=6). (C) Anti-Ac-AP responses (n=l).

Figure 35. Female and male adult A. caninum hookworms recovered from the colons of either vaccinated or alum-injected dogs.

Figure 36A and B. Spearman rank order correlations between hookworm burden and anti-MTP-1 antibody titer

Figure 37A and B. A) Relationship between anti-TTR IgE antibodies and hookworm burden reductions; B) Relationship between anti-TTR IgGl antibodies and hookworm burden reductions

Figure 38A and B. HV-4 Canine hemoglobin (B) and hematocrit (A) changes following L3 challenge

Figure 39. Statistically significant reduction in worm size (between 1 and 2 mm) among the TTR vaccinated group relative to the adjuvant control group.

Figure 40. CD4+ lymphocytes from hookworm-infected (egg positive) individual post- stimulation with Ancylostoma L3 antigen. Figure 41. CD4+ lymphocytes from hookworm-infected (egg positive) individual post- stimulation with Pichia-expresses recombinant Na-ASP-1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides compositions for use in eliciting an immune response to hookworm in a mammal. Such compositions may be utilized as vaccines for use in the treatment and/or prevention of hookworm infection. The vaccines comprise purified preparations of antigens which are derived from hookworm, and a pharmacologically acceptable carrier. By "derived from" we mean that the antigen is a biomolecule that originated from (i.e. was isolated from) a hookworm. For example, the antigen may be a protein, a polypeptide, or an antigenic fragment of a protein, or polypeptide, which constitutes part of a hookworm organism. Typically, such an antigen is isolated and at least partially purified from a hookworm by methods which are well known to those of skill in the art (for example, see Examples section below). When manufactured for use in eliciting an immune response or as a vaccine, such antigens may be "synthetic" i.e. obtained synthetically (e.g. by peptide synthesis in the case of polypeptides and protein fragments), or "recombinant" i.e. obtained by genetic engineering techniques (e.g. by production in a host cell which harbors a vector containing DNA which encodes the antigen). Those of skill in the art will recognize that many such suitable expression systems are available, including but not limited to those which employ E. coli, yeast (e.g. Pichia pastor is), baculovirus/insect cells, plant cells, and mammalian cells, and. In preferred embodiments of the invention, the antigens are expressed in a yeast or baculovirus/insect cell expression system.

Examples of specific antigens, their amino acid primary sequences, and nucleic acid sequences which encode them are given herein. For ease of reference, Table I lists some exemplary antigens and their corresponding SEQ ID NOS. However, those of skill in the art will recognize that many variants of the sequences presented herein may exist or be constructed which would also function as antigens in the practice of the present invention. For example, with respect to amino acid sequences, variants may exist or be constructed which display: conservative amino acid substitutions; non-conservative amino acid substitutions; truncation by, for example, deletion of amino acids at the amino or carboxy terminus, or internally within the molecule; or by addition of amino acids at the amino or carboxy terminus, or internally within the molecule (e.g. the addition of a histidine tag for purposes of facilitating protein isolation, the substitution of residues to alter solubility properties, the replacement of residues which comprise protease cleavage sites to eliminate cleavage and increase stability, the addition or elimination of glycosylation sites, and the like, or for any other reason). Such variants may be naturally occurring (e.g. as a result of natural variations between species or between individuals); or they may be purposefully introduced (e.g. in a laboratory setting using genetic engineering techniques). All such variants of the sequences disclosed herein are intended to be encompassed by the teaching of the present invention, provided the variant antigen displays sufficient identity to the described sequences. Preferably, identity will be in the range of about 50 to 100%, and more preferably in the range of about 75 to 100%), and most preferably in the range of about 80 to 100% of the disclosed sequences. The identity is with reference to the portion of the amino acid sequence that corresponds to the original antigen sequence, i.e. not including additional elements that might be added, such as those described below for chimeric antigens.

TABLE I. Hookworm antigens, description, and corresponding SEQ ID NOS.

The invention also encompasses chimeric antigens, for example, antigens comprised, of the presently described amino acid sequences plus additional sequences which were not necessarily associated with the disclosed sequences when isolated but the addition of which conveys some additional benefit. For example, such benefit may be utility in isolation and purification of the protein, (e.g. histidine tag, GST, and maltose binding protein); in directing the protein to a particular intracellular location (e.g. yeast secretory protein); in increasing the antigenicity of the protein (e.g. KHL, haptens). All such chimeric constructs are intended to be encompassed by the present invention, provided the portion of the construct that is based on the sequences disclosed herein is present in at least the indicated level of homology.

Those of skill in the art will recognize that it may not be necessary to utilize the entire primary sequence of a protein or polypeptide in order to elicit an adequate antigenic response to the parasite from which the antigen originates. In some cases, a fragment of the protein is adequate to confer immunization. Thus, the present invention also encompasses antigenic fragments of the sequences disclosed herein, and their use in vaccine preparations. In general, such a fragment will be at least about 10-13 amino acids in length. Those of skill in the art will recognize that suitable sequences are often hydrophilic in nature, and are f equently surface accessible.

Likewise, with respect to the nucleic acid sequences disclosed herein, those of skill in the art will recognize that many variants of the sequences may exist or be constructed which would still function to provide the encoded antigens or desired portions thereof. For example, due to the redundancy of the genetic code, more than one codon may be used to code for an amino acid. Further, as described above, changes in the primary sequence of the antigen may be desired, and this would necessitate changes in the encoding nucleic acid sequences. In addition, those of skill in the art will recognize that many variations of the nucleic acid sequences may be constructed for purposes related to cloning strategy, (e.g. for ease of manipulation of a sequence for insertion into a vector, such as the introduction of restriction enzyme cleavage sites, etc.), for purposes of modifying transcription (e.g. the introduction of promoter or enhancer sequences, and the like), or for any other suitable purpose. All such variants of the nucleic acid sequences disclosed herein are intended to be encompassed by the present invention, provided the sequences display about 50 to 100% identity to the original sequence and preferably, about 75 to 100% identity, and most preferably about 80 to 100%) identity. The identity is with reference to the portion of the nucleic acid sequence that corresponds to the original sequence, and is not intended to cover additional elements such as promoters, vector-derived sequences, restriction enzyme cleavage sites, etc. derived from other sources.

The antigens of the present invention may be derived from any species of hookworm, examples of which include but are not limited to Necator americanus, Ancylostoma canium, Ancylostoma ceylancium and Ancylostoma duodenale.

Examples of suitable hookworm antigens include but are not limited to Na-ASP-1, Na-ACE, Na-CTL, Na- APR- 1 , NA-APR-2, Ac-TMP, Ac-MEP- 1 , Ac-MTP- 1 , Ac-ASP- 1 , Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac- CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac- APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR.

In some embodiments of the invention, the antigenic entity is an activation associated secretory protein, examples of which include but are not limited to Na-ASP-1, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ay-ASP-1, and Ay-ASP-2.

In other embodiments of the invention, the antigenic moiety is a protease, examples of which include but are not limited to metalloproteases (e.g. Ac-MTP-2, Ac-MTP-3; cysteine proteases; aspartic proteases (e.g. Ac-APR-1 and Ac-APR-2); and serine proteases.

In yet other embodiments of the invention, the antigen may be a lectin (e.g. Na-CTL, Ac-CTL).

In other embodiments of the invention, the antigen may be a protease inhibitor (e.g. Ac-API-1, Ay-API-1, Ac-AP, Ac-KPI-1).

In a preferred embodiment, the antigen utilized in the practice of the present invention is Ac-TMP, the DNA encoding sequence of which is given in Figure 6 A (SEQ ID NO. 11), and the amino acid sequence of which is given in Figure 6B (SEQ ID NO. 12).

In another preferred embodiment, the antigen utilized in the practice of the present invention is Ac-MEP-1, the DNA encoding sequence of which is given in Figure 7 A (SEQ ID NO. 13, and the amino acid sequence of which is given in Figure 7B (SEQ ID NO. 14).

In another preferred embodiment, the antigen utilized in the practice of the present invention is Ac-MTP-1, the DNA encoding sequence of wliich is given in Figure 8 A (SEQ ID NO. 15, and the amino acid sequence of which is given in Figure 8B (SEQ ID NO. 16).

Other preferred antigens include but are not limited to Na-CTL (SEQ ID NOS. 5-6); Na-APR-1 (SEQ ID NOS. 7-8); Na-APR-2 (SEQ ID NOS. 9-10); Ac-TMP (SEQ ID NOS. 11-12); Ac-ASP-3 (SEQ ID NOS. 21-22); Ac-ASP-4 (SEQ ID NOS. 23-24); Ac-ASP-5 (SEQ ID NOS. 25-26); Ac-ASP-6 (SEQ ID NOS. 27-28); Ac-TTR (SEQ ID NOS. 29-30); Ac-103 (SEQ ID NOS. 31-32); Ac-VWF (SEQ ID NOS. 33-34); Ac-CTL (SEQ ID NOS. 35-36); Ac- API-1 (SEQ ID NOS. 37-38); Ac-MTP-1 (SEQ ID NOS. 39-40); Ac-MTP-2 (SEQ ID NOS. 41-42); Ac-MTP-3 (SEQ ID NOS. 43-44); Ac-KPI-1 (SEQ ID NOS. 47-48); Ac-APR-1 (49- 50); Ac-APR-2 (SEQ ID NOS. 51-52); Ay-ASP-1 (SEQ ID NOS. 55-56); Ay-ASP-2 (SEQ ID NOS. 57-58); Ay-MTP-1 (SEQ ID NOS. 59-60); Ay-API-1 (SEQ ID NOS. 61-62); Ay- TTR (SEQ ID NOS. 63-64).

The present invention provides compositions for use in eliciting an immune response which may be utilized as a vaccine against hookworm. By "eliciting an immune response" we mean that an antigen stimulates synthesis of specific antibodies at a titer of about >1 to about 1 x 10⁶ or greater. Preferably, the titer is from about 10,000 to about 1 x 10⁶ or more, and most preferably, the titer is greater than 1 x 10⁶, as measured by, for example, ³H thymidine incorporation. By "vaccine" we mean an antigen that elicits an immune response that results in a decrease in hookworm burden of a least about 30% in an organism in relation to a non- vaccinated (e.g. adjunct alone) control organism. Preferably, the level of the decrease is about 50%, and most preferably, about 60 to about 70%) or greater.

The present invention provides compositions for use in eliciting an immune response which may be utilized as a vaccine against hookworm. The compositions include a substantially purified hookworm antigen or variant thereof as described herein, and a pharmacologically suitable carrier. The preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the composition may contain other adjuvants. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of hookworm antigen in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%.

The vaccine preparations of the present invention may further comprise an adjuvant, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc.

The preparations of the present invention may contain a single hookworm antigen. Alternatively, more than one hookworm antigen may be utilized in a preparation, i.e. the preparations may comprise a "cocktail" of antigens.

The present invention also provides method of eliciting an immune response to hookworm and methods of vaccinating a mammal against hookworm. By eliciting an immune response, we mean that administration of the antigen causes the synthesis of specific antibodies (at a titer in the range of 1 to 1 x 10⁶, preferably 1 x 10³, more preferable in the range of about 1 x 10³ to about 1 x 10⁶, and most preferably greater than 1 x 10⁶) and/or cellular proliferation, as measured, e.g. by ³H thymidine incorporation. The methods involve administering a composition comprising a hookworm antigen in a pharmacologically acceptable carrier to a mammal. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including bu not limited to by injection, orally, intranasally, by ingestion of a food product containing the antigen, etc. In preferred embodiments, the mode of administration is subcutaneous or intramuscular.

The present invention provides methods to elicit an immune response to hook worn and to vaccinate against hookworm in mammals. In one embodiment, the mammal is a human. However, those of skill in the art will recognize that other mammals exist for which it would also be of benefit to vaccinate against hookworm, i.e. the preparations may also be used for veterinary purposes. Examples include but are not limited to companion "pets" such as dogs, cats, etc.; food source, work and recreational animals such as cattle, horses, oxen, sheep, pigs, goats, and the like.

Those of skill in the art will recognize that, in general, in order to vaccinate (or elicit an immune response in) a species of interest (e.g. humans) against hookworm, the antigen which is utilized will be derived from a species of hookworm which parasitizes the species of interest. For example, in general, antigens from Necator americanus may be preferred for the immunization of humans, and antigens from Ancylostoma canium may be preferred for the immunization of dogs. However, this may not always be the case. For example, Ancylostoma canium is known to parasitize humans as well as its primary canine host. Further, cross- species hookworm antigens may sometimes be highly effective in eliciting an immune response in a non-host animal, i.e. in an animal that does not typically serve as host for the parasite from which the antigen is derived. Rather, the measure of an antigen's suitability for use in an immune-stimulating or vaccine preparation is dependent on its ability to confer protection against invasion and parasitization by the parasite as evidenced by, for example, hookworm burden reduction or inhibition of hookworm associated blood loss (e.g. as measured by hematocrit and/or hemoglobin concentration. For example, for use in a vaccine preparation, an antigen upon administration results in a reduction in worm burden of at least about 30%o, preferably at least about 50%, and most preferably about 60 to about 70%>.

In one embodiment of the present invention, a method for enabling vaccination of a patient against infectious diseases is provided. The method involves treating hookworm infection to a degree sufficient to increase lymphocyte proliferation, followed by vaccinating the patient against said infectious disease. The method is based on evidence provided in Example 10 which shows that hookworm infestation causes anergy to hookworm and possibly other antigen stimulation. Therefore, by chemically treating hookworm infected patients prior to vaccination against hookworm or any infectious agent, the response to the vaccination will be improved. Examples of infectious diseases against which vaccination outcomes may be improved include but are not limited to HIN, tuberculosis, malaria, and routine childhood vaccinations (e.g. measles, tetanus, diphtheria, pertussis, polio, and the like).

Examples of agents with which hookworm may be chemically treated include but are not limited to albendazole and other antihelminthic drugs. Certain of the antigens described herein may also be useful in the treatment of other neoplastic, autoimmune, and cardiovascular conditions, as well as for the treatment of pro- inflammatory states. Such uses of other hookworm antigens have been described in, for example, United States patent 5,427,937 to Capello et al. and United States patent 5,753,787 to Hawdon.

The present invention also provides the following nucleic acid and amino acid sequences: SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63 and SEQ ID NO. 64. The sequences represent cDNA sequences and the amino acid sequences (open reading frames) wliich they encode. While the sequences themselves are being claimed, other sequences with a high level of identity in comparison to those described are also contemplated, e.g. sequences having at least about 65 to 100%) identity, or preferably about 75 to 100% identity, or most preferably at least about 80 to 100% identity, to the sequences that are given.

In particular, the sequences for Ac-APR-2 (SEQ ID NOS. 51 and 52) and Ay-TTR (SEQ ID NOS. 63 and 64) are partial sequences which represent the majority of the antigen sequence. Thus, the present invention encompasses the entire Ac-APR-2 antigen and the entire Ay-TTR antigen.

Further, those of skill in the art will recognize that the Ay-TTR antigen which is provided in the present application is representative of the Ay-TTR family of antigens present in many species of nematodes. As such, an Ay-TTR antigen from any nematode is intended to be encompassed by the present invention. In particular, any Ay-TTR antigen derived from a hookworm species including but not limited to Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale, are encompassed. EXAMPLES Example 1. Molecular Cloning and Purification of Ac-TMP Materials and Methods.

Immunoscreening of adult A. caninum library Preparation of anti- A. caninum secretory product antibody. One hundred living adult stage Ancylostoma caninum hookworms were recovered from the intestines of an infected dog, at necropsy (6 weeks post-infection), as described previously (Hotez and Cerami, 1983). The adult worms were washed three times in sterile PBS, then maintained in 15 ml RPM 1640 containing 25 mM HEPES, 100 units/ml of ampicillin and 100 μg/ml streptomycin at 37C (5%> CO²) for 24 hours. The supernatant was collected, concentrated with PEG6000, and dialyzed against 1 L phosphate buffered saline (pH 7.2) overnight at 4C. Following dialysis, the secreted products were centrifuged at 10,000xg for 10 min, and the supernatant was recovered.

A rabbit was immunized by subcutaneous injection with the hookworm-secreted proteins (400 ug) emulsified with complete Freund's adjuvant. Subsequently, the rabbit was immunized at two week intervals with the same quantity of hookworm secreted proteins emulsified with incomplete Freund's adjuvant for a total of three immunizations. The final bleed was obtained 10 days after the final immunizations, and the serum was separated from whole blood and stored at -20C.

Construction of the cDNA expression ZapII (Stratagene, La Jolla CA) library was reported previously (Capello et al, 1996)). An estimated 5 x 10⁵ plaques were screened with the rabbit anti-A. caninum adult secretory product antibody according to manufacturer's instructions. Briefly, 5 x 10⁴ plaques were plated on an LB agar plate. A. caninum antigen expression was induced by covering the plaques with nitrocellulose membranes soaked with 10 mM IPTG. Four hours after incubation at 37C, the membranes were lifted, blocked with 5% non-fat milk in PBS, and then incubated with the rabbit antibody (1 :500 dilution) for 1 hour at 24C. The membranes were washed three times with PBS buffer containing 0.1% Tween-20 (PBS-Tween) and then incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (Sigma) at a 1 : 1000 dilution at 24C for another hour. The membranes were washed again three times with PBS-Tween and then developed with 3,3'-diaminobenzidine (DAB) substrate and hydrogen peroxide. The putative positive clones were scored and isolated for secondary screening. The immunopositive clones were excised into pBluscript phage according to manufacturer's instructions (Stratagene), Phagemid DNA was extracted using the alkaline lysis method (Qiagen) and double strand sequencing was performed using flanking vector primers (T₃ and T₇). Nucleotide and deduced amino acid sequences were compared to existing sequences in GenBank by BLAST searching. ESEE 3.1 software was used for sequence analysis. Reverse transcription polymerase chain reaction fRT-PCR') amplification.

RT-PCR was used to determine the developmental stage specificity of Ac-tmp mRNA transcription. A. caninum eggs, L I and L2 larval stages, and L3 infective larvae were obtained as described previously (Hawdon et al, 1999). The total RNA was isolated from each life history stage using TRIzol reagent (GIBCO BRL). Single-strand cDNA was synthesized using oligo d(T) primer and MMLV-RT(GIBCO BRL). Specific primers (TIMP3'-1HR and TIMP5'-2ER based on the sequence of Ac-tmp from 60bp to 440bp were used to amplify the Ac-tmp cDNA. PCR reaction parameters were comprised of 94C denaturing for 1 min, 55C annealing for 1 min, 72C extension for 2 min. A total 30 cycles were performed.

Purification of Ac-TMP natural product. Optimization of semi-preparative reverse phase chromatographic conditions for the fractionations of A. caninum adult secretory products was carried out on a 510 HPLC system (Waters), equipped with a 490 E multiwavelength detector with a semi-preparative flow-cell, set at 214, 280, 260 and 254mm and a 250mm x 4.6 I.D.

YMC-Pack Protein-RP, 200A, 5μm C₄ Column (Waters). The adults, caninum secretory products used as starting material were collected over 15 hr from 1260 adult hookworms in 15 ml RPMI 1640 containing 25 mM HEPES, 100 units/ml ampicillin, 100 μg/ml streptomycin and 100 μg/ml gentamicin at 37C. The supernatant was concentrated by ultrafiltration in a Centricon-3 microconcentrator (Amicon) to 0.3 vol. before centrifugation for 1 hr at 7,500 x g. Approximately 0.6 mg of the parasite secretory protein was chromatographed. Eluent A was 0.01% Trifluoroacetic acid (TFA) in water, and eluent B was 0.01%) TFA in acetonitrile. A 40-min linear aradient from 0-80% B was run at a flow-rate of 1 ml/min. Fractions of 0.5 min were collected, lyophilized, and were used for further purification and analysis by SDS-PAGE (Laemmli, 1970). For SDS-PAGE, 2 μl of secretory products as well as the 10 μl of HPLC isolated fraction number 51 were mixed with the same volume of 2X SDS-PAGE sample buffer (4% SDS, 2.5% 2- mercapto ethanol, 15% glycerol) and boiled for 5 min. The samples were run on a 4-20%> gradient SDS-PAGE gel at 100 V for 2 hours. The gel was stained with silver according to manufacturer's instruction (BIO-RAD).

RP-HPLC of Fraction 51, the fraction that contained the most abundant^, caninum. secretory protein from the semi-preparative separation, was carried out on a 510 HPLC system equipped as described above using a 250 mm. x 3.0 I.D. YMC protein RP, 200 A, 5 μm C4 column. Eluent A was 0.01% TFA in water, and B was 0.01% TFA in acetonitrile. A 30-min linear gradient from 0-60% B was run at a flow-rate of 1 ml/min. Fractions of 0.5 min were collected and lyophilized. The major protein peak collected from this separation was subjected to amino acid sequence analysis and SDS-PAGE (Laemmli, 1970). Amino acid sequence analysis based on the Edman degradation of protein was performed on procise 494 model protein sequencer (Applied Biosystems) equipped with a 785A programmable detector and a 140C pump system, by ProSeq, Inc. (Boxford MA). The sequencer products were identified using standard procise 610A software.

To confirm that the N-terminal sequence corresponded to Ac-TMP, degenerate oligonucleotide primers were synthesized in both orientations that corresponding to the partial N-terminal peptides sequence of fraction number 51. Paired flanking degenerate vector primers were used to amplify the product from DNA obtained from the adult cDNA library constructed in ZapII . The "hot start" PCR conditions were 10 mM Tris-HCl (pH 8.5) containing 50 mM KC1, 2.0 mM MgCl₂, 0.2 mM of each dNTP, and 1 μl cDNA library, in 20 μl reaction. The reactions were heated at 94C for 5 min, then lowered to 85C for 5 min, then 1 unit of Taq DNA polymerase (GIBCO BRL) was added. This was followed by 30 cycles of 1 min of denaturation at 94C, 1 min of annealing at 55C, and 2 min of extension at 72C. The PCR products were run on an agarose gel and stained with ethidium bromide. The PCR products were gel purified with the QIAEX II Gel Extraction kit (Qiagen, Valencia, CA), and sequenced.

Results for Example 1

Ac-TMP cDNA. Ac-TMP cDNA was cloned from an adult hookworm cDNA library by immunoscreening with rabbit antibody directed against whole A.caninum adult secretory products. Two positive identical clones were isolated. The full-length cDNA consists of 559 bps (SEQ ID NO. 11) encoding an open reading frame (ORF) of 140 amino acids (SEQ ID NO. 12) and a poly-A tail at the 3' end. The predicted ORF has a calculated molecular weight of 16,100 daltons and a theoretical pi of 7.55. There is a hydrophobic signal peptide sequence with a signal peptidase cleavage site between amino acids 16 and 17. Ac-TMP has a signature N terminal Cys-X-Cys sequence immediately following the signal peptide. One putative N linked glycosylation site (N-X-T) exists between amino acids 119 and 122 (Fig.6B).

GenBank database searching revealed that the predicted amino acid sequence of this molecule shares 33 percent identity and 50 percent similarity to the N-terminal domain of human tissue inhibitor of metalloproteinase 2 (TIMP-2). Both Ac-TMP and a putative TIMP from the free-living nematode Caenorhabditis elegans are comprised of a single domain and lack a second, C-terminal domain that is characteristic of vertebrate TIMPs (data not shown). RT-PCR amplification. To identify the life-history stage specific expression of Ac-TMP, mRNAs were extracted from different developmental stages of A.caninum and reverse transcribed to cDNA with Ac-TMP specific primers. RT-PCR produced a 380 bp specific band that was only amplified from adult cDNA. No amplification was seen from the cDNA of eggs, Li-L2 and L3 life history stages. Amplification of A. caninum genomic DNA revealed two bands suggestive of a possible intron or the existence of a second, related Ac-TMP gene (data not shown).

Identification of Ac-TMP in secretory products of A. caninum adult worm. To confirm that Ac-TMP is released by adult A. caninum hook-worms, the protein was identified in and purified from parasite secretory products via RP-HPLC. Each of the major peaks were subjected to amino acid sequence analysis as part of a larger A. caninum proteomics study (data not shown). The peak of protein corresponding to "Fraction 51" was selected for further study and re-chromatographed. Fraction 51 was comprised of one predominant band after silver staining that migrated with an apparent molecular weight of Mr =16,000. The N- terminal peptide sequence (20 amino acids) of this fraction was an identical match with the sequence of the predicted ORF of Ac-TMP after the predicted signal peptidase cleavage site. Based on the calculated area under the curve of HPLC peak 51 relative to the total area of the entire secretory product profile, Ac-TMP was determined to comprise approximately 6.3 percent of the total A. caninum secretory products. This identified the molecule as one of the most abundant proteins released by adult caninum. The abundance of Ac-TMP in hookworm secretory products was confirmed by visual inspection on SDS-PAGE. Paired degenerate primers based on the sequence of the first seven amino acids were used to construct PCR products from the adult hookworm cDNA library. DNA sequence of the PCR products confirmed the identity to Ac-TMP cDNA (data not shown).

This example demonstrates that TMP is the most abundant protein secreted by hookworms and that the protein has been cloned and expressed, and the recombinant protein isolated.

Example 2. Molecular cloning and characterization of Ac-mep-l. Materials and Methods.

Parasites. A. caninum parasites were maintained in beagles as described previously (Schad 1982). Third stage infective larvae (L3) were isolated from charcoal copro-cultures and stored in BU buffer (Hawdon et al. 1995). Adult caninum worms were collected from infected dogs upon necropsy. These worms were washed three times in PBS, snap frozen in liquid nitrogen, and stored at -80 ^°C.

Nucleic acids Genomic DNA was isolated from adults, caninum by standard methods (Ausubel et al. 1993). A. caninum RNA was isolated by grinding previously frozen (-80 °C) adult worms in the presence of Trizol reagent (Gibco BRL) and following manufacturers protocol. cDNA was prepared from RNA by the ProSTAR First Strand RTPCR Kit (Stratagene) according to the manufacturer's instructions.

A. caninum genomic and cDNA libraries An A. caninum genomic DNA library was constructed as follows: 3Q ugA. caninum genomic DNA was partially digested (37 °C for 5 min) by 8 U Sau3A restriction enzyme (NEB) in a 100 ul volume with recommended buffer. The digested DNA was then ethanol precipitated and pelleted by standard methods. The resulting pellet was dried, dissolved in water, and ligated into the Lambda-FIXII vector (Stratagene) according to manufacture's protocol. This ligation reaction was then packaged with Gigapack Gold packaging extract (Stratagene) and amplified. An A. caninum adult cDNA library was constructed previously (Capello et al. 1996) in lambda ZAPII (Stratagene) vector.

Metalloprotease cloning Cloning the Ac-mep-l cDNA began with PCR on adult hookworm library cDNA using a degenerate primer and oligo-dT. A degenerate primer was designed against a conserved sequence containing the zinc binding motif observed in an BLAST alignment of two hypothetical zinc metalloprotease genes from C. elegans (GenBank™ accession numbers T22668 and Q22523) The reaction conditions were as follows: 85 ng template DNA, IX thermophillic DNA buffer (Promega), 2.5mM MgC12, 0.2 mM dNTP's, 2 uM each primer, 1 U taq DNA polymerase (Promega), in 20 μl total volume. The reactions were cycled at 94 °C for 1 min, 55C for 1 min, and 72C for 1 min 35 times. This PCR yielded a fragment which when cloned (pGEM-T, Promega) and sequenced represented 458 bp (including 21 residues of the poly A tail) of the 3 ''Ac-mep-l cDNA (Clone MP-1). Utilizing the MP-1 as the basis for specific primer design additional sequence of Ac-mep-l (Clone MP- 2) was identified by PCR on library DNA with T3 (vector) and MEP-R1 gene specific primers. Reactions were conducted on serial dilutions of library DNA until a unique product was amplified and then cloned. Reaction conditions were as described above.

In a similar clone MP-3 was amplified with T3 and MEP-R2 primers. The 5 '-RACE kit from GibcoBRL was employed to identify the 5' end of Ac-mep-l . Briefly, first strand cDNA was produced in a reverse transcription reaction with the Ac-mep-l specific primer RACE-Rl on freshly prepared RNA. This cDNA was then poly C tailed at its 3' end with terminal deoxytransferase and used as template in a PCR reaction with anchor primer AAP (GibcoBRL) and gene specific reverse primer MEP-R2. The resulting products were diluted and used as template in a hemi-nested PCR reaction with anchor primer UAP (GibcoBRL) and gene specific primer MEP-R3. The PCR product generated was cloned and termed MP-4.

More 5' sequence was identified from a genomic DNA clone (G-MEP) of Ac-mep-l like sequence. Multiple clones were sequenced to confirm the Ac-mep-l cDNA and the full length coding region of Ac-mep-l was PCR amplified (clone FL-1) under the conditions described above as a single fragment utilizing suitable primers. Sequence analysis Alignment of the partial Ac-mep-l clones was conducted using MEGALIGN software from DNASTAR Inc. (version 3.7.1). BLAST analysis of the initial sequences used for degenerate primer design and the predicted open reading frame (ORF) of Ac-mep-l was conducted using the National Center for Biotechnology Information BLAST utility. Sequence analysis of Ac- mep-1 was conducted using the Curatools sequence analysis utility (Curagen Corp., New Haven, CT.). The FGENESH gene finder utility (CGG WEB server (genomic.sanger.ac.uk) with settings to analyze C. elegans DNA was utilized for gene predictions from the genomic DNA clone G-MEP. Identification of potential exon sequences in GMEP was accomplished with the Wise2 sequence analysis utility (sanger.ac.uk/Software Wise2/).

Northern blotting Northern blot analysis was conducted on Trizol (GibcoBRL) isolated total RNA from ten adult worms. This RNA was fractionated on a 1.2% formaldehyde gel and blotted to Hybond-N membrane (Amersham) by standard methods. The blot was probed with a ³²P random prime labeled DΝA fragment representing bp 780-2688 of the Ac-mep-l cDΝA. Developmental RT-PCR RT-PCR was used to investigate Ac-mep-l transcription in A. caninum life history stages. For these reactions cDΝA from egg, LI, non-activated and activated L3 and adult worms were tested vfiihAcmep-1 specific primers MEP-F1 and MEP- Rl. The quality if these cDΝAs was verified in separate reactions using primers PKA-F and PKA-R, which are specific fox A. caninum protein kinase A (Hawdon et al. 1995). The reaction conditions were identical to those defined in Section 2.4. Anti-Ac-mep-1 antibody A cDΝA fragment representing 610 amino acids from the C- terminal portion of Ac-MEP-1 was amplified from the adults, caninum cDΝA lambda library by PCR using suitable primers. This fragment was T/A cloned into pGEM (Promega) from which it was cloned into pET28c expression vector (Νovagen) at the Hindlll site by standard methods (Sambrook and Russell, 2001). Bacterial protein expression of truncated Ac-MEP-1 (tAc-MEP-1) was induced by the addition of 1 mM IPTG to a culture of BL21(DE3)PlysS (Stratagene) cells transformed with the tAc-MEP-1 /pET28c construct. The expressed protein was insoluble. In order to purify XAc-mep- the induced cell pellets were frozen (BL21(DE3)PlysS cells lyse after freezing), resuspended in one-tenth vol. of 50 mM tris pH 8.0, 2 μM EDTA, sonicated until no longer viscous and then centrifuged at 12, 000 xg for 15 min (Sorvall RC5B, GSA rotor). The resulting pellet was resuspended in 15 ml 1% SDS, 0.5%> B-mercaptoethanol, sonicated, boiled for 5 min, and then incubated at room temperature for 2 h. Undissolved debris was removed by repeat centrifugation. The supernatant was dialyzed exhaustively against phosphate buffered saline (pH 7.4) to remove the BME. The protein was purified on HisBind (Νovagen) nickel resin affinity column according to the manufacturer's protocol without denaturant. Groups of five male Balb/c mice (6-week-old) were immunized intraperitoneally with 20 ug of alum-precipitated tAc- MEP-1 or alum alone as control. The mice were subsequently boosted twice at 2-week intervals. One week after the third and final immunization, sera was collected, pooled, and used as a primary antibody in the western blot and immunostaining analysis. Western blotting Proteins separated by 10%> SDS-PAGE were transferred to methanol charged Immobilon-P PNDF membranes (Millipore) in transfer buffer (39 mM glycine, 48 mM tris base, 0.037% SDS, pH 8.3) for 18 h at 30N. The membrane was blocked in 5% nonfat milk in PBS (blocking buffer), for 1 h at room temperature (RT) with gentle shaking and incubated with E. coli absorbed primary mouse anti-t Ac-MEP-1 antibody (1:1500) diluted in blocking buffer for 1 h at RT. The membrane was then washed three times in blocking buffer (10 min each), and incubated for 1 h at RT with horseradish peroxidase- conjugated goat anti-mouse IgG secondary antibody (1 :5000) in blocking buffer with shaking. Finally, the membrane was washed three times in PBS for 15 min and developed with Renaissance (ΝEΝ Life Science Products) chemiluminescent reagents. Immunόlocalization Adult A. caninum worms were paraffin embedded and sectioned by standard methods. In situ immunolocalization of Ac-MEP-1 was accomplished by incubating de-parrafinized worm sections in a 1:100 dilution (in PBS, pH 7.4) of mouse anti-tAc-MEP-1 or control sera (see above) for 1 h at RT. The sections were washed three times in PBS and incubated in a 1 :200 dilution of goat anti-mouse IgG at 25 °C for 1 h followed by washing in PBS (three times). Sections were then visualized with a Olympus IX-50 inverted fluorescence microscope (U-MWIG filter) and photographed. Results for Example 2 cDΝA structure of Ac-mep- 1 The cloning strategy employed in obtaining the complete coding sequence of Ac-mep-l was as follows: About 2.6 kb o ^'the Ac-mep-l transcript was identified by sequencing degenerate PCR clone MP-1, PCR derived clones MP-2, MP-3 and the 5' RACE clone MP-4. Although there was a methionine codon close to the 5' end of the RACE product, this codon was preceded by 58 in-frame amino acids that contained no stop, suggesting that MP-4 did not represent the actual 5' end of Ac-mep-l. In addition, we have been unable to obtain a cDΝA clone (by PCR) that included a spliced leader sequence. Therefore, G-MEP, a genomic DΝA clone of Ac-mep- 1 like sequence (98.7%) exon identity), was examined with a gene prediction program for C. elegans DΝA and a different potential transcription start site than was identified by 5' RACE was identified . This prediction extended 158 bp beyond the 5' RACE sequence and increased the deduced coding region by 91 amino acids. Utilizing this prediction the entire coding region of Ac-mep-l was amplified as a single product of 2.7 kb product and the clone was confirmed by partially sequencing both its ends. The total length of the Ac-mep-l transcript is -2.8 kb as verified by Northern blot (non-coding portions of the 5' and 3' ends were not amplified in the full length PCR). The deduced amino acid sequence of this transcript encodes a single ORF of 870 amino acids with four potential N-linked glycosylation sites (predicted pl=5.5, m.w.=98.7 kDa). The N- terminal amino acids of Ac-MEP-1 comprise a hydrophobic signal peptide sequence with a predicted cleavage after residue 22 (SEE Figure with AC-MEP-1 sequence). Two signature zinc-binding motifs characteristic of the Endopeptidase 24.11 family of metalloproteases (Hooper, 1994) were identified.

Ac-mep-l is 66% similar and 48% identical to a metalloprotease (Hc-MEPlb) from the related trichostrongyle blood feeding nematode H. contortus. It is also equally similar to a metalloprotease (T25B6.2) from the non-parasitic nematode C. elegans (Gen-Bank™ T28906). Fourteen cysteine residues are highly conserved between these three molecules. Two additional cysteines (only one is conserved) are present in both Ac-MEP-1 and Hc- MEPlb.

Northern blot and developmental analysis of Ac-mep-l expression Northern blot analysis reveals a single mRNA transcript of approximately 2.8 kb in adult hookworm mRNA (not shown). RT-PCR was employed to investigate the developmental specificity of Ac-mep-l transcription. Of the cDNAs tested it was possible to identify transcription only in the adult stage of the parasite and not in hookworm eggs, LI or activated and non-activated L3 larvae. In contrast, positive control PCR conducted on the same cDNAs with primers specific for A. caninum protein kinase A revealed amplification from all template cDNAs. Thus, Ac-mep-l appears to be expressed exclusively in adult worms.

Western blot analysis and immunolocalization of Ac-mep- 1 in adult worm sections By western blotting, the mouse anti-MEP-1 antiserum strongly recognizes adult caninum proteins of -90 and 100 kDa. Immunohistochemical analysis of adult worm sections localizes Ac-MEP-1 to the microvillar surface of the hookworm gut. The antiserum reacts strongly to the gut microvilli in sections of adult worm as compared with sections incubated with control sera. Weaker staining in the tegument of the adult worm was also occasionally noted. Although the function of Ac-MEP-1 is not known, its location along the microvillar surface of the parasite gut would suggest that the enzyme is in direct contact with the blood meal, and may, therefore, have a role in nutrient digestion.

This example demonstrates that MEP-1 is an important enzyme which allows hookworms to digest blood, and therefore is an attractive vaccine target. The recombinant MEP-1 protein has been cloned and expressed. Example 3. AC-MTP Antigen Studies

Infective third-stage Ancylostoma hookworm larvae (L3) release a zinc-dependent metalloprotease that migrates with an apparent molecular weight of 50 kDa (Hawdon et al 1995a). The enzyme is released specifically in response to stimuli that induce feeding and development in.the L3 (Hawdon et al, 1995b), and probably functions either in parasite skin and tissue invasion or ecdysis (Hotez et al, 1990). Because of its role in parasite-derived tissue invasion and molting, an anti-enzyme antibody response directed against Ac-MTP-1 might block larval migrations and parasite entry into the intestine. Ac-MTP- 1 is stage specific, and released by hookworm L3activated under hostlike conditions to resume feeding in vitro. Release of Ac-MTP- I during activation makes this molecule an attractive vaccine target.

Example 3A. Isolation of a cDNA from an A. caninum L3expression library that encodes a zinc-metalloprotease (Ac-mtp-1) of the astacin family. Material and Methods

Antisera: Sera used for immunoscreening of the A. caninum L3 expression library were collected from 5 residents of Nanlin county in Anhui Province, China, under an IRB- approved human investigations protocol. Ancylostoma duodenale is the predominant hookworm in this region, with a ratio of A. duodenale to Necator americanus of greater than 20:1 based on the recovery of larval and adult hookworms from infected patients (Yong et al. 1999). Sera were obtained from Anhui residents who had high titers of circulating antibodies to A. caninum L3 whole lysate antigens, as described elsewhere (Xue et al., 2000). Two of the residents were hookworm egg-negative, whereas the remaining 3 harbored quantitative fecal egg counts of less than 400 eggs per gram of feces. Because of their high antibody titer and low intensity of infection, these individuals were considered putatively resistant, and their sera were pooled and used for immunoscreening. Negative control sera were collected from college students in Shanghai.

Expression library screening: An A. caninum (Baltimore strain) L3 cDNA library constructed in X Zapll (Stratagene, La Jolla, CA) (Hawdon et al. 1995) was screened using the pooled antisera according to the manufacturer's instructions. Briefly, 5 x 10⁴ plaques were induced to express protein by applying a nitrocellulose membrane soaked in 10 mM IPTG for 4 hr at 37 C. Following incubation, the membrane was incubated in 5% non-fat dry milk in PBS for 1 hr. The blocked membrane was incubated with a 1 :100 dilution of pooled human sera in PBS for 1 hr at 22 C, washed 3 times in PBS for 10 min at 22 C, and incubated with a 1:1000 dilution of horseradish peroxidase conjugated anti-human IgG (Sigma, St. Louis MO). The membrane was developed with substrate of 3,3'-diaminobenzidine (DAB) and 0.0 15 %> hydrogen peroxide. Positive plaques were subjected to several rounds of plaque purification by re-plating and re-screening. Plasmids were rescued by in vivo excision (Short and Sorge, 1992) and both strands sequenced using primers complementary to flanking vector sequence. Nucleotide and deduced amino acid sequences were compared to existing sequences in the GeneBank database by BLAST searching (Altschul et al., 1997).

Cloning of full-length Ac-MTP cDNA: All of the positive clones isolated were truncated at the 5' end. To obtain the 5' end, a PCR using a gene specific primer P I and a primer corresponding to the conserved nematode spliced leader was used to amplify the 5' end from first strand cDNA of A. caninum L3. Twenty μL reactions containing 100 ng of each primer, 1 U of Taq polymerase (Promega, Madison WI), and 1 μL of cDNA was denatured for 2 min at 95 C, followed by 30 cycles of 1 min at 94 C, 1 min at 55 C, and 2 min at 72 C. Amplicons were gel purified and cloned into pGEM Easy-T vector (Promega, Madison, WI) by standard methods.

Stage Specificity: The stage-specificity ofmtp-1 transcription was determined by RT-PCR (Hawdon et al, 1995). A. caninum eggs were isolated from the feces of infected dogs by sucrose floatation (Nolan et al, 1994), axenized by treatment with NaOCl, and plated on nematode growth medium agar plates (Sulston et al. 1988). Following incubation at 26 C for 24-30 h, the hatchlings (mixed L^L,) were washed from the plates with BU buffer (Hawdon and Schad, 1991) and snap-frozen in a dry ice/ethanol bath. Unhatched eggs were also snap frozen to make cDNA. A. caninum adults were collected from the small intestine of an infected dog at necropsy. RT-PCR was performed on A. caninum eggs, mixed Lj/L₂ serum- stimulated and nonstimulated L₃ (see below), and adult A. caninum samples as follows. Samples were ground to a powder in a pre-chilled (liquid N₂) mortar, and total RNA isolated using the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. The RNA was treated with 10 U DNAse 1 (RNase free, Boehringer Mannheim, Indianapolis, IN) and reextracted with TRIzol. Total egg RNA was isolated by mechanical disruption with glass beads in the presence of TRIzol using a BeadBeater machine (BioSpec, Bartlesville, OK), DNAse treated, and re-extracted as above. First strand cDNA was synthesized from each sample in a 50 μL reaction containing 50 mM Tris HCl, pH 8.3, 75 mM KCI, 3mM MgCl₂, 10 mM DTT, 500 ng oligo(dT) primer, 1 μg of total RNA, and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies) at 37 C for 1 hr. The reaction was incubated at 94 C for 5 min, and brought to 100 μL with dH₂0. One μL of the first strand cDNA was used in a PCR with primers MTP5'- I (5'-CTTCTCATGATCAACAAACACTACG) SEQ ID NO. 65 and MTP3'-1 (5'- AATCTAACTCCAACATCTTCTGGTG) SEQ ID NO. 66. The reaction was cycled 30 times for 1 min at 94 C, I min at 55 C, and 1 min at 72 C. Amplicons were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide.

Expression and Purification of Recombinant Protein: The full-length Ac-mtp-1 cDNA was cloned in-frame in the expression vector pET28 (Novagen) and transformed into competent BL-21 E. coli cells using standard techniques. Expression of the recombinant protein, containing 6 vector-encoded histidine residues (His-Tag) at the 5' end, was induced by the addition of 1 mM IPTG for 3 hours at 37C. One ml of cells expressing rMTP- 1 were sedimented by centrifugation at 5000 x g for 5 min, the supernatant discarded, and the cells lysed in 100 mis of TE (pH 8.0) containing 100 μg/ml lysozyme and 0.1 % Triton X- 100. After incubation at 30 C for 20 min, the sample was sonicated (power level 2-3, 20-30%) duty cycle) on ice for 10 bursts of 5 sec each until the sample was no longer viscous. Soluble and insoluble cell fractions were separated by electrophoresis in a 12% SDS-PAGE under reducing conditions, and the resolved proteins visualized with Cooomassie blue staining. For purification of rMTP-1, a cell pellet from 2 1 of induced bacterial culture was suspended in 60 ml of 1.0% SDS, 0.5% 2-mercaptoethanol, boiled for 5 min, and cooled to room temperature. The extract was dialyzed against 2 liters of 0.1 %SDS in PBS for 48 hr with 2 changes of buffer, and applied to a 10 ml HisBind nickel resin column (Novagen). Chromatography was conducted according to the manufacturer's instruction except that 0.1%) SDS was added to all buffers.

In an effort to increase solubility and investigate the domain structure of MTP-1, 3 constructs lacking the amino HisTag sequences were made by PCR. The full length Ac-MTP cDNA (1-1642 bp), the cDNA without the 5'-propeptide (408-1642 bp), and the putative catalytic domain (408-1101 bp) were cloned in frame into pET28 at the upstream Neo I site, thereby removing the HisTag coding sequence from the vector. The recombinant proteins were expressed under the same conditions as described above. Antiserum Production Anti-rMTP polyclonal antiserum. was obtained by immunizing BABL/C mice with purified rMTP. Twenty μg of column purified rMTP was co-precipitated with alum (Ghosh et al. 1996) and injected subcutaneously. Additional boosts with alum precipitated rMTP (20 μg each) were administered at 3, 6, and 9 weeks.

Mouse antiserum was adsorbed against bacterial lysates of E. coli strain BL21 to remove antibodies reacting with bacterial proteins. Twenty-five ml of induced cells were centrifuged, dissolved in 25 mis of 2X sample buffer (100 mM Tris, pH6.8, 2% SDS, 2.5% 2- mercaptoethanol), and centrifuged at 12,000 x g for 10 min. Nitrocellulose membranes (4 cm x 8 cm) were soaked in the supernatant for 20 min, followed by incubation in transfer buffer (48 mM Tris, 39 mM glyine, 0.037% SDS, 20% methanol) for 30 min. The membranes were washed 3 times in PBS containing 0.1% Tween-20 and incubated with a 1 :100 dilution of the mouse antiserum for 1 hr at 22 C. The incubation was repeated 2 times with fresh membranes. To confirm specificity of the antibody, an aliquot of the adsorbed mouse antiserum was adsorbed a second time against bacterial lysates of BL21 (DE3) cells expressing full length rMTP-1. The adsorbed antiserum was used for Western blotting. In vitro activation of L3 and collection of ES products: A. caninum L₃ were activated under host-like conditions as described previously (Hawdon et al, 1999). Briefly, L3 collected from coprocultures were decontaminated with 1% HCl in BU buffer (Hawdon and Schad, 1991) for 30 min at 22 C. Approximately 5000 L₃ were incubated at 37 C, 5% CO₂ for 24 hr in 0.5 ml RPM₁₆₄₀, tissue culture medium supplemented with 25 mM HEPES pH 7.0, and antibiotics (Hawdon et al., 1999) in individual wells of 24-well tissue culture plates. L3 were activated to resume feeding by including 15% (v/v) of a <10 kD ultrafiltrate of canine serum and 25 mM S-methyl-glutathione (Hawdon et al, 1995). Non-activated L₃ were incubated in RPMI without the stimuli. The percentage of feeding larvae was determined as described (Hawdon et al, 1996).

Medium containing activated and non-activated L₃ were transferred to separate microcentrifuge tubes and centrifuged for 5 min at 14,000 rpm. Supernatants from identical treatment groups were pooled, filtered through a 0.45 μm syringe filter to remove any L₃ and cast cuticles, and stored at -20 C. Prior to electrophoresis, the supernatants were concentrated by ultrafiltration using Centricon 10 cartridges (Amicon, Beverley, MA). Concentrated ES were washed with 1 ml of BU, ultrafiltered, and lyophilized.

To collect adult ES, 1260 adult worms were incubated in RPMI_]640, tissue culture medium (Hawdon et al, 1999) for 15 hrs at 37 C, 10%) CO₂. The supernatant was concentrated 3-fold by ultrafiltration in Centricon 3 spin columns. Western blotting: Lysates of bacterial cells expressing rMTP- 1 fusion proteins and lyophilized ES products were re-suspended in 2x SDS-PAGE sample buffer (4%>SDS, 5%> 2- mercaptoethanol, 15% glycerol) and separated on a 4-20% gradient SDS-PAG (Invitrogen, Carlsbad, CA). Separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA) by electroblotting at 25V for 1 hr (Towbin et al, 1979). The membrane was blocked with 5% non-fat dry milk in wash buffer (PBS, pH7.4, 0.1% Tween 20) for 1 hour at 22 C. The blocked membrane was incubated for 1 hr at 22 C with a 1 :5000 dilution of mouse rMTP antisermn which has been preabsorbed against bacterial lysates expressing full length rMTP. The membrane was washed 3 times with wash buffer for 10 min at 24 C, followed by incubation with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse Ig (Boehringer Mannheim, Indianapolis, IN) for 1 hour at 22 C. Bands were visualized using chemiluminescent detecting reagents (ECL+, Amersham. Pharmacia Biotech, Piscataway, NJ). Results for Example 3A.

Cloning of A. caninum MTP cDNA An _^4. caninum L3 cDNA expression library was screened using pooled sera with high anti-hookworm L3 titer collected from human patients in endemic regions of China. Twelve positive clones were identified, 6 of which were identical as determined by DNA sequencing. Each clone contained a 3'poly-A tail, but was truncated at the 5' end. The 5' end was isolated from A. caninum L₃ cDNA by PCR using a primer derived from the nematode spliced leader (Hawdon et al., 1995; Bektech et al., 1988) together with the gene-specific primers PI.

The full length cDNA, without the poly(dA) tail, is 1703 bp (see Figure 8 A, SEQ ID NO. 15) and encodes a 547 amino acid open reading frame (see Figure 8B, SEQ ID NO. 16) with a calculated molecular weight of 61,730 and a pi of 8.72. The ATG start codon begins 2 nt downstream from the end of the spliced leader sequence, resulting in a total of 23 untranslated nt at the 5' end of the Ac-mtp-1 cDNA. A TAA stop codon is located at nt 1666- 1668, followed by a 35 bp 3' UTR containing an AATAAA polyadenylation signal (Blumenthal and Steward, 1997) 12 bp upstream (bases 1687-1692) from the poly(dA) tail. Amino acids 1 through 16 of the deduced protein sequence are predicted to represent a hydrophobic signal peptide, with a potential cleavage site between Ala,6and Gly,7 (Nielson et al, 1995). The deduced sequence contains 2 potential N-linked glycosylation sites (N-X-S/T) at Asn39 and Asnl59.

A BLAST search (Altschul et al, 1997)of GenBank using the Ac-MTP-1 predicted amino acid sequence indicated significant homology to members of a family of zinc metalloproteinases called the astacins (Bond and Benyon, 1995), named for the digestive protease astacin from the crayfish Astacus astacus. A search of the protein structure databases (Apweiler et al, 2000) with the Ac-MTP-1 deduced amino acid sequence revealed the presence of characteristic astacin fingerprints, including the extended zinc binding domain and a conserved Met turn located 37 amino acids downstream. The catalytic domain containing the zinc binding site is followed by a domain with homology to epidermal growth factor (EGF), from amino acids 334 to 368. From amino acids 374 to 484 is a domain with weak homology to the CUB domain, named for its occurrence in complement subcomponents Clr/Cls, embryonic sea urchin protein C/egf, and 2?MP- 1. The EGF and CUB domains are common in astacin metalloproteinases, and are believed to be involved in protein-protein interactions (Bond and Benyon, 1995).

Following the N-terminal signal peptide is a 119 amino acid, helix-rich pro-peptide domain. The C-terminal end of the propeptide domain contains a 4 basic amino acid sequence (R-E-K-R) from amino acids 132 to 135 that is a potential recognition site for furin or other trypsin-like processing enzymes (Bond and Benyon, 1995). Proteolysis at this site would activate Ac-MTP- 1 to a putative 412 amino acid processed form with a calculated MW of 46419 and a pi of 8.04.

RT-PCR analysis of stage specificity: The stage-specificity of Ac-mtp-l expression was investigated by qualitative RT-PCR of cDNA from several developmental stages of A. caninum. Ac-mtp-l specific primers were designed to amplify a 434 bp portion of the Ac-mtp-l cDNA corresponding to nt 985 -1419 of the complete sequence. The product of the predicted size was amplified from both non-activated and activated L₃ cDNA, but not from A. caninum egg or L,/L₂ mixed stage cDNA. A band of lesser intensity was seen in adult cDNA. A longer fragment was amplified from genomic DNA, indicating that the primers spanned an intron, and confirming that the amplicons from the cDNAs were derived from amplification of cDNA rather than contaminating genomic DNA. Control primers that amplify a portion of the constitutively expressed^, caninum protein kinase A catalytic subunit (Hawdon et al., 1995) successfully amplified product from all DNA samples, indicating that amplifiable template was present.

Expression of recombinant MTP and immunoblotting: Recombinant MTP- 1 was produced in E. coli, purified by Ni column chromatography, and used to immunize BALB/c mice for the production of specific antiserum. The antiserum was adsorbed against E. coli lysates and used to determine if Ac-MTP-1 is secreted by A. caninum L₃ in vitro. ES products from 10,000 non-activated (non-feeding) and activated (feeding) L₃ were analyzed by Western blotting using the rMTP-1 antiserum. The antiserum recognizes both the full length and processed (i.e. without the pro-peptide domain) forms of rMTP-1 expressed in E. coli BL21 (DE3) cells but fails to recognize any bands in lysates of induced cells containing the vector alone.

The rMTP antiserum recognized bands of MW, of 47.5 and 44.5 in the ES products of 10,000 A. caninum L3 that had been activated to resume feeding in vitro. The antiserum failed to recognize any bands in ES from 10,000 non-activated L₃ in culture medium alone, or in adult A. caninum ES products or worm lysates (not shown). A slower migrating band in activated ES has a MW similar to that of the processed form of rMTP (47.5 versus 46.5), indicating that A. caninum L₃ release processed MTP-1 during in vitro activation. The lower MW band was also recognized by pre-immune mouse serum (not shown), suggesting that the antiserum recognized a protein unrelated to Ac-MTP-1. To confirm that this recognition was non-specific, the mouse antiserum was adsorbed against BL21 (DE3) cells expressing full length MTP-1 and used to probe the Western blot. Adsorbed antiserum failed to recognize any rMTP-1, but recognized a band of MWr = 44.5 in activated ES products, suggesting that recognition of the lower MW band by the antiseruin is non-specific.

Recombinant MTP-1 was recognized by the pooled sera used to screen the library, but sera from individuals living in a non-endemic area (Shanghai) failed to recognize rMTP-1 (not shown). Example 3B. Isolation and characterization of a MTP-1 cDNA

Serum from hookworm-infected patients in China was used as a probe to carry out the isolation and characterization of a cDNA from an caninum L3 expression library that encodes a zincmetalloprotease (Ac-mtp-l) of the astacin family. An A. caninum (Baltimore strain) L3 cDNA expression library constructed in 1 ZapII (Stratagene, La Jolla, CA) (Hawdon et al., 1995) was screened according to the manufacturer's instructions using pooled antisera from patients in Anhui Province, China, where A. duodenale is the predominant hookworm species (Yong et al., 1999). Sera from patients with low fecal egg counts and high titers of circulating antibodies to A. caninum L3 whole lysate antigens, suggesting that they might be resistant to hookworm infection, were used. Six identical, truncated clones were recovered following plaque purification. The 5' end was isolated from A. caninum L3 cDNA by nested PCR using the nematode spliced leader sequence together with two gene-specific primers (Hawdon et al., 1995), and two independent 5' end clones were sequenced. Results from Example 3B.

The amplified sequence is believed to represent the complete 5' end of the transcript because the predicted ATG start codon is the first methionine following the spliced leader, the first 16 deduced amino acids encode a signal peptide characteristic of secreted proteins (Nielson et al., 1997), and alignments with similar metalloproteases suggest that this is the complete amino acid sequence. The full length cDNA, without the poly(dA) tail, is 1703 bp and encodes a 547 amino acid open reading frame with a calculated molecular weight of 61,730 and apl of 8.72. Amino acids 1 through 16 of the deduced protein sequence are predicted to represent a hydrophobic signal peptide, with a potential cleavage site between Alal6 and Glyl7 (Nielson et al., 1997). The protein sequence contains two potential N-linked glycosylation sites (NX- S/T) at Asn39 and Asnl59. A BLAST search (Altschul et al, 1997) of GenBank using the Ac-MTP-1 predicted amino acid sequence indicated significant homology to members of a family of zinc metalloproteinases called the astacins (Bond and Beynon, 1995), named for a digestive protease from the crayfish Astacus astacus. Members of this family are characterized by a short -terminal signal peptide that targets them for secretion, followed by a pro-peptide, and a catalytic domain containing the characteristic zinc-binding region and 'Met turn'. Unlike astacin, most other members of the family contain C-terminal domains, including variable numbers of EGF and CUB domains (Bond and Beynon, 1995). A search of the protein structure databases (Apweiler et al, 2000) with the Ac- MTP-1 deduced amino acid sequence revealed the presence of characteristic astacin fingerprints, including an extended zinc binding region, and a conserved Met turn located 37 amino acids downstream. The catalytic domain containing the zinc binding site is followed by a domain with homology to epidermal growth factor (EGF), from amino acids 334 to 368. From amino acids 374 to 484 is a domain with weak homology to the CUB domain, named for its occurrence in complement subcomponents Clr/Cls, embryonic sea urchin protein Uegf, and BMP-1 (Bork and Beckman, 1993).

Astacin metalloproteinases are synthesized as inactive proenzymes. Removal of the pro-peptide by a processing enzyme activates the enzyme (Bond and Beynon, 1995). Ac- MTP-1 contains a 119 amino acid N-terminal domain with a predicted four amino acid recognition site (R₁₃₂ E_]33 K_]34 R₁₃₅) for a trypsin- or furin-type processing enzyme at its C- terminus (Bond and Beynon, 1995). Proteolysis at this site would activate Ac-MTP-1 to a putative 412 amino acid processed form with a calculated MW of 46,419 and a pi of 8.04. The pro-peptide is also predicted to contain four amphipathic α-helices separated by a short linker region (amino acids 23-86) (Kelley et al, 2000). The stage-specificity of Ac-mtp-l expression was investigated by qualitative RT-PCR of cDNA from several developmental stages of A. caninum. Specific primers were designed to amplify a 434 bp portion of the Ac-mtp-l cDNA corresponding to nucleotides 985-1419 of the complete sequence. A product of the predicted size was amplified from both non- activated and activated L3 cDNA, but not from A. caninum egg or L1/L2 mixed stage cDNA, indicating that Ac-mtp-l is expressed primarily in the L3 stage. A band of lesser intensity was seen in adult cDNA. Although this band was weak, conclusions regarding the amount of gene expression are not possible, as the RT-PCR is qualitative only. However, a Western blot of adult lysates using mouse anti-rMTP serum failed to recognize any proteins in adult ES or lysates (not shown). This suggests that expression of Ac-MTP-1 is restricted to the L3 stage, and that the message present in the adult stages is untranslated or possibly partially degraded.

Recombinant MTP-1 was produced in Escherichia coli, purified by Ni column chromatography, and used to immunize BALB/c mice for the production of specific antiserum. The antiserum was adsorbed against E. coli lysates and used to determine if Ac- MTP-1 is secreted by A. caninum L3 in vitro. ES products collected from 10,000 non- activated (non-feeding) and activated (feeding) L3 (Hawdon and Schad, 1993) were analyzed by Western blotting using the rMTP-1 antiserum. The antiserum recognizes both the full length and processed (i.e. without the pro-peptide domain) forms of rMTP-1 expressed in E. coli BL21(DΕ3) cells, but fails to recognize any bands in lysates of induced cells containing the vector alone. A lower MW band was observed and is similar in size to the processed rMTP (i.e. lacking the pro-sequence), suggesting that some of the rMTP expressed in E. coli undergoes in vitro cleavage at the C-terminal end of the pro-peptide. This is probably the result of autocatalytic cleavage, although non-specific cleavage by a bacterial protease is also a possibility. Autocatalysis might also represent the physiological activation mechanism of Ac-MTP-1 in vivo.

The rMTP antiserum recognized bands of Mr of 47.5 and 44.5 in the ES products of 10,000 A. caninum L3 that had been activated to resume feeding in vitro. The antiserum failed to recognize any specific bands in ES from non-activated L3, in culture medium alone, or in adult A caninum ES products or worm lysates (not shown). A slower migrating band in activated ES had a Mr similar to that of the processed form of rMTP (47.5 vs. 46.5), indicating that caninum L3 release processed MTP-1 during in vitro activation. Furthermore, MTP-1 is released only in response to stimuli that activate L3 to resume feeding, and therefore, most likely functions at some stage of the infective process (Hawdon et al, 1996). The metalloproteolytic activity described previously was also released specifically during activation, and was of similar molecular size (Hawdon et al., 1995), suggesting that Ac-MTP-1 might be responsible for at least a portion of this activity.

A lower MW band (Mr 44.5 kDa) in activated ES products was also recognized by pre-immune mouse serum (not shown), suggesting that the antiserum recognized a protein unrelated to Ac-MTP-1. To confirm that this recognition was non-specific, the mouse antiserum was adsorbed against E. coli cells expressing full length MTP-1 and used to probe the Western blot. Adsorbed antiserum failed to recognize any rMTP-1, but recognized a band of Mr 44.5 in activated ES products, suggesting that recognition of the lower MW band by the antiserum is non-specific. Recombinant MTP-1 was recognized by the pooled sera used to screen the library, but sera from individuals living in a non-endemic area (Shanghai) failed to recognize rMTP-1 (not shown).

While the exact function of Ac-MTP-1 is unknown, the stage specificity of expression and the specific release during activation suggest a critical role in the infective process. Thus, interruption of Ac-MTP-1 function in vivo offers a useful strategy for the development of a vaccine to control hookworm disease.

This example demonstrates that MTP-1 is an important enzyme used by the hookworm parasite for invasion, and the protein is an immunodominant antigen because it is recognized by serum from patients with low hookworm burden despite repeated exposure to hookworm. MTP is therefore an attractive candidate for a vaccine antigen. Example 3C. Canine vaccine trials with Ac-MTP-1 antigen

To test whether Ac-MTP-1 could be an effective vaccine, two groups of five (5) purpose-bred male beagles 8 + 1 wk of age were vaccinated either with the recombinant (expressed and isolated from Escherichia coli) fusion protein formulated with AS02A adjuvant, or adjuvant alone. The composition of AS02A, which has been successfully used in several malaria vaccine clinical studies, is described elsewhere (Lalvani et al, 1999; Bojang et al, 2001 ; Kester et al, 2001). Details of the animal husbandry and housing conditions were reported previously (Hotez et al, 2002a). The recombinant fusion protein containing a polyhistidine tag was purified from washed E. coli inclusion bodies that were solubilized in 6 M guanidine-HCl in 10 mM Tris HCl, pH 8.0. The solubilized inclusion bodies were processed in 5-10 ml batches by gel filtration chromatography (Sephacryl S-300, 26/60 gel filtration column [Amersham Pharmacia] pre-equilibrated in a buffer containing 0.1 NaH2PO4, 10 mM Tris-HCl and 6 M guanidine) at room temperature (flow rate of 2 ml/minute). Selected fractions containing Ac-MTP-1 (as determined by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGΕ]) were pooled, refolded according to the method of Singh et al (2001), and then loaded onto a 5 ml Hi-Trap IMAC column (Amersham Pharmacia) charged with ZnCl₂ and equilibrated in 50 mM sodium phosphate pH 7.2, 1 M urea, and 0.5 M NaCI. The column was subsequently washed with 15 column volumes of equilibration buffer, and the bound protein was eluted with 50 mM sodium phosphate pH7.2, 1 M urea, 0.5 M NaCI, and 50 mM ethylenediamine tetraacetic acid (ΕDTA). Eluted samples containing protein were pooled and dialyzed against 10 mM Tris-HCl pH 8.0, 5% glycerol, 1 mM dithiothreitol, and 2 mM EDTA. The purified recombinant Ac-MTP-1 did not exhibit enzymatic activity (data not shown).

The recombinant Ac-MTP-1 fusion protein was mixed with SBAS2 adjuvant and administered to each of five dogs in four intramuscular injections on days 1, 4, 43, and 50. Each dog received approximately 140 μg of recombinant fusion protein and 0.5 ml of ASO2A per dose. Five dogs were also injected intramuscularly with AS02A on the same schedule. Following immunization, blood was collected weekly by venipuncture and the serum was separated and stored frozen at -20°C. Antigen-specific canine IgG2 and IgE antibodies were measured by indirect enzyme-linked immunosorbent assay (ELISA) as described previously (Hotez et al, 2002a). Immunoblotting of secretory products from nonactivated L3 and L3 activated under host stimulatory conditions was done as described previously (Zhan et al, 2002) using pooled sera from the Ac-MTP-1 -vaccinated dogs. Fourteen days following the final immunization, each dog in the study was subcutaneosly infected with 500 A. caninum L3. The origin of the hookworm strain used for the study is described elsewhere (Hotez et al, 2002c). Validation of the hookworm species used in the study was confirmed by a polymerase chain reaction followed by restriction fragment length polymorphism (Hawdon, 1996). Following infection, the dogs were bled weekly by venipuncture to obtain a complete blood count (CBC). Serum chemistries were also obtained at the end of the vaccination schedule and prior to necropsy. Quantitative hookworm egg counts (McMaster technique) on each dog were obtained 3 days per wk beginning on day 12 post-infection (PI). Five wk post-infection, the dogs were killed by intravenous barbituate injection, and the adult hookworms were recovered and counted from the small and large intestines at necropsy (Hotez et al., 2002c). The statistical significance of differences between adult hookworm burdens was determined using the Anova test, as were differences in hematological parameters and in quantitative hookworm egg counts. Comparisons of hookworm burden and egg counts with antibody titers were measured using Spearman rank order (nonparametric) correlations.

SDS-PAGE analysis of the Ac-MTP-1 recombinant fusion proteins followed by Coomassie blue staining revealed that the protein migrates with an apparent MW of 61 kDa - the predicted mass of the proenzyme. Also present is a triplet of bands that migrate with a lower apparent molecular weight, which probably corresponds to the partially processed Ac-MTP-1. Following immunization, each of the vaccine-recipient dogs developed high titers of IgG2 anti-Ac-MTP-1 -specific antibody ranging between 1 :40,500 and 1 :364,500; the anti-Ac-MTP-1 -specific IgE antibody responses ranged between 1:500 and 1:1,500. Sera from the vaccinated dogs recognized a triplet of closely migrating proteins with the predicted molecular weight of the proenzyme and processed form of Ac-MTP-1 in secretory products of host-activated L3, but not in those of non-activated L3. The additional bands may also correspond to other related metalloproteases secreted by A. caninum L3; at least 3 closely related expressed sequence tags from A. caninum L3 were found in a dbEST database (ncbi.nim.nih.gov/dbEST/index.html).

Overall, there were no statistically significant differences in the number (mean + standard deviation) of adult hookworms recovered from the vaccinated dogs (154 + 34 hookworms) compared to the number of adult hookworms recovered from control dogs (143 + 30 hookworms). However, as shown in Fig. 33A there was a statistically significant reduction in the number of adult hookworms recovered from the intestines of vaccinated dogs that had high mύ-A.caninum IgG2 antibody titers. The Spearman correlation between antibody titers and adult hookworm burden was -0.89 (P = 0.04). The number of hookworms recovered from the dog with the highest antibody titer (98 hookworms) was equivalent to a 50 percent reduction in worm burden compared to the number of adult hookworms recovered from the dog with the lowest antibody titer (189 hookworms). An identical relationship was noted between IgG2 antibody titers and median quantitative egg counts (Fig. 33B).

These studies suggest that Ac-MTP-1 might offer downstream promise as an anti-hookworm vaccine antigen. EXAMPLE 4. Canine vaccine trials with Ac-TMP, Ac-AP, and Ac-APR-1 antigens

To evaluate whether antibodies directed against parasite enzymes and enzyme inhibitors have therapeutic potential for ancylostomiasis, canine vaccine trials employing recombinant fusion proteins that encode adult A. caninum proteases or protease inhibitors were conducted. Because small quantities of proteins are available from living hookworms, testing these molecules as vaccine candidates requires recombinant vector expression in prokaryotic or eukaryotic host systems, followed by canine immunization with the purified recombinant fusion protein. Material and Methods for Example 4.

Study dogs and animal husbandry: Following protocol approval by The George Washington University Institutional Animal Care and Use Committee (IACUC), purpose bred, parasite naϊve, male beagles 8 + 1 week of age were purchased, identified by ear tattoo, and maintained in the AALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited George Washington University Animal Research Facility. The dogs were housed in a room dedicated for the study, at a room temperature of 70 + 4°F, with 10-15 air changes per hour comprised of 100 percent fresh air, and 12 hr light cycles alternating with 12 hr dark cycles. The airflow and timer functions were monitored daily. The dogs were fed on a diet of Teklad Certified Dog Chow #8727, supplemented with a canned soft diet in the event of anorexia. The drinking water was piped from a filter plant and delivered via automate water system; water analysis was performed by the U.S. Army Corps of Engineers. Water from the facilities automatic system is cultured for bacteria and fungi annually. The pens were flushed daily and sanitized every two weeks. Dogs within a given study group were permitted to live together and socialize prior to the hookworm larval challenge, but were caged individually post-infection. All dogs were quarantined for approximately one week before beginning the vaccine trial. Prior to vaccination a complete blood count (CBC), serum chemistries, and a pre-vaccination serum sample were obtained.

Vaccine study design and sample size: The vaccine trial was designed to test three different antigens, each formulated with alum, as well as an alum adjuvant control. A total of 24 dogs were randomly assigned into four groups comprised of 6 dogs each. The canine sample size was selected on the ability to detect a 40-50 percent reduction in the numbers of adult hookworms in the small intestines of the vaccinated group relative to control dogs, at a statistical power of 80 percent (alpha = 0.05, two-tailed). The data were derived from the mean and standard deviation of adult hookworms previously recovered from age-matched dogs infected with 400 A. caninum L₃ (Hotez et al, 2002).

Recombinant Antigens: Each group of 6 dogs was vaccinated with recombinant hookworm proteins expressed as fusion proteins either in Escherichia coli or in an insect cell line with baculovirus. Ac-AP (Cappello et al, 1995; 1996) and Ac-TMP, were expressed in E.coli as pET 28 (Novagen) fusion proteins containing a polyhistidine tag (Cappello et al, 1996). Ac- APR-1 (Harrop et al, 1996) was expressed in a baculovirus pBacPAK6 vector (Clontech), modified to contain a polyhistidine-encoding sequence and additional restriction enzyme sites (Brindley et al, 2001). Recombinant Ac-AP and Ac-TMP fusion proteins were then purified by nickel affinity chromatography, followed by a second step of purification. In the case of Ac-AP (Cappello et al, 1995; 1996), the recombinant protein was purified by mono-S (Amersham-Pharmacia) ion exchange chromatography, while Ac-TMP (Zhan et al, 2002) was purified by superdex 75 (Amersham-Pharmacia) gel filtration chromatography. Ac-APR-1 (Harrop et al, 1996) was purified by substrate affinity chromatography using pepstatin agarose (Brindley et al, 2001). The antigen stock protein concentration was determined by Pierce Micro BCA assay (Pierce Chemicals) or by the absorbance of the sample at 289 nm using an extinction coefficient that was calculated from the deduced amino acid composition of the fusion protein. The amount of alum adsorbed protein in each dose of antigen was measured by the Pierce Micro BCA assay using a bovine serum albumin standard. The relative purity of each of the antigens relative to contaminating E. coli or insect cell proteins was determined by analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Adjuvant formulations : Recombinant Ac-TMP and Ac- APR fusion proteins were alum precipitated with a combination of aluminum potassium sulfate dodecahydrate and sodium bicarbonate as described previously (Ghosh et al, 1996). The method requires the precipitation of an aqueous solution of the protein with aluminum salt under alkaline conditions, followed by centrifugation and washing (Ghosh and Hotez, 1999). Using this method, recombinant Ac-AP fusion protein was not detected in the alum precipitate. Therefore, the first two doses of Ac-AP were administered without adjuvant. However, the final two doses of Ac-AP were adsorbed to an amorphous, non-crystalline calcium phosphate gel.

Canine Immunizations: A four-dose immunization schedule was selected (Table II). Each of the dogs was vaccinated by subcutaneous immunization at two sites in the shoulder, through a 22 gauge needle. The volume of the injections ranged between 0.5 and 1.0 ml. Four doses of each antigen were administered over a 38-day period. The first two injections (primary immunization) were administered on days 1 and 4, and the final two immunizations (boosts) were administered on days 34 and 38. Dogs in the control group were injected with an equivalent amount of alum.

Canine antibody measurements: Blood was collected weekly by venipuncture and the serum was separated and stored frozen at -20°C. Antigen-specific canine IgGl antibodies were measured by indirect enzyme-linked immunosorbent assay (ELISA). Other IgG subclasses were not measured due to the unavailability of suitable high-quality canine- specific reagents. The optimal concentrations of sample sera and enzyme-linked detection antibody were determined by checkerboard titrations. Optimal antigen concentrations were determined by using a saturation technique. NUNC Maxisorp F96 certified plates (Rosklide, Denmark; Batch no. 045638) were coated with 0.1 ml per well of antigen in 0.05M carbonate bicarbonate buffer (pH 9.6). Sealed plates were incubated overnight (ON) at 4°C and then washed 3 times with PBS (pH 7.2) using a DYNEX Opsys™ plate washer (Chantilly, VA). The plates were treated for 1.5 hours with 0.25 ml per well of 0.15M PBS (pH 7.2) containing 0.5% Tween 20 (PBS-Tween 20) at room temperature (RT), decanted, and blotted on paper towels. Various serial dilutions of test sera were prepared in 0.1 ml PBS-Tween 20 and incubated ON at 4°C. After washing, 0.1ml of anti-canine IgGl conjugated to alkaline phosphatase (Bethyl Laboratories, Montgomery, TX) at a dilution of 1:1000 were added to each well. After 1.5 hours at RT, the plates were washed 10 times with PBS-Tween 20, before 0.1 ml of 2.5 mM of para-nitro phenylphosphate (Sigma St. Louis, MO) in a solution of lOmM diethanolamine (Sigma, St. Louis, MO) and 0.5 mM magnesium chloride (Sigma, St.Louis, MO) (pH 9.5) were added to each well. The plates were incubated in the dark for 30 minutes and read at a wavelength of 405 nm on a SpectraMax 240 PC reader (Molecular Devices, Sunnyvale, CA) with SOFTmax Pro software (Molecular Devices, Sunnyvale, Ca). The mean optical density of control canine sera was used as a baseline. The last serum dilution greater than 3 times above baseline was considered the titration endpoint. The geometric mean of these endpoints was calculated for the six canines from each group. Canine hookworm infections and parasite recovery: Fourteen days following the final immunization, each dog in the study was orally infected with 400 A. caninum L₃ administered in a gelatin capsule. The origin of the hookworm strain used for the study is described elsewhere (Hotez et al, 2002). Validation of the hookworm species used in the study was confirmed by a polymerase chain reaction followed by restriction fragment length polymorphism (Hawdon, 1996). Following infection, the dogs were bled weekly by venipuncture in order to obtain a complete blood count (CBC). Serum chemistries were also obtained at the end of the vaccination schedule and prior to necropsy. Quantitative hookworm egg counts (McMaster technique) on each dog were obtained three days per week beginning on day 12 post-infection. Five weeks post-infection, the dogs were euthanized by intravenous barbituate injection, and the adult hookworms were recovered and counted from the small and large intestines at necropsy (Hotez et al, 2002). The sex of each of the adult hookworms was determined by visual inspection. The necropsies were performed over a period of three days when 8 dogs per day (two dogs from each of the four groups) were euthanized. Approximately 1-2 cm of small intestine was separated and placed into formalin for future histopathological analysis.

Statistical methods: The percentage reduction or increase in adult hookworm burden in the vaccinated group was expressed relative to the control group by the following formula:

(mean hookworms in control group - mean hookworms in vaccinated group) XI 00

(mean hookworms in control group)

The statistical significance of differences in adult hookworm burdens was determined using nonparametric tests; the Kruskal-Wallis with Dunn procedures, and Mann- Whitney U tests.

Differences between groups in hematological parameters and in quantitative hookworm egg counts were assessed by the ANOVA test. When more than two tests were computed on the same variable, the level of significance was adjusted for the number of tests. The sex differences of the adult hookworms recovered were statistically compared by the Wilcoxon-

Signed Ranks test for two dependent groups. Differences were considered statistically significant if the calculated P value was equal to or less than 0.10 (two sided) or - 0.05 (one sided).

Results for Example 4.

Adult A caninum antigens: Three recombinant A. caninum antigens were selected for canine vaccinations. Two of them, Ac-AP and Ac-TMP are protease inhibitors secreted only by adult stage hookworms. Ac-AP is a 91 amino acid factor Xa inhibitor anticoagulant (Cappello et al, 1995; 1996), and Ac-TMP is a 140 amino acid putative tissue inhibitor of metalloproteinase, and the most abundant protein secreted by A. caninum. The third antigen selected, was Ac-APR-1, a 422 amino acid aspartic acid cathepsin (Harrop et al, 1996). SDS- PAGE analysis of the recombinant fusion proteins followed by Coomassie blue staining was carried out. As expected, the recombinant fusion proteins Ac-APR-1 and Ac-TMP migrated on SDS-PAGE with apparent molecular weights of M_r = 45,000 and 18,000, respectively. The predicted molecular mass of Ac-AP expressed as a pET 28 fusion protein with an N- terminal poyhistidine tag is 12,191 Da (Cappello, 1996). On SDS-PAGE, the recombinant Ac-AP fusion protein was visualized as a band with a predominant Mr of 22,000 and a minor band that migrates at approximately 15,000 Da. This observation may correspond to polypeptide oligomer formation. This was shown previously to occur during purification of the Ac-AP natural product (Cappello et al, 1995). Factor Xa inhibitory activity, DNA sequence analysis of the pET 28 plasmid encoding the recombinant Ac-AP fusion protein, and amino terminal peptide sequence analysis by Edman degradation of the 22 kDa band confirmed the identity of the gene product (data not shown).

Canine antibody responses. A canine vaccination schedule was selected that provided for a primary immunization to be administered in two subcutaneous doses over an initial 4-day period (day 1 and day 4), followed by two subsequent subcutaneous immunization boosts that were administered beginning 30 days after the primary immunizations (day 34 and day 38). Ac-TMP and Ac-APR-1 were injected as alum-precipitated proteins. In contrast, Ac-AP did not form a precipitate with alum. Therefore, for the first two doses, Ac-AP was administered subcutaneously without adjuvant. However, during the 30-day time period between the second and third immunization, a protocol that employed calcium phosphate gel was shown to effectively precipitate Ac-AP (data not shown). For that reason, calcium phosphate was selected as the adjuvant for the final two immunizing doses of Ac-AP.

Geometric mean IgGl antibody titers to the three vaccine antigens are shown in Fig. 34A-C. Among the dogs vaccinated against Ac-APR-1 (Fig. 34A), there was a rise in antigen-specific IgGl following the final two immunization boosts at approximately 6 weeks after the primary immunization. In contrast, anti- Ac-TMP IgGl antibody responses were more robust (Fig. 34B), and began to increase 2 weeks following the primary immunization, prior to the third and fourth doses. Following the final two boosts there was a second increase in anti- Ac-TMP antibody titer that exceeded 1 : 10,000. Five of the six dogs vaccinated against Ac-AP failed to respond immunologically to the antigen. As shown in Fig. 34C, the single canine who responded to Ac-AP vaccination exhibited an antigen- specific antibody response following the final two doses.

Adult A. caninum hookworm recovery from the small intestine. The numbers of adults. caninum hookworms recovered from the small intestines of the vaccinated dogs is shown in Table III.. Hookworm burden reductions in the vaccinated dogs relative to dogs injected with alum alone ranged between 4.5 to 18 percent. The above reduction was not sufficient to show statistical significance between groups (Kruskal-Wallis test, P = 0.19). However, the probability (P) of 18 percent reduction in the number of hookworms recovered from the small intestines of the dogs vaccinated with Ac-APR-1 (the biggest reduction in one group) was less than 0.05 by the Dunn procedure, and 0.03 by Mann- Whitney U one sided test. Dogs vaccinated against Ac-TMP also exhibited a reduction in the adult hookworm burden (10.8 percent) although this was not statistically significant. The five dogs that did not exhibit an antibody response against Ac-AP, also exhibited no significant hookworm burden reduction. However, the single dog with a significant anti- Ac-AP antibody response, exhibited a 34.7 percent reduction in adult hookworm burden. As shown in Table III, data did not provide sufficient evidence for statistically significant reductions ^"in quantitative hookworm egg counts between the vaccinated and control dogs. Similarly, vaccination did not affect the hematological parameters of the dogs, including hematocrit, hemoglobin, white blood cell count, and eosinophilia (data not shown). As expected, the challenge dose of hookworm used in this study did not produce anemia in the control alum-injected dogs (data not shown). Adult A. caninum hookworm recovery from the colon. TABLE III. Reduction of adult hookworms in the small intestines of vaccinated relative to alum-injected dogs.

* Positive immune response ** P < 0.05 (Dunn procedure)

Whereas there was a reduction in the numbers of adult hookworms recovered from the small intestines of vaccinated dogs, there was a corresponding increase in the number of adult hookworms that were recovered from the colon (Table IV).

TABLE IV. Increase of adult A caninum hookworms in the colons of vaccinated dogas relative to alum-injected dogs.

* Positive immune response ** P < 0.05 (Dunn procedure)

The increase in the number of adult hookworms recovered from the large intestines was statistically significant (Kruskal-Wallis test, P = 0.07). The dogs vaccinated with either Ac- TMP (500 percent increase) or Ac-APR-1 (300 percent increase), exhibited a statistically significant increase relative to the dogs injected with alum (Dunn procedure, P<0.05). Dogs that were vaccinated with Ac-AP but did not exhibit an antigen-specific antibody response did not have a statistically significant increase in the number of adult hookworms recovered from the colon. However, the single dog with a significant anti- Ac-AP antibody response exhibited a 1083 percentage increase in the number of adult hookworms in its colon.

Overall, there were no statistically significant differences between the vaccinated and control dogs with respect to the total numbers of adult hookworms recovered from small and large intestines combined (data not shown). Instead, antibody responses to the recombinant hookworm antigens resulted in significant migration of adult hookworms away from the small intestine and into the colon. The ratio of adult hookworms in the small intestine relative to the colon decreased from 43.9 in the alum-injected dogs down to ratios between 6.6 and 7.3 in the Ac-TMP and Ac-APR-1 vaccinated dogs, respectively. The single dog exhibiting an anti- Ac-AP antibody response had a small intestine to colon hookworm burden ratio of 1.6, indicating that almost one-half of this dog's hookworm burden had shifted to the colon. Sex-dependent differences. Hookworms of either sex did not migrate away from the small intestine and into the colon in equal numbers. As shown in Fig.35, it was more common to recover female adult hookworms from the colon than males. The greater numbers of female hookworms residing in the colon was statistically significant for dogs vaccinated with Ac- APR-1 (P = 0.04) and Ac-AP (P =0.06). Male hookworms were more likely than female hookworms to be recovered from the small intestines, although the differences were not statistically significant. Sex determinations were not made on the hookworms attached to a 1-2 cm segment of small intestine that was saved for histopathological analysis. The mean number of hookworms in this segment ranged between 5 and 6 worms. This small number of worms did not contribute significantly to the sex-dependent difference score (data not shown).

This example demonstrates that it is feasible to vaccinate mammals with recombinant fusion proteins to elicit an antigen specific response, and that the antibody response is associate either with a hookworm burden reduction in the gut or in a shift in hookworm habitat in the gut.

EXAMPLE 5. Canine Vaccine Trials of Ac-MTP-1 and Ac-TTR Example 5A. Antibody titers and hookworm reduction.

E. coli derived antigens Ac-MTP-1 and Ac-TTR were tested in vaccine trials in dogs. Antigens were administered with adjuvant SBAS2. The vaccinated animals exhibited high levels of canine IgG2 antigen-specific antibodies, and a modest increase in antigen-specific IgE. Subsequently the dogs were challenged by subcutaneous injection of L3 hookworm larvae.

As shown in Fig. 36A and B, there was a statistically significant reduction in the number of adult hookworms recovered from the intestines of vaccinated dogs that had high a ti-A. caninum IgG2 anti-MTP-1 antibody titers. The Spearman correlation between antibody titers and adult hookworm burden was -0.89 (P = 0.04). The number of hookworms recovered from the dog with the highest antibody titer (98 hookworms) was equivalent to a 50 percent reduction in worm burden compared to the number of adult hookworms recovered from the dog with the lowest antibody titer (189 hookworms). An identical relationship was noted between IgG2 antibody titers and median quantitative egg counts.

SDS-PAGE analysis of the Ac-MTP-1 recombinant fusion proteins followed by Coomassie blue staining revealed that the protein migrates with an apparent MW of 61 kDa - the predicted mass of the proenzyme. Also present is a triplet of bands that migrate with a lower apparent molecular weight, which probably corresponds to the partially processed Ac-MTP-1. Following immunization, each of the vaccine-recipient dogs developed high titers of IgG2 anti-Ac-MTP-1 -specific antibody ranging between 1:40,500 and 1:364,500; the anti-Ac-MTP-1 -specific IgE antibody responses ranged between 1 :500 and 1:1,500. Sera from the vaccinated dogs recognized a triplet of closely migrating proteins with the predicted molecular weight of the proenzyme and processed form of Ac-MTP-1 in secretory products of host-activated L3, but not in those of non-activated L3.

With respect to the use of the TTR antigen, as can be seen in Figures 37A and B, one dog with high IgE and IgGl antibody to TTR exhibited reduced (6%ι) hookworm burden.

This example demonstrates that vaccination of mammals with either MTP or with TTR elicit a protective antibody response, and that with high antibody titers a reduction in worm burden is observed.

Example 5B. Protection against blood loss and decrease in hookworm size due to vaccination with hookworm antigen

Animals were also tested to ascertain whether vaccination with hookworm antigens protected against blood loss. Vaccination with Ac-TTR was shown to confer significant protection against blood loss (Fig. 38A and B).Using the Mann- Whitney test, the differences in both hemoglobin (38B) concentration (P = 0.036) and hematicrit (38A) concentration (P=0.009) between the TTR and adjuvant control groups were statistically significant.

Further, the differences in hemoglobin concentration translated to a statistically significant reduction in worm size. Data was collected using an imaging system based on scans of the worms recovered from a host. Worms were photographed with a CoolSnapPro digital camera (Media Cybernetics), and the images measured in ImagePro Software using a macro to determine worm length (in mm) compared between treatments. As shown in Fig. 39 there was a statistically significant reduction in worm size (between 1 and 2 mm) among the TTR vaccinated group relative to the adjuvant control group.

This example demonstrates that vaccination with TTR, in addition to reducing worm burden, will also reduce blood loss.

EXAMPLE 6. Chimeric hookworm antigens

The protective effect of two different hepatitis B core particles expressing a peptide epitope that corresponds to amino acids 291-303 of Na-ASP-1 (also found in Ac-ASP-1) were investigated. Previously by investigation of relative hydropathy (hydrophobicity and hydrophilicity) of the predicted amino acid sequence of Na-ASP-1 and Ac-ASP-1 it was discovered that both molecules exhibit a hydrophilic sequence that modeling predicted could represent a looped-out region of the molecule. Covalent attachment of the peptide to KLH (keyhole limpet hemocyanin) confirmed that the chimeric molecule could protect mice against challenge infections.

Two different chimeric molecules expressing ASP-1 were constructed. ICC- 1546 expresses ASP-1 amino acids 291-303 as a "looped out" tethered structure, whereas ICC- 1564 expresses the same peptide as an N-terminal structure. Previous studies had demonstrated that mouse anti-L3 antibody recognizes ICC-1546, but not ICC-1564.

The antigenic chimeras were administered as described above with alhydrogel as adjuvant. DSM (detergent solubilized membrane extract of adults, caninum) served as a negative control. Larval challenge was carried out by subcutaneous injection of L3 stage larvae.

The results showed that vaccination of dogs with either particle produced high levels of anti-particle antibody. Most of the antibody was directed against the hepatitis core antigen constituent. However, in one dog vaccinated with ICC-1546, there was a high level of anti-ASP-1 (and anti-peptide) antibody. This dog exhibited a significant reduction in hookworm burden (Table V).

This example demonstrates that high antibody titers to a specific epitope associated with ASP-1 will result in reduced worm burden.

EXAMPLE 7. Antigen expression in baculovirus/insect cells and yeast

Expression of hookworm antigens in eukaryotic expression systems, such as baculovirus/insect cells and the yeast Pichia pastoris, have been carried out to afford maximum opportunities for obtaining soluble and bioactive recombinant proteins.

A. Expression in Pichia pastoris

The antigens Na-ASP-1, Ac-TTR, Ac- API, and Ay-ASP-2 have been successfully expressed with Pichia fermentation systems. Antigens were isolated with polyhistidine tags for ease of isolation.

B. Expression in Baculovirus/insect Cell System

Antigens Na-CTL, Ac-MEP-1, Ac-ASP-2 and Ac-MTP-1 have been successfully expressed in a baculovirus/insect cell expression system. Antigens were isolated with polyhistidine tags for ease of isolation. EXAMPLE 8. Cloning of cDNAs of A. ceylancium Orthologous Antigens Ay-ASP-1,

Ay-ASP-2 and Ay-MTP

Orthologous antigens from the hamster parasite hookworm A. ceylancium were successfully cloned following the construction of an A. ceylancium larval cDNA library.

The ceylanicum orthologue of ASP-1 was cloned by screening the A. ceylanicum L3 cDNA library using a 900bp ³²P- labeled Ac- ASP 1 cDNA fragment as a probe. Screening of approximately 500,000 clones resulted in 85 positive clones. Of these 21 clones were sequenced of which 19 encoded identical cDNAs. No other orthologues of ASP-1 were found. The clones exhibited 85% identity and 92%) similarity with Na-ASP-1.

By screening approximately 100,000 plaques of the ceylanicum L3 cDNA library using a 600bp ³²P- labeled Ac-asp-2 cDNA fragment as a probe, 30 positive clones were obtained, of which 10 were sequenced and found to be identical to Ay-ASP-2 predicted ORFs (orthologous clones).

By screening approximately 500,000 A. ceylanicum L3 cDNA library using a 590bp ³²P- labeled Ac-MTP cDNA fragment as a probe, 700 positive clones were obtained and 8 sequenced. Seven of the 8 encoded Ay-MTP-1, while one other encoded a putative isoform (Ay-MTP-2).

This example demonstrates that there is a high degree of similarity between antigens from A. caninum and A. ceylancium hookworm species, and the data suggests a high degree of identity (>80%>) amongst most of hookworm antigens.

EXAMPLE 9. Immunolocalization

Immuno localization of some of the major vaccine antigens was carried out in sections of adult hookworms. The immunolocalizations were determined to be as follows: Ac-103 as a hookworm surface antigen, Ac-FAR-1 and Ac- API as components of the pseudocoelomic fluid, (Ac- API is also a pharyngeal protein), Ac-CP-1 as an amphidial gland protein, Ac-TMP in the excretory glands, and ASP-3 as an amphidial and esophageal protein. In addition the total proteins of the hookworm ES products localized to amphidial and excretory glands, and to the brush border membrane of the hookworm alimentary canal.

This example demonstrates that many of the hookworm antigens are exposed either on the surface of the worm or secreted by worm and are therefore susceptible to targeting by host antibodies or host immunocompetent cells.

Example 10. Human investigations conducted in Minas Gerais State, Brazil

It has been previously reported that in China and elsewhere, human hookworm infection exhibits a unique epidemiology compared with the other soil-transmitted helminthiases (e.g., ascariasis and trichuriasis) and schistosomiasis (Gandhi et al, 2001). Whereas the prevalence and intensity of these other helminth infections peak during childhood and adolescence and subsequently decline into adulthood, the prevalence and intensity of hookworm infection increases with age. In many Chinese and Brazilian villages (and presumably elsewhere) middle aged and even elderly residents exhibit the most severe infections.

The underlying immunological mechanisms accounting for this observation has been investigated. Shown in Figs. 40 and 41, CD-4 + lymphocytes were gated from the whole blood of hookworm infected residents and stimulated with either L3 soluble hookworm antigen Fig. 40) or Pichia-expressed recombinant Na-ASP-1 (Fig. 41). Host cytokine production was measured by an intracellular cytokine staining technique. Both antigens stimulated high levels of IL-10 and IL-5, but not IL-4. IL-10 is a strong immunomodulator with downregulatory, anti-inflammatory properties, and IL-4 is associated with antibody production and TH-2 type immunity. The findings suggest that hookworm infected individuals might be anergic to hookworm and possibly other antigen stimulation.

In contrast, it was shown that individuals treated for hookworm produce IL-4. This observation indicates that removal of hookworms from the intestine helps to reconstitute a patient's immunity. This is a critical observation since it suggests that in the absence of treatment a recombinant hookworm vaccine may be unlikely to function as a therapeutic vaccine in patients who are actively infected, and that anthelminthic chemotherapeutic treatment may be necessary prior to vaccination.

Further, these observations also suggest that hookworm infection might thwart otherwise successful vaccinations against such etiological agents as HIV and malaria. In regions of Subsaharan Africa where hookworm overlaps with HIV and malaria, it may become essential to monitor a study participant's hookworm status prior to HIV or malaria vaccination, and to treat those that are found to be actively infected prior to immunization.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

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Claims

CLAIMSWe claim:

1. A composition comprising: a recombinant or synthetic antigen or a fragment thereof derived from hookworm, and, a pharmacologically acceptable carrier.

2. The composition of claim 1 wherein said recombinant or synthetic antigen displays at least about 80%) identity to an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac- API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac- AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

3. The composition of claim 2 wherein said antigen is Ac-TMP.

4. The composition of claim 2 wherein said antigen is Ac-MEP-1.

5. The composition of claim 2 wherein said antigen is Ac-MTP-1.

6. The composition of claim 1 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

7. A method of eliciting an immune response to hookworm in a mammal, comprising the step of, administering to said mammal an effective amount of a composition comprising a recombinant or synthetic antigen or a fragment thereof derived from hookworm, and a pharmacologically acceptable carrier.

8. The method of claim 7 wherein said recombinant or synthetic antigen displays at least about 80%) identity to an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac- API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac- AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

9. The method of claim 8 wherein said antigen is Ac-TMP.

10. The method of claim 8 wherein said antigen is Ac-MEP-1.

11. The method of claim 8 wherein said antigen is Ac-MTP-1.

12. The method of claim 7 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

13. A method of vaccinating a mammal against hookworm, comprising the step of, administering to said mammal an effective amount of a composition comprising a recombinant or synthetic antigen or fragment thereof derived from hookworm, and a pharmacologically acceptable carrier.

14. The method of claim 13 wherein said recombinant or synthetic antigen displays at least about 80% identity with an antigen selected from the group consisting of Na-ASP-1, Na- ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac- ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac- CTL, Ac- API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac- APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

15. The method of claim 14 wherein said antigen is Ac-TMP.

16. The method of claim 14 wherein said antigen is Ac-MEP-1.

17. The method of claim 14 wherein said antigen is Ac-MTP-1.

18. The method of claim 13 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

19. A composition comprising: a recombinant or synthetic antigen or fragment thereof derived from hookworm, wherein said recombinant or synthetic antigen displays at least about 80%> identity with an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA- APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac- ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR, and, a pharmacologically acceptable carrier.

20. The method of claim 19 wherein said antigen is Ac-TMP.

21. The method of claim 19 wherein said antigen is Ac-MEP-1.

22. The method of claim 19 wherein said antigen is Ac-MTP-1.

23. The method of claim 19 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

24. A vaccine comprising: a recombinant or synthetic antigen or fragment thereof derived from hookworm, wherein said recombinant or synthetic antigen displays at least about 80%) identity with an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA- APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac- ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR, and, a pharmacologically acceptable carrier.

25. The method of claim 24 wherein said antigen is Ac-TMP.

26. The method of claim 24 wherein said antigen is Ac-MEP-1.

27. The method of claim 24 wherein said antigen is Ac-MTP-1.

28. The method of claim 24 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

29. A composition for eliciting an immune response, comprising: a recombinant or synthetic antigen or fragment thereof derived from hookworm, wherein said recombinant or synthetic antigen displays at least about 80% identity with an antigen selected from the group consisting of Na-ASP-1, Na-ACE, Na-CTL, Na-APR-1, NA- APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac-ASP-2, Ac-ASP-3, Ac-ASP-4, Ac- ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac-CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac-APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay- API, and Ay-TTR, and, a pharmacologically acceptable carrier.

30. The method of claim 29 wherein said antigenic protein, polypeptide, or fragment thereof is Ac-TMP.

31. The method of claim 29 wherein said antigenic protein, polypeptide, or fragment thereof is Ac-MEP-1.

32. The method of claim 29 wherein said antigenic protein, polypeptide, or fragment thereof is Ac-MTP-1.

33. The method of claim 29 wherein a species of said hookworm is selected from the group consisting of Necator americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

34. A method for enabling vaccination of a patient against infectious diseases, comprising the steps of: a) treating hookworm infection to a degree sufficient to increase lymphocyte proliferation; and b) vaccinating said patient against said infectious disease.

35. The method of claim 34 wherein said infectious disease is selected from the group consisting of HIV, tuberculosis, malaria, measles, tetanus, diphtheria, pertussis, and polio.

36. A method for enabling hookworm vaccination, comprising the steps of: a) chemically treating a hookworm infected patient to ameliorate hookworm infection; and b) vaccinating said patient with a recombinant or synthetic antigen or fragment thereof derived from hookworm after amelioration of hookworm infection.

37. The method of claim 36 wherein said recombinant or synthetic antigen displays at least about 80% identity with an antigen is selected from the group consisting of Na-ASP-1, Na- ACE, Na-CTL, Na-APR-1, NA-APR-2, Ac-TMP, Ac-MEP-1, Ac-MTP-1, Ac-ASP-1, Ac- ASP-2, Ac-ASP-3, Ac-ASP-4, Ac-ASP-5, Ac-ASP-6, Ac-TTR-1, Ac-103, Ac-VWF, Ac- CTL, Ac-API, Ac-MTP-1, Ac-MTP-2, Ac-MTP-3, Ac-FAR-1, Ac-KPI-1, Ac-APR-1, Ac- APR-2, Ac-AP, Ay-ASP-1, Ay-ASP-2, Ay-MTP-1, Ay-API, and Ay-TTR.

38. A method of reducing blood loss in a patient infected with hookworm, comprising the step of administering to said patient a composition comprising a recombinant or synthetic antigen or fragment thereof derived from hookworm, and, a pharmacologically acceptable carrier.

39. A method of reducing hookworm size in a patient infected with hookworm, comprising the step of administering to said patient a composition comprising a recombinant or synthetic antigen or fragment thereof derived from hookworm, and, a pharmacologically acceptable carrier.

40. A method of reducing hookworm burden in a patient infected with hookworm, comprising the step of administering to said patient a composition comprising a recombinant or synthetic antigen or fragment thereof derived from hookworm, and, a pharmacologically acceptable carrier.

41. SEQ ID NO. i l.

42. SEQ ID NO. 12.

43. SEQ ID NO. 13.

44. SEQ ID NO. 14 .

45. SEQ ID NO. 15.

46. SEQ ID NO. 16.

47. SEQ ID NO. 5.

48. SEQ ID NO. 6.

49. SEQ ID NO. 7.

50. SEQ ID NO. 8.

51. SEQ ID NO. 9.

52. SEQ ID NO. 10.

53. SEQ ID NO. i l

54. SEQ ID NO. 12.

55. SEQ ID NO. 21.

56. SEQ ID NO. 22.

57. SEQ ID NO. 23.

58. SEQ ID NO. 24.

59. SEQ ID NO. 25.

60. SEQ ID NO. 26.

61. SEQ ID NO. 27.

62. SEQ ID NO. 28.

63. SEQ ID NO. 29.

64. SEQ ID NO. 30.

65. SEQ ID NO. 31.

66. SEQ ID NO. 32.

67. SEQ ID NO. 33.

68. SEQ ID NO. 34.

69. SEQ ID NO. 35.

70. SEQ ID NO. 36.

71. SEQ ID NO. 37.

72. SEQ ID NO. 38.

73. SEQ ID NO. 39.

74. SEQ ID NO. 40.

75. SEQ ID NO. 41.

76. SEQ ID NO. 42.

77. SEQ ID NO. 43.

78. SEQ ID NO. 44.

79. SEQ ID NO. 47.

80. SEQ ID NO. 48.

81. SEQ ID NO. 49.

82. SEQ ID NO. 50.

83. SEQ ID NO. 51.

84. SEQ ID NO. 52.

85. SEQ ID NO. 55.

86. SEQ ID NO. 56.

87. SEQ ID NO. 57.

88. SEQ ID NO. 58.

89. SEQ ID NO. 59.

90. SEQ ID NO. 60.

91. SEQ ID NO. 61.

92. SEQ ID NO. 62.

93. SEQ ID NO. 63.

94. SEQ ID NO. 64.

95. An Ac-APR-2 antigen.

96. An Ay-TTR antigen derived from a nematode.

97. The Ay-TTR antigen of claim 96, wherein said nematode is a hookworm.