CN1571677A - Hookworm vaccine - Google Patents

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

Background of the invention

field of invention

The present invention generally relates to vaccines for hookworms. In particular, the invention provides vaccines based on antigens of parasite origin.

Background of the invention

Hookworm infection is an important public health problem in developing countries worldwide, causing enteritis, intestinal blood loss, anemia, stunting 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 certain areas of China, such as Hainan Province in the South China Sea, more than 60% of the population carries hookworms (Gandhi et al., 2001).

Most of the pathological symptoms caused by hookworms arise from mature stages of the parasite in the human gut. Adsorption of mature Ancylostoma hookworms to the mucosa and submucosa of the vertebrate small intestine is one of the most well-defined examples of host-parasite relationships in all of parasitology. Several cubic millimeters of host mucosal and submucosa tissue are included in the parasite's oral sheath, making it possible to actually touch the host-parasite association during autopsy or necropsy (Kalkofen, 1970; Kalkofen, 1974).

Ancylostoma caninum is a major cause of morbidity and mortality in dogs throughout the world, including subtropical regions of North America. Hookworm-associated ischemia, causing severe anemia and even death, can occur between 2 and 3 weeks after a single initial infection in dogs (Soulsby, 1982; Jones and Hotez, 2002). Notably, Ancylostoma canis (A. canium) has recently been identified as an important human pathogen. Zoonotic infection with mature H. canis parasites can cause eosinophilic enteritis syndrome, an inflammatory state of the gut in response to parasitic attack (Prociv and Croese, 1990). The pathogenesis of Ancylostoma canium infection is associated with intestinal ischemia, which can occur at the stage of adsorption and food uptake by the mature parasite in the small intestine of mammals (Kalkofen, 1970; Kalkofen, 1974 ).

Current treatment and control efforts for hookworm infestations focus on the periodic removal of mature hookworms from patients with anthelmintics. This approach has several limitations, including rapid superinfection after treatment, requiring multiple clinic visits, and eventual development of anthelmintic-resistant strains of hookworm after years of heavy anthelmintic treatment (Savioli et al., 1997; Geerts and Gryseels, 2000). Therefore, it would be highly beneficial if additional methods of treatment and prevention of hookworm infection in mammals were available. For example, it would be very beneficial to have a useful vaccine to treat or prevent hookworm infection.

Summary of the invention

The present invention provides formulations that elicit an immune response against hookworms. The formulation contains various hookworm antigens that have been shown to be useful in eliciting an immune response. These formulations can be used as vaccines against hookworms in mammals, such as humans. Following administration of the formulation, the vaccinated mammal may develop an immune response against hookworms, develop immunity to parasitic infection, or may exhibit reduced worm load, remission of ischemia, or reduction in the size of parasitic hookworms. To this end, the present invention provides a composition comprising recombinant or synthetic antigens or fragments thereof from hookworms and a pharmaceutically acceptable carrier. Recombinant or synthetic antigens may exhibit at least 80% identity to antigens 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 a preferred embodiment, the antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1. Antigens may be derived from hookworm species such as Nector americanus, Ancylostoma canium, Ancylostoma ceylancium, and Ancylostoma duodenale.

The invention also provides methods of eliciting an immune response to hookworms 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 fragment of the antigen) derived from hookworm and a pharmaceutically acceptable carrier. Recombinant or synthetic antigens exhibit at least about 80% identity to antigens 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. Antigens may be derived from hookworms of species such as Ancylostomum americanum, Ancylostoma canis, Ancylostoma ceylonensis, and Ancylostoma duodenale.

The invention further provides a method of immunizing a mammal against hookworms. The method includes the step of administering to the mammal an effective amount of a composition comprising a recombinant or synthetic antigen (or fragment of the antigen) derived from hookworm and a pharmaceutically acceptable carrier. Recombinant or synthetic antigens exhibit at least about 80% identity to antigens 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. Antigens may be derived from hookworms of species such as Ancylostomum americanum, Ancylostoma canis, Ancylostoma ceylonensis, and Ancylostoma duodenale.

The present invention further provides a composition comprising recombinant or synthetic antigens (or fragments of antigens) derived from hookworms. Recombinant or synthetic antigens exhibit at least about 80% identity to antigens 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 composition further includes a pharmaceutically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1. Antigens may be derived from hookworms of species such as Ancylostomum americanum, Ancylostoma canis, Ancylostoma ceylonensis, and Ancylostoma duodenale.

The present invention further provides a vaccine comprising recombinant or synthetic antigens (or fragments of antigens) derived from hookworms. Recombinant or synthetic antigens exhibit at least about 80% identity to antigens 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 includes a pharmaceutically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1. Antigens may be derived from hookworms of species such as Ancylostomum americanum, Ancylostoma canis, Ancylostoma ceylonensis, and Ancylostoma duodenale.

The present invention further provides a composition for eliciting an immune response, said composition comprising a recombinant or synthetic antigen (or a fragment of an antigen) derived from hookworm. Recombinant or synthetic antigens exhibit at least about 80% identity to antigens 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 includes a pharmaceutically acceptable carrier. In preferred embodiments, the antigen is Ac-TMP, Ac-MEP-1 or Ac-MTP-1. Antigens may be derived from hookworms of species such as Ancylostomum americanum, Ancylostoma canis, Ancylostoma ceylonensis, and Ancylostoma duodenale.

The invention further provides methods enabling the vaccination of a patient against an infectious disease. The method comprises the steps of: treating hookworm infection to a level sufficient to increase lymphocyte proliferation and immunizing the patient against an infectious disease such as HIV, tuberculosis, malaria, measles, tetanus, diphtheria, whooping cough or polio .

The invention also provides methods enabling vaccination against hookworms. The method comprises the steps of: chemotherapy treating a hookworm-infected patient to alleviate the hookworm infection, and inoculating the patient with a recombinant or synthetic antigen (or an antigenic fragment thereof) derived from hookworm after the hookworm infection is alleviated. In such methods, hookworm infection may be completely eradicated by treatment or may be reduced to such an extent that hookworm immunization is effective. Recombinant or synthetic antigens may exhibit at least about 80% identity to antigens 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 invention also provides a method of reducing ischemia in a patient infected with hookworm. The method comprises the step of administering to a patient a composition comprising a recombinant or synthetic antigen (or fragment thereof) derived from hookworm and a pharmaceutically acceptable carrier.

The present invention also provides methods of reducing the size of hookworms in a patient infected with hookworms. The method comprises the step of administering to a patient a composition comprising a recombinant or synthetic antigen (or fragment thereof) derived from hookworm and a pharmaceutically acceptable carrier.

The present invention further provides a method of reducing the number of hookworms in a patient infected with hookworms. The method comprises the step of administering to a patient a composition comprising a recombinant or synthetic antigen (or fragment thereof) derived from hookworm and a pharmaceutically 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. 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. 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. ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64.

Description of 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 number 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 number 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 number 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 number 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 number AF132291.

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 number 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).

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).

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).

21A and B. Ac-MTP-2: A, cDNA (SEQ ID NO. 41) and B, amino acid sequence (SEQ ID NO. 42).

22A and B. Ac-MTP-3: A, cDNA (SEQ ID NO. 43) and B, amino acid sequence (SEQ ID NO. 44).

23A and B. Ac-FAR-1: A, cDNA (SEQ ID NO. 45) and B, amino acid sequence (SEQ ID NO. 46). GeneBank accession number AF529181.

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.

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).

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).

Figures 33A and B. Spearman's rank correlation between hookworm numbers and anti-MTP-1 antibody titers. A) total number of insects; B) median EPG.

34A-C. Antigen-specific geometric mean IgGl antibody titers as a function of time in dogs immunized with L. canis recombinant fusion protein. The geometric mean was calculated for 6 dogs per group, except for the Ac-AP group, in which only one dog developed an antigen-specific antibody response. Arrows show timed immunizations. (A) Anti-Ac-APR-1 responses (n=6). (B) Anti-Ac-TMP responses (n=6). (C) Anti-Anti-Ac-AP responses (n=1).

Figure 35. Male and female mature Ancylostoma canis larvae recovered from the colon of immunized or alum-injected dogs.

Figures 36A and B. Peelman's rank correlation between hookworm numbers and anti-MTP-1 antibody titers.

Figure 37A and B. A) Association between anti-TTR IgE antibodies and reduction in hookworm population; B) association between anti-TTR IgG1 antibody and reduction in hookworm population.

Figures 38A and B. Changes in HV-4 canine hemoglobin (B) and hematocrit (A) after L3 challenge.

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

Figure 40. CD4+ lymphocytes from hookworm-infected (egg positive) individuals following stimulation with Ancylostoma L3 antigen.

Figure 41. CD4+ lymphocytes from hookworm infected (egg positive) individuals after stimulation with Pichia expressing recombinant Na-ASP-1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides compositions for eliciting an immune response in a mammal against hookworms. This composition can be used as a vaccine for the treatment and/or prevention of hookworm infection. The vaccine comprises a purified antigen preparation and a pharmaceutically acceptable carrier, and the antigen is derived from hookworm. The term "derived from" means that the antigen is derived from (ie, isolated from) a biomolecule of hookworms. For example, an antigen may be a protein, polypeptide or antigenic fragment of a protein or polypeptide that forms part of a hookworm organism. Typically, such antigens are isolated and at least partially purified from hookworms, isolation and purification methods being well known to those skilled in the art (see eg the Examples section below). When manufactured for eliciting an immune response or as a vaccine, such antigens may be "synthetic", i.e. obtained synthetically (for example, in the case of polypeptides and protein fragments by peptide synthesis), or "recombinant", That is, obtained by genetic engineering techniques (for example, by production in a host cell containing a vector having DNA encoding an antigen). Those skilled in the art will recognize that a variety of such suitable expression systems can be employed, including but not limited to those using E. coli, yeast (e.g., Pichia pastoris), baculovirus/insect cells, plant cells, and mammalian cells. expression system. In a preferred embodiment of the invention, the antigen is expressed in a yeast or baculovirus/insect cell expression system.

Examples of specific antigens, their amino acid primary sequences, and nucleic acid sequences encoding them are given here. For ease of reference, Table 1 lists some exemplary antigens and their corresponding SEQ ID NOS. However, those skilled in the art will recognize that various variants of the sequences recited herein may exist or be constructed which may also serve as antigens in the practice of the present invention. For example, depending on the amino acid sequence, variants may exist or be constructed which exhibit: conservative amino acid substitutions; non-conservative amino acid substitutions; Terminal or carboxy-terminal or intramolecular addition of amino acids (e.g., addition of a histidine tag to facilitate protein separation), substitution of residues to alter solubility properties, substitution of residues including protease cleavage sites to eliminate cleavage sites and to improve stability, add or remove glycosylation sites, etc. or for any other reason). Such variants may be naturally occurring (eg, as a result of natural variation between species or between individuals); or may be purposefully introduced (eg, in experimental devices using genetic engineering techniques). All variants of the sequences disclosed herein are intended to be included in the teaching of the present invention if the antigenic variants show sufficient identity to the described sequences. Preferably, the identity to the disclosed sequence is in the range of about 50 to 100%, more preferably in the range of about 75 to 100%, and most preferably in the range of 80 to 100%. Identity is by reference to that portion of the amino acid sequence that corresponds to the sequence of the original antigen, ie excluding other elements that may have been added, such as the chimeric antigens described below.

Table I. Hookworm antigens, description, and corresponding SEQ ID NOS. source antigen describe SEQ ID NOs. cDNA open reading frame Lastostomum americana Na-ASP-1 secreted protein SEQ ID NO.1 SEQ ID NO.2 Na-ACE Cholinesterase SEQ ID NO.3 SEQ ID NO.4 Na-CTL C-lectin SEQ ID NO.5 SEQ ID NO.6 Na-APR-1 aspartic protease SEQ ID NO.7 SEQ ID NO.8 Na-APR-2 aspartic protease SEQ ID NO.9 SEQ ID NO.10 Ancylostoma caninum Ac-TMP met protease inhibitor SEQ ID NO.11 SEQ ID NO.12 Ac-MEP-1 metalloendopeptidase SEQ ID NO.13 SEQ ID NO.14 Ac-MTP-1 Astaxanthin SEQ ID NO.15 SEQ ID NO.16 Ac-ASP-1 secreted protein SEQ ID NO.17 SEQ ID NO.18 Ac-ASP-2 secreted protein SEQ ID NO.19 SEQ ID NO.20 Ac-ASP-3 secreted protein SEQ ID NO.21 SEQ ID NO.22 Ac-ASP-4 secreted protein SEQ ID NO.23 SEQ ID NO.24 Ac-ASP-5 secreted protein SEQ ID NO.25 SEQ ID NO.26 Ac-ASP-6 secreted protein SEQ ID NO.27 SEQ ID NO.28 Ac-TTR-1 transerythrin SEQ ID NO.29 SEQ ID NO.30 Ac-103 surface protein SEQ ID NO.31 SEQ ID NO.32 Ac-VWF surface lectins SEQ ID NO.33 SEQ ID NO.34 Ac-CTL C-lectin SEQ ID NO.35 SEQ ID NO.36 Ac-API Aspartyl protease inhibitors SEQ ID NO.37 SEQ ID NO.38 Ac-MTP-1 Astaxanthin SEQ ID NO.39 SEQ ID NO.40 Ac-MTP-2 Astaxanthin SEQ ID NO.41 SEQ ID NO.42 Ac-MTP-3 Astaxanthin SEQ ID NO.43 SEQ ID NO.44 Ac-FAR-1 conjugated retinol SEQ ID NO.45 SEQ ID NO.46 Ac-KPI-1 Protease inhibitor SEQ ID NO.47 SEQ ID NO.48 Ac-APR-1 aspartic protease SEQ ID NO.49 SEQ ID NO.50 Ac-APR-2 Pepsinogen SEQ ID NO.51 SEQ ID NO.52 Ac-AP anticoagulant SEQ ID NO.53 SEQ ID NO.54 Ancylostoma ceylonis Ay-ASP-1 secreted protein SEQ ID NO.55 SEQ ID NO.56 Ay-ASP-2 secreted protein SEQ ID NO.57 SEQ ID NO.58 Ay-MTP-1 Astaxanthin SEQ ID NO.59 SEQ ID NO.60 Ay-API-1 Aspartyl protease inhibitors SEQ ID NO.61 SEQ ID NO.62 Ay-TTR class-transthyretin SEQ ID NO.63 SEQ ID NO.64

The invention also includes chimeric antigens, eg, antigens consisting of the amino acid sequences described herein plus other sequences which, when isolated, are not necessarily linked to the disclosed sequences, but which confer some other advantage with the addition of these amino acids. For example, these advantages can be used to isolate and purify proteins (e.g., histidine tag, GST, maltose-binding protein); direct proteins to specific intracellular locations (e.g., yeast secreted proteins); improve protein antigenicity (e.g., KHL, hapten). All such chimeric constructs are intended to be included within the scope of the invention so long as the presence of partial constructs based on the sequences disclosed herein at least demonstrates a level of homology.

Those skilled in the art will recognize that it is not necessary to use the entire primary sequence of a protein and or polypeptide in order to elicit an adequate antigenic response to the parasite from which the antigen originates. In some cases, fragments of the protein are sufficient to confer immunity. Accordingly, the invention also includes antigenic fragments of the sequences disclosed herein, and their use in vaccine formulations. Typically, such fragments are at least about 10-13 amino acids in length. Those skilled in the art will recognize that suitable sequences are generally hydrophilic and often surface accessible.

Likewise, based on the nucleic acid sequences disclosed herein, those skilled in the art will recognize that various variants of the sequences can exist or be constructed which still provide the encoded antigen or a desired portion thereof. For example, due to the redundancy of the genetic code, more than one codon may be used to encode an amino acid. Furthermore, as noted above, it may be desirable to make changes to the primary sequence of the antigen, and this is necessary for changes in the coding nucleic acid sequence. In addition, those skilled in the art will recognize that various variants of nucleic acid sequences can be constructed for purposes involving cloning strategies (e.g., insertion of sequences into vectors for ease of manipulation, such as introduction of restriction enzyme cleavage sites, etc.), for modification The purpose of transcription (eg, introducing a promoter or enhancer sequence, etc.), or for any other suitable purpose. All such variants of the nucleic acid sequences disclosed herein are intended to be included within the scope of the present invention provided that the sequences exhibit approximately 50 to 100% identity, and preferably, approximately 75 to 100% identity, to the original sequence, And most preferably, about 80 to 100% identity. The identity refers to the partial nucleic acid sequence corresponding to the original sequence, and should not include other elements, such as promoters, vector-derived sequences, restriction sites derived from other sources, etc.

The antigens of the present invention may be from any species of hookworm, examples include but not limited to Ancylostomum americanum, Ancylostoma caninum, Ancylostoma ceylonis 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 a secreted protein associated with activation, examples of these proteins 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 (eg, Ac-APR-1 and Ac-APR-2); and serine proteases.

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

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

In a preferred embodiment, the antigen used in implementing the present invention is Ac-TMP, its DNA coding sequence is listed in Figure 6A (SEQ ID NO.11), and its amino acid sequence is listed in Figure 6B (SEQ ID NO. 12).

In another preferred embodiment, the antigen used in implementing the present invention is Ac-MEP-1, its DNA coding sequence is listed in Figure 7A (SEQ ID NO.13), and its amino acid sequence is listed in Figure 7B ( In SEQ ID NO.14).

In another preferred embodiment, the antigen used in implementing the present invention is Ac-MTP-1, its DNA coding sequence is listed in Figure 8A (SEQ ID NO.15), and its amino acid sequence is listed in Figure 8B ( In 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.23-24); 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); 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 eliciting an immune response that can be used as vaccines against hookworms. The term "eliciting an immune response" means that an antigen stimulates the synthesis of specific antibodies in titers of about >1 to about 1 x ¹⁰⁶ or greater. Preferably, the titer is from about 10,000 to about 1 x ¹⁰⁶ or greater, and most preferably, the titer is greater than 1 x ¹⁰⁶ , as determined, for example, by incorporation ^of3H thymidine. The term "vaccine" refers to an antigen that elicits an immune response that results in at least about a 30% reduction in the number of hookworms in an organism compared to a control organism that is not vaccinated (eg, adjuvanted only). Preferably, the reduction level is about 50%, more preferably about 60 to about 70% or higher.

The present invention provides compositions for eliciting an immune response that can be used as a vaccine against hookworms. The composition comprises a substantially purified hookworm antigen as described herein, or a variant thereof, and a pharmaceutically acceptable carrier. The preparation of such compositions for use as vaccines is well known to those skilled in the art. In general, such compositions will be prepared 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, liquid prior to administration may also be prepared. The preparation can also be in emulsified form. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., 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. Additionally, the compositions may contain other adjuvants. If it is desired to administer the composition in oral form, various thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders, etc. may be added. The compositions of the present invention may contain any such additional ingredients to provide the composition in a form suitable for administration. The final amount of hookworm antigen in the formulation can vary. Typically, however, the amount in the formulation will be from about 1% to about 99%.

The vaccine formulation of the present invention may further include an adjuvant, suitable examples include but not limited to Seppic, Quil A, Alhydrogel and the like.

The formulations of the invention may contain a single hookworm antigen. Alternatively, more than one hookworm antigen may be used in the formulation, ie, the formulation may contain a "cocktail" of antigens.

The invention also provides methods of eliciting an immune response against hookworms and methods of immunizing mammals against hookworms. The term eliciting an immune response means that the administration of the antigen elicits specific antibodies (titers ranging from 1 to 1×10 ⁶ , preferably 1×10 ³ , more preferably 1×10 ³ to about 1×10 ⁶ , and most preferably greater than 1×10 ⁶ ) Synthesis and/or cell proliferation, eg measured by incorporation of ³ H thymidine. The method comprises administering to the mammal a composition comprising a hookworm antigen in a pharmaceutically acceptable carrier. The vaccine formulations of the present invention can be administered in any number of suitable ways known to those skilled in the art, including but not limited to injection, oral, intranasal, by ingestion of foods containing antigens, and the like. In preferred embodiments, the administration is subcutaneous or intramuscular.

The present invention provides methods of eliciting an immune response against hookworms and methods of immunizing mammals against hookworms. In one embodiment, the mammal is a human. However, those skilled in the art will recognize that there are other mammals for which it is also beneficial to be immunized against hookworms, ie the preparations are also used for veterinary purposes. Such examples include, but are not limited to, companion "pets," such as dogs, cats, etc.; food sources, working and recreational animals, such as livestock, horses, cows, sheep, pigs, goats, and the like.

Those skilled in the art will recognize that generally for immunization (or eliciting an immune response) of a target species (eg, human) against hookworms, the antigens used should be derived from the hookworm species that parasitize the target species. For example, generally antigens derived from E. americanum are preferred for immunization of humans, and antigens derived from H. canis are preferred for immunization of dogs. But this is not always the case. For example, Ancylostoma canis is known to parasitize humans and their original canine hosts. Furthermore, cross-species hookworm antigens may sometimes be highly effective at eliciting an immune response in a non-host animal, ie a host of parasites that are not normally the source of the antigen. Furthermore, determination of the suitability of an antigen for use in immunostimulatory or vaccine formulations relies on its ability to confer protection against parasite challenge and parasitism, as indicated by, for example, a reduction in hookworm numbers or inhibition of hookworm-associated ischemia (e.g., by hematocrit Volume and/or concentration of hemoglobin are measured) as shown. For example, for use in vaccine formulations, the antigen upon administration results in a reduction in hookworm populations of at least about 30%, preferably at least about 50%, and most preferably at least about 60% to about 70%.

In one embodiment of the invention there is provided a method capable of immunizing a patient against an infectious disease. The method comprises treating a hookworm infection to a level sufficient to increase lymphocyte proliferation and then vaccinating the patient against the infection. The method is based on the facts presented in Example 10 showing that hookworm infestation causes cells to be immunoreactive to challenge with hookworm and possibly other antigens. Thus, by chemically treating hookworm-infected patients prior to immunization against hookworms or any infectious agent, the response to immunization can be enhanced. Examples of infectious diseases for which the outcome of immunization against said infectious diseases is improved include, but are not limited to, HIV, tuberculosis, malaria, and routine childhood immunizations (eg, measles, tetanus, diphtheria, pertussis, polio, etc.).

Examples of hookworm agents by which chemical treatment may be performed include, but are not limited to, albendazole and other anthelmintics.

The specific antigens described here are also useful in the treatment of other neoplastic, autoimmune and cardiovascular diseases, as well as in the treatment of proinflammatory states. This use of other hookworm antigens has been described, for example, in US Patent 5,427,937 to Capello et al. and US 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.21, SEQ ID NO. 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. 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. ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64. The sequences represent the cDNA sequence and its encoded amino acid sequence (open reading frame). Although the sequences themselves have been claimed, other sequences having a high level of identity to those described are contemplated, for example at least 65% to 100% identity to the given sequence, or preferably about 75% % to 100% identity, or most preferably at least about 80% to 100% sequence identity.

In particular, Ac-APR-2 (SEQ ID NOS.51 and 52) and Ay-TTR (SEQ ID NOS.63 and 64) are partial sequences representing most of the antigenic sequences. Thus, the present invention includes intact Ac-APR-2 antigen and intact Ay-TTR antigen.

In addition, those skilled in the art will recognize that the Ay-TTR antigens provided in this application represent antigens of the Ay-TTR family present in many species of nematodes. Likewise, Ay-TTR antigens from any nematode should be included within the scope of the present invention. In particular, any Ay-TTR from hookworms of the species including, but not limited to, Ancylostoma americana, Ancylostoma canis, Ancylostoma ceylonis, and Ancylostoma duodenale is also included within the scope of the present invention .

Example

Molecular cloning and purification of embodiment 1.Ac-TMP

Materials and methods

Immunoscreening of a library of mature Leptostomum canis to generate antibodies against secreted products of Leptostomum canis. One hundred live mature-stage Ancylostomacaninum hookworms were recovered from the intestines of infected dogs at necropsy (6 weeks post-infection) as described above (Hotez and Cerami, 1983). Mature hookworms were rinsed 3 times in sterile PBS and then stored in 15 ml RPM 1640 containing 25 mM HEPES, 100 units/ml ampicillin and 100 μg/ml streptomycin for 24 hours at 37°C (5% CO ₂ ) . The supernatant was collected, concentrated with PEG6000, and dialyzed against 1 L of phosphate buffer (pH 7.2) at 4° C. overnight. After dialysis, the precipitated product was centrifuged at 10,000×g for 10 minutes, and the supernatant was recovered.

Rabbits were immunized by subcutaneous injection of hookworm-secreted protein (400 μg) emulsified in complete Freund's adjuvant. Subsequently, the rabbits were immunized with the hookworm-secreted protein emulsified with the same amount of Freund's incomplete adjuvant at intervals of 2 weeks for a total of three immunizations. Final blood was obtained 10 days after the final immunization, and serum was isolated from whole blood and stored at -20°C.

The construction of a cDNA expression ZapII (Stratagene, La Jolla CA) library was previously reported (Capello et al., 1996). An estimated 5 x ¹⁰⁵ plaques were screened with rabbit anti-H. canis mature secretion product antibody according to the manufacturer's instructions. Briefly, 5 x ¹⁰⁴ plaques were plated on LB agar plates. Expression of H. canis antigens was induced by covering plaques with nitrocellulose membrane infiltrated with 10 mM IPTG. After 4 hours of incubation at 37°C, the membrane was lifted, blocked with 5% skimmed milk in PBS, and incubated with rabbit antibody (diluted 1:500) for 1 hour at 24°C. The membrane was washed three times with PBS containing 0.1% Tween-20 (PBS-Tween) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) at a dilution ratio of 1:1000 for 1 hour at 24°C . The membrane was washed again three times with PBS-Tween, and then developed with 3,3'-diaminobenzidine (DAB) substrate and hydrogen peroxide. Potentially positive clones were scored and isolated for secondary screening.

Immunopositive clones were excised into pBluscript phage according to the manufacturer's instructions (Stratagene), phagemid DNA was extracted by alkaline lysis (Qiagen), and double-strand sequencing was performed using flanking vector primers (T ₃ and T ₇ ). Nucleotide and deduced amino acid sequences were compared to existing sequences in GenBank by BLAST searches. Sequence analysis was performed using ESEE 3.1 software.

Reverse transcription polymerase chain reaction (RT-PCR) amplification

RT-PCR was used to identify the developmental stage specificity of Ac-tmp mRNA transcription. A. Caninum eggs, L1 and L2 larval stages, and L3 infectious larval stage were obtained as described above (Hawdon et al., 1999). Total RNA was isolated from each life history stage using TRIzol reagent (GIBCO BRL). Single-stranded cDNA was synthesized using oligo d(T) primer and MMLV-RT (GIBCOBRL). Specific primers (TIMP3'-1HR and TIMP5'-2ER) based on the Ac-tmp sequence from 60bp to 440bp were used to amplify Ac-tmp cDNA. The PCR reaction parameters were: denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes. A total of 30 cycles are performed.

Optimization of semi-preparative reversed-phase chromatographic conditions for purification of Ac-TMP natural product for fractionation of mature secretory products of Ancylostoma canis is carried out on a 510 HPLC system (Waters) equipped with a semi-preparative flow cell A 490E multi-wavelength detector with wavelengths set at 214, 280, 260 and 254 mm, and a 250 mm×4.6 IDYMC-Pack Protein-RP, 200 Å, 5 μm C ₄ column (Waters). After 15 hours, 1260 mature hookworms were collected from 1260 mature hookworms as the starting material. Mature H. canisinum secretion products were added to 37 °C containing 25 mM HEPES, 100 units/ml ampicillin, 100 μg/ml streptomycin and 100 μg/ml streptomycin. Gentamicin in 15ml RPMI 1640. The supernatant was concentrated to 0.3 volumes by ultrafiltration in a Centricon-3 microconcentrator (Amicon) before centrifugation at 7,500 xg for 1 hour. Approximately 0.6 mg of the parasite's secreted protein was subjected to chromatographic analysis. Eluent A was 0.01% trifluoroacetic acid (TFA) in water and eluent B was 0.01% TFA in acetonitrile. Elution was performed with a linear gradient from 0 to 80% B over 40-min at a flow rate of 1 ml/min. Fractions of 0.5 min were collected and lyophilized before further purification and analysis by SDS-PAGE (Laemmli, 1970). For SDS-PAGE, 2 μl of the secreted product as well as 10 μl of the HPLC fraction no. 51 were mixed with the same volume of 2×SDS-PAGE sample buffer (4% SDS, 2.5% 2-mercaptoethanol, 15% glycerol) and boiled for 5 minute. The samples were electrophoresed on a 4-20% gradient SDS-PAGE gel at 100V for 2 hours. Gels were silver stained according to the manufacturer's instructions (BIO-RAD).

Fraction 51 was subjected to RP-HPLC on a 510 HPLC system equipped with a 250 mm. x 3.0 I.D. YMC protein RP, 200A, 5 μm C4 column as described above, said fraction containing most of the canines isolated from the semi-prep Nematode secreted protein. Eluent A was 0.01% TFA in water and B was 0.01% TFA in acetonitrile. A linear gradient from 0 to 60% B was eluted at a flow rate of 1 ml/min for 30 min. Fractions of 0.5 min were collected and lyophilized. Fractions of the major protein peak collected from this separation were subjected to amino acid sequence analysis and SDS-PAGE (Laemmli, 1970). Amino acid sequence analysis based on protein Edman degradation was performed on an Accurate Model 494 Protein Sequencer (Applied Biosystems) equipped with a 785A Programmable Detector and 140C Pump System provided by ProSeq, Inc. (Boxford MA). Sequencing products were identified using standard Accurate 610A software.

To confirm the N-terminal sequence corresponding to Ac-TMP, degenerate oligonucleotide primers were synthesized in both directions corresponding to the partial N-terminal peptide sequence of fraction No. 51. Pairs of flanking degenerate vector primers were used to amplify products from DNA obtained from mature cDNA libraries constructed in ZapII. "Hot start" PCR conditions were 10 mM Tris-HCl (pH 8.5) containing 50 mM KCl, 2.0 mM _MgCl2 , 0.2 mM of each dNTP, and 1 μl of cDNA library for a total of 20 μl reactions. The reaction was heated at 94°C for 5 minutes and then lowered to 85°C for 5 minutes before adding 1 unit of Taq DNA polymerase (GIBCO BRL). Thirty cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes were followed. PCR products were electrophoresed on agarose gels and stained with ethidium bromide. PCR products were gel purified with QIAEX II Gel Extraction Kit (Qiagen, Valencia, CA) and sequenced.

The result of embodiment 1

Ac-TMP cDNA . Ac-TMP cDNA was cloned from a mature hookworm cDNA library by immunoscreening with rabbit antibodies directed against all Ancylostoma canium mature secretion products. Two positive identical clones were isolated. The full-length cDNA is 559 bp (SEQ ID NO. 11), which encodes an open reading frame (ORF) of 140 amino acids (SEQ ID NO. 12) and a polyA tail at the 3' end. The predicted ORF has a calculated molecular weight of 16,100 Daltons and a theoretical pi of 7.55. The hydrophobic signal peptide sequence has a signal peptidase cleavage site between amino acids 16 and 17. Immediately after the signal peptide, Ac-TMP has a signature N-terminal Cys-X-Cys sequence. A putative N-linked glycosylation site (NXT) exists between amino acids 119 and 122 (Figure 6B).

A GenBank database search revealed that the predicted amino acid sequence of this molecule shares 33% identity and 50% similarity with the N-terminal domain of human tissue inhibitor of metalloproteinase 2 (TIMP-2). Ac-TMPs and putative TIMPs from the spontaneous nematode Caenorhabditiselegans contain a single domain and lack a second C-terminal domain characteristic of vertebrate TIMPs (data not shown).

RT-PCR amplification. To identify Ac-TMP expressed specifically for life cycle stages, mRNAs were extracted from Ancylostoma canium at different developmental stages and reverse transcribed into cDNA using Ac-TMP-specific primers. RT-PCR yielded a specific band of 380 bp amplified only from the mature cDNA. No amplification results were observed for cDNA from egg, Li-L2 and L3 life cycle stages. Amplification of A. caninum genomic DNA revealed two bands, indicating a possible intron or the presence of a second related Ac-TMP gene (data not shown).

Identification of Ac-TMP in secreted products of adult Ancylostoma canis. To confirm that Ac-TMP is released by mature Ancylostoma canis, the protein was identified in and secreted from parasites by RP-HPLC Purified. Each major peak was subjected to amino acid sequence analysis as part of a larger H. canis proteomics study (data not shown). The protein peak corresponding to "fraction 51" was selected for further investigation and the chromatographic analysis was repeated. Fraction 51 after silver staining included a prominent band with a migration apparent molecular weight Mr=16,000. The N-terminal peptide sequence (20 amino acids) of this fraction perfectly matched the ORF sequence 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 total secretion product profile, Ac-TMP was determined to comprise approximately 6.3% of the total A. caninum secretion products. This identified the molecule as the most abundant protein released by mature Ancylostoma canis (A. caninum). The abundance of Ac-TMP in hookworm secretions was confirmed by visual inspection on SDS-PAGE. Pairs of degenerate primers based on the sequence of the first seven amino acids were used to construct PCR products from the mature hookworm cDNA library. The DNA sequence of the PCR product confirmed identity to the Ac-TMP cDNA (data not shown).

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

Example 2. Molecular cloning and identification of Ac-mep-1.

Materials and methods

Parasites The H. canis parasite lives in beagle dogs as described above (Schad 1982). Infected larvae of the third stage (L3) were isolated from charcoal dung cultures and maintained in BU buffer (Hawdon et al., 1995). Mature A. caninum hookworms were collected from infected dogs after necropsy. The hookworms were washed three times in PBS, snap frozen in liquid nitrogen, and stored at -80°C.

Nucleic Acids Genomic DNA was isolated from mature H. caninum by conventional methods (Ausubel et al., 1993). H. caninum RNA was isolated by grinding previously frozen (-80° C.) mature hookworms in the presence of Trizol reagent (Gibco BRL) according to the manufacturer's protocol. cDNA was prepared from RNA by the ProSTAR First Strand RTPCR Kit (Stratagene) according to the manufacturer's instructions.

Genomic and cDNA libraries of Ancylostoma caninum (A. caninum) genomic DNA library was constructed according to the following operation: In the recommended buffer solution with a volume of 100 μl, 30 μg of genomic DNA of Ancylostoma caninum (A. caninum) was composed of 8 units The Sau3A restriction enzyme (NEB) was partially degraded (37°C for 5 minutes). The degraded DNA was then subjected to ethanol precipitation and the precipitate collected by standard methods. The resulting precipitate was dried, dissolved in water, and ligated into λ-FIXII vector (Stratagene) according to the manufacturer's instructions. The ligation reactions were then packaged and amplified with Gigapack Gold packaging extract (Stratagene). A cDNA library of mature H. canis was constructed previously (Capello et al., 1996) in the lambda ZAPII (Stratagene) vector.

Metalloprotease Cloning The cloning of the Ac-mep-1 cDNA started with PCR on the mature hookworm library cDNA using degenerate primers and oligo-dT. Degenerate primers were designed against conserved sequences containing the zinc-binding motif observed in a BLAST alignment of two putative zinc metalloprotease genes from C. elegans ( GenBank ^™ , accession numbers T22668 and Q22523). The reaction conditions were as follows: 85 ng of template DNA, 1X thermophilic DNA buffer (Promega), 2.5 mM _MgCl2 , 0.2 mM dNTPs, 2 μM of each primer, 1 U of taq DNA polymerase (Promega), a total volume of 20 μl . The reaction was performed for 35 cycles as follows: 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. This PCR yielded a fragment which when cloned (pGEM-T, Promega) and sequenced represented 458 bp (21 residues including the polyA tail) of the 3'Ac-mep-1 cDNA (clone MP-1). Using MP-1 as the basis for specific primer design, additional Ac-mep-1 (clone MP-2) sequences were identified on library DNA by PCR with T3 (vector) and MEP-R1 gene-specific primers. Reactions are run on serially diluted library DNA until unique products are amplified and cloned. The reaction conditions are as described above.

In similar clones, MP-3 was amplified with T3 and MEP-R2 primers. The 5' end of Ac-mep-1 was identified using the 5'-RACE kit from GibcoBRL. Briefly, the first cDNA strand was generated in a reverse transcription reaction on freshly prepared RNA using the Ac-mep-1 specific primer RACE-R1. This cDNA was then polycytosine-tailed at the 3' end using terminal deoxytransferase and used as template in a PCR reaction containing anchor primer AAP (GibcoBRL) and gene-specific reverse primer MEP-R2. The resulting product was diluted and used as template for a semi-nested PCR reaction containing anchor primer UAP (GibcoBRL) and gene-specific primer MEP-R3. The resulting PCR product was cloned and named MP-4.

Additional 5' sequences were identified from an Ac-mep-1-like sequenced genomic DNA clone (G-MEP). Multiple clones were sequenced to confirm that the Ac-mep-1 cDNA and the full-length coding region of Ac-mep-1 were PCR amplified under the conditions described above as separate fragments using appropriate primers (clone FL-1).

Sequence analysis The DNASTAR MEGALIGN software (version 3.7.1) was used to align some Ac-mep-1 clones. BLAST analysis of the original sequence and predicted Ac-mep-1 open reading frame (ORF) for degenerate primer design was performed using the BLAST application of the National Center for Biotechnology Information. Sequence analysis of Ac-mep-1 was performed using the Curatools sequence analysis application (CuragenCorp., New Haven, CT). The FGENESH finder utility (CGG web server (genomic.sanger.ac.uk)) with settings for analyzing C. elegans DNA was used for gene prediction from genomic DNA clone G-MEP. Identification of probable exon sequences in GMEP was done with the Wise2 sequence analysis application (sanger.ac.uk/Software Wise2/).

Northern Blot Northern blot analysis was performed on total RNA isolated from Trizol (GibcoBRL) of ten mature hookworms. RNA was fractionated on a 1.2% formaldehyde gel and blotted on Hybond-N membranes (Amersham) by standard methods. The blot was probed with ³² P randomly initially labeled DNA fragments representing base pairs 780 to 2688 of the Ac-mep-1 cDNA.

RT-PCR of developmental stages RT-PCR was used to study the transcription of Ac-mep-1 in various life cycle stages of H. canis. For these reactions, cDNA from eggs, L1, inactive and activated L3, and mature hookworms were tested with the Acmep-1 specific primers MEP-F1 and MEP-R1. These cDNAs will be specific for A. caninum protein kinase A if the quality of these cDNAs is verified in an isolation reaction using primers PKA-F and PKA-R (Hawdon et al., 1995) . These reaction conditions correspond to those defined in Section 2.4.

Anti-Ac-mep-1 antibody representing a cDNA fragment of 610 amino acids from the C-terminal portion of Ac-MEP-1 was amplified by PCR from a cDNA lambda library of mature L. caninum using appropriate primers. This fragment was T/A cloned into pGEM (Promega) and cloned into the Hindlll site of the pET28c expression vector (Novagen) by standard methods (Sambrook and Russell, 2001). The bacterial protein of truncated Ac-MEP-1 (tAc-MEP-1) was induced by adding 1 mM of IPTG to cultures of BL21(DE3)PlysS (Stratagene) cells transformed with the tAc-MEP-1/pET28c construct Express.

The expressed protein is insoluble. To purify tAc-mep-1, the induced cell pellet was frozen (after freezing, BL21(DE3)PlysS cells were lysed), resuspended in one-tenth volume of 50 mM tris pH 8.0, 2 μM EDTA, and sonicated until free viscous, then centrifuge at 12,000xg for 15 minutes (Sorvall RC5B, GSA rotor). The resulting pellet was resuspended in 15 ml of 1% SDS, 0.5% B-mercaptoethanol, sonicated, boiled for 5 minutes, and incubated at room temperature for 2 hours. Insoluble debris was removed by repeated centrifugation. The supernatant was dialyzed extensively against phosphate buffered saline (pH 7.4) to remove BME. Proteins were purified on HisBind (Novagen) nickel resin affinity columns without denaturing agents according to the manufacturer's protocol. Groups of 5 male Balb/c mice (6-week old) were immunized intraperitoneally with 20 μg of alum-precipitated tAc-MEP-1 or alum alone as a control. Mice were subsequently boosted twice at 2-week intervals. One week after the third and final immunization, sera were collected, pooled and used as primary antibodies for western blot and immunostaining analyses.

Western blotting. Proteins separated by 10% SDS-PAGE were transferred to methanol-charged Immobilon-P PVDF in transfer buffer (39 mM glycine, 48 mM tris base, 0.037% SDS, pH 8.3) at 30 V for 18 hours. membrane (Millipore). Primary mouse anti-tAc-MEP-1 antibody (1:1500) was used to block the membrane with 5% skimmed milk in PBS (blocking buffer) for 1 hour at room temperature (RT) with gentle shaking and absorbed by E. coli After incubation for 1 hour at room temperature, the mouse anti-tAc-MEP-1 antibody was diluted in blocking buffer. The membrane was then washed three times (10 min each) in blocking buffer and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5000) in blocking buffer at RT with shaking 1 hour. Finally, the membrane was rinsed in PBS for 15 minutes and developed with Renaissance (NEN Life Science product) chemiluminescent reagent.

Immunolocalization of mature Ancylostoma canis worms by paraffin embedding and sectioning by standard methods. Ac-MEP-1 in situ was accomplished by incubating deparaffinized hookworm sections in mouse anti-tAc-MEP at a 1:100 dilution (in PBS, pH 7.4) or control serum (supra) for 1 hr at RT. Immunolocalization. Sections were washed three times in PBS and incubated in a 1 :200 dilution of goat anti-mouse IgG for 1 hour at 25°C, followed by washing in PBS (three times). Sections were then observed and photographed with an Olympus IX-50 inverted fluorescence microscope (U-MWIG filter).

The result of embodiment 2

The cDNA structure of Ac-mep-1 The cloning strategy used to obtain the complete Ac-mep-1 coding sequence was as follows: degenerate PCR clone MP-1 by sequencing, PCR-derived clones MP-2, MP-3 and 5′ RACE Clone MP-4 identified an Ac-mep-1 transcript of approximately 2.6 kb. Although there is a methionine codon near the 5' end of the RACE product, this codon is preceded by 58 in-frame amino acids excluding the terminator, showing that MP-4 does not represent the actual Ac-mep-1 5' end. Furthermore, we were unable to obtain cDNA clones (by PCR) including the splice leader sequence. Therefore, the examination of the genomic DNA clone G-MEP of Ac-mep-1 similar sequence (98.7% exon identity) used the gene prediction program of C. Potential transcription initiation site. This prediction extends 158 bp beyond the 5' RACE sequence and adds 91 amino acids to the deduced coding region. Using this prediction, the entire coding region of Ac-mep-1 was amplified as a single product of 2.7 kb and the clone was verified by partial sequencing of both ends. The total length of the Ac-mep-1 transcript was confirmed to be approximately 2.8 kb by Northern blotting (noncoding regions at the 5' and 3' ends were not amplified in the full-length PCR). The deduced amino acid sequence of this transcript encodes a unique ORF of 870 amino acids with four possible N-linked glycosylation sites (predicted pi = 5.5, mw = 98.7 kDa). The amino-terminal amino acid of Ac-MEP-1 contains a hydrophobic signal peptide sequence with a predicted restriction enzyme cleavage site after residue 22 (see figure with AC-MEP-1 sequence). Two signature zinc-binding motifs characterizing the endopeptidase 24.11 family of metalloproteases were identified.

Ac-mep-1 shares 66% similarity and 48% identity with a metalloprotease (Hc-MEP 2b) from the related Trichostrongylus blood-fed nematode Haemonchus contortus (H. Contortus). It is also similar to the metalloprotease (T25B6.2) from the non-parasitic nematode C. elegans (Gen-Bank ^™ T28906). Fourteen cysteine residues are highly conserved among these three molecules. The other two cysteines (only one is conserved) are present in Ac-MEP-1 and Hc-MEP1b.

Northern blot and chromogenic analysis of Ac-mep-1 expression Northern blot analysis revealed a single mRNA transcript of approximately 2.8 kb in mature hookworm mRNA (not shown). The developmental specificity of Ac-mep-1 transcription was investigated by RT-PCR. In the tested cDNA it was possible to identify transcripts of only the mature stage of the parasite and not hookworm eggs, L1 or activated and non-activated L3 larvae. In contrast, positive control PCR performed on the same cDNA with primers specific for Ancylostoma canium protein kinase A showed amplification from all template cDNAs. Thus, Ac-mep-1 appears to be expressed exclusively in mature hookworms.

Western Blot Analysis and Immunolocalization of Ac-mep-1 in Mature Hookworm Sections Mouse anti-MEP-1 antiserum significantly recognized mature Ancylostoma canium proteins of approximately 90 and 100 kDa by Western blotting. Immunohistochemical analysis of mature hookworm sections localizes Ac-MEP-1 to the surface of the microvilli of the hookworm gut. Antisera reacted strongly with visceral microvilli of mature hookworm sections compared to sections incubated with control serum. Faint staining in the skin of mature hookworms was also occasionally noted. Although the function of Ac-MEP-1 is unknown, the location along the microvilli surface of the parasite's gut would suggest that the enzyme is in direct contact with dried blood and may thus have a role in nutrient digestion.

This example shows that MEP-1 is an important enzyme that enables hookworms to digest blood and is therefore an attractive vaccine target. Recombinant MEP-1 protein has been cloned and expressed.

Example 3. AC-MTP Antigen Research

Ancystoma larvae (L3) in the third infectious stage release a zinc-dependent metalloprotease that migrates with an apparent molecular weight of 50 kDa (Hawdon et al. 1995a). The enzyme is specifically released in response to L3 stage-induced feeding and developmental stimuli (Hawdon et al., 1995b) and may play a role in parasite skin and tissue invasion or molt (Hotez et al., 1990). Because of its role in parasite-derived tissue invasion and desquamation, antienzyme antibody responses directed against Ac-MTP-1 may block larval migration and parasite entry into the gut. Ac-MTP-1 is stage-specific and is released by hookworm L3 activated in a host-like environment to resume feeding in vitro. The release of Ac-MTP-I during activation makes this molecule an attractive vaccine target.

Example 3A. Isolation of cDNA from an A. caninum L3 expression library encoding a zinc-metalloprotease of the astaxanthin family (Ac-mtp-1).

Materials and methods

Antiserum : Sera for immunoscreening of the A. caninum L3 expression library were collected from 5 residents of Nanlin County, Anhui Province, China, following an IRB-approved human study protocol. Ancylostoma duodenale is the predominant hookworm in this area, with a ratio of greater than 20:1 for N. duodenale to E. americana based on the recovery of larvae and mature hookworms from infected patients (Yong et al. 1999). Sera were obtained from Anhui residents with high titers of circulating antibodies against all cleaved antigens of H. canisum L3 as described elsewhere (Xue et al., 2000). Two residents were negative for hookworm eggs, while the remaining 3 individuals had a quantitative fecal worm egg count of less than 400 eggs per gram of feces. Because of their higher antibody titers and lower infection intensity, these individuals were considered as presumed resistant and their sera were pooled for immune screening. Negative control serum was collected from college students in Shanghai.

Screening of the expression library : A cDNA library (Stratagene, La Jolla, CA) (Stratagene, La Jolla, CA) (Hawdon et al. 1995). Briefly, 5 × ¹⁰ plaques expressing protein were induced by immersing nitrocellulose membranes in 10 mM IPTG for 4 h at 37 °C. After incubation, the membrane was incubated in 5% non-fat dry milk in PBS for 1 hour. The blocked membrane was incubated in PBS with a 1:100 dilution of pooled human serum for 1 hour at 22°C, washed three times with PBS for 10 minutes at 22°C, and incubated with a 1:1000 dilution of horseradish peroxidase Conjugated anti-human IgG (Sigma, St. Louis MO) was co-cultured. The membrane was developed with the substrate 3,3'-diaminobenzidine (DAB) and 0.015% hydrogen peroxide. Positive plaques were subjected to multiple rounds of plaque purification by re-plating and re-screening. The plasmid was rescued by in vivo excision (Short and Sorge, 1992), and both strands were sequenced using primers complementary to the flanking vector sequences. Nucleotide and deduced amino acid sequences were compared to existing sequences in the GeneBank database by BLAST searches (Altschul et al., 1997).

Cloning of full-length Ac-MTP cDNA : All isolated positive clones were truncated at 5'. To obtain the 5' end, PCR using the gene-specific primer PI and a primer corresponding to the conserved nematode splice leader sequence was used to amplify the 5' end from the first cDNA strand of A. caninum L3. 20 μl of a reaction containing 100 ng of each primer, 1 U of Taq polymerase (Promega, Madison WI) and 1 μl of cDNA was denatured at 95° C. for 2 minutes, followed by 30 cycles of: 1 minute at 94° C., 1 minute at 55° C. 1 min, and 2 min at 72 °C. Amplicons were gel purified and cloned into pGEM Easy-T vector (Promega, Madison, WI) by conventional methods.

Stage specificity : Stage specificity of mtp-1 transcription was determined by RT-PCR (Hawdon et al., 1995). Ancylostoma canis eggs were isolated from feces of infected dogs by sucrose flotation (Nolan et al., 1994), axenized with NaOCl, and plated on nematode growth medium agar plates ( Sulston et al., 1988). Following incubation at 26°C for 24-30 hours, the incubation was washed from the plate (mixed L ₁ /L ₂ ) with BU buffer (Hawdon and Schad, 1991) and snap frozen in a dry ice/ethanol bath. Unhatched eggs are also snap frozen for cDNA production. Mature hookworms of A. caninum were collected from the small intestine of infected dogs during necropsy. RT-PCR on H. caninum eggs, pooled L ₁ /L ₂ serum-stimulated and unstimulated L ₃ (described below), and mature H. canis samples were performed as follows. Samples were ground to a powder in a pre-chilled (liquid _N2 ) mortar and total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. RNA was treated with 10 units of DNAse 1 (RNase-free, BoehringerMannheim, Indianapolis, IN) and re-extracted with TRIzol. Total egg RNA was isolated by mechanical disruption of glass beads using a BeadBeater machine (BioSpec, Bartlesville, OK) in the presence of TRIzol, DNAse-treated and re-extracted as described above. First strand cDNA was synthesized from each sample in a 50 μL reaction containing the following components for 1 hour at 37°C: 50 mM Tris HCl, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT, 500 ng oligo(dT) primers, 1 μg of total RNA, and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies). Reactions were incubated at 94°C for 5 minutes and made up to 100 μL with dH ₂ O. 1 μL of the first cDNA strand was used in PCR with primers MTP5'-I (5'-CTTCTCATGATCAACAAACACTACG) SEQ ID NO.65 and MTP3'-1 (5'-AATCTAACTCCAACATCTTCTGGTG) SEQ ID NO.66. The reaction was subjected to 30 cycles of 1 minute at 94°C, 1 minute at 55°C, and 2 minutes 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 into the expression vector pET28 (Novagen) in a frame-compliant manner using conventional techniques and transformed into competent BL-21 Escherichia coli (E.coli) cells middle. Expression of the recombinant protein encoded in a vector containing 6 histidine residues encoding the 5' end (His-tag) was induced by adding 1 mM IPTG at 37°C for 3 hours. 1 ml of cells expressing rMTP-1 were pelleted by centrifugation at 5000 x g for 5 minutes, the supernatant was discarded, and the cells were lysed in 100 ml of TE (pH 8.0) containing 100 μg/ml of lysozyme and 0.1% Triton X-100. After incubation at 30°C for 20 minutes, the samples were sonicated (power level 2-3, 20-30% duty factor) on ice, with 10 breaks for 5 seconds per sample until the samples were no longer sticky. Soluble and insoluble cell fractions were separated by electrophoresis on 12% SDS-PAGE under reducing conditions, and soluble proteins were visualized by Coomassie brilliant blue staining. To purify rMTP-1, cell pellets from 2 liters of induced bacterial cultures were 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 hours with two buffer changes and applied to a 10 ml HisBind nickel resin column (Novagen). Chromatography was performed according to the manufacturer's instructions, except that 0.1% SDS was added to all buffers.

In an effort to improve solubility and study the domain structure of MTP-1, three constructs lacking the aminoHis tag sequence were prepared by PCR. Full-length Ac-MTP cDNA (1-1642bp), cDNA without 5′-propeptide (408-1642bp), and putative catalytic domain (408-1101bp) were cloned in-frame into pET28 at the Nco I site Click upstream to remove the His tag coding sequence from the vector. Express the recombinant protein under the same conditions as described above. Antiserum Production Anti-rMTP polyclonal antisera were obtained by immunizing BABL/C mice with purified rMTP. 20 μg of column-purified rMTP were co-precipitated with alum (Ghosh et al., 1996) and injected subcutaneously. Additional boosters were administered at weeks 3, 6 and 9 along with alum-precipitated rMTPs (20 μg each).

Mouse sera were adsorbed against bacterial lysates of E. coli strain BL21 to remove antibodies reactive with bacterial proteins. 25 ml of induced cells were centrifuged, dissolved in 25 ml of 2X sample buffer (100 mM Tris, pH 6.8, 2% SDS, 2.5% 2-mercaptoethanol), and centrifuged at 12,000 xg for 10 minutes. The nitrocellulose membrane (4cm×8cm) was immersed in the supernatant for 20 minutes, and then incubated in transfer buffer (48mM Tris, 39mM glycine (glyine), 0.037% SDS, 20% methanol) for a total of 30 minutes. minute. Membranes were washed 3 times in PBS containing 0.1% Tween-2 and incubated with mouse antiserum at a dilution of 1:100 for 1 hour at 22°C. The incubation was repeated twice with fresh membranes. To confirm the specificity of the antibody, adsorbed mouse antisera were second adsorbed against an aliquot of BL21(DE3) cell lysate expressing full-length rMTP-1. Absorbed antisera were used for Western blotting.

In vitro activation of L3 and collection of ES product: A. caninum _L3 was activated as described above (Hawdon et al. 1999) under conditions similar to those in the host. Briefly, L3 collected from fecal cultures were purged with 1% HCl in BU buffer (Hawdon and Schad, 1991) for 30 min at 22°C. Approximately 5000 _L3 were cultured at 37°C, 5% _CO2 for 24 hours in a single well of a 24-well tissue culture plate in 0.5 ml RPM ₁₆₄₀ tissue culture medium supplemented with 25 mM HEPES pH 7.0 and antibiotics (Hawdon et al. 1999). Feeding was restarted by including 15% (v/v) <10 kD ultrafiltered canine serum and 25 mM S-methyl-glutathione (Hawdon et al. 1995) to activate L3. Unactivated _L3 was incubated in RPMI without stimulation. The percentage of fed larvae was determined as described above (Hawdon et al. 1996).

The medium containing activated and non-activated _L3 was transferred to a separation microcentrifuge tube and centrifuged at 14,000 rpm for 5 minutes. Supernatants from the same treatment groups were pooled, filtered through a 0.45 μm syringe filter to remove any _L3 and exfoliated epidermis, and stored at -20°C. Prior to electrophoresis, supernatants were concentrated by ultrafiltration using Centricon 10 cartridges (Amicon, Beverley, MA). Concentrated ES was washed with 1 ml of BU, ultrafiltered and lyophilized.

To collect mature ES, 1260 mature hookworms were cultured in RPMI ₁₆₄₀ tissue culture medium (Hawdon et al. 1999) for 15 hours at 37°C, 10% _CO2 . The supernatant was concentrated 3-fold by ultrafiltration in a Centricon 3 spin column.

Western blot : Lysates of bacterial cells expressing rMTP-1 fusion protein and lyophilized ES products were resuspended in sample buffer for 2x SDS-PAGE (4% SDS, 5% 2-mercaptoethanol, 15% glycerol) And the separation was performed on a 4-20% gradient SDS-PAG (Invitrogen, Carlsbad, CA). Separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) by electroblotting at 25V for 1 hour (Towbin et al., 1979). The membrane was blocked with 5% nonfat dry milk in wash buffer (PBS, pH 7.4, 0.1% Tween 20) for 1 hour at 22°C. Blocked membranes were incubated for 1 hour at 22°C with a 1 :5000 dilution of mouse rMTP antiserum that had been pre-spiked against a bacterial lysate expressing full-length rMTP. Membranes were washed three times with wash buffer for 10 min at 24°C and then incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN) at 22°C for 1 Hour. Bands were visualized using chemiluminescent detection reagents (ECL+, Amersham. Pharmacia Biotech, Piscataway, NJ).

Results of Example 3A

Cloning of A. caninum MTP cDNA The pooled serum collected from patients in mainland China with high anti-hookworm L3 titers was used to screen the expression library of A. caninum L3 cDNA. Twelve positive clones were identified, 6 of which were confirmed to be identical by DNA sequencing. Each clone contained a 3' polyA tail but was truncated at the 5' end. The 5' end was isolated from A. caninum _L3 cDNA by PCR using primers from the nematode splicing leader together with the gene-specific primer P1 (Hawdon et al. 1995; Bektech et al. 1988).

The full-length cDNA without a polyA tail is 1703bp (see Figure 8A, SEQ ID NO.15) and encodes an open reading frame of 547 amino acids (see Figure 8B, SEQ ID NO.16) with a calculated molecular weight of 61,730, and The pI is 8.72. The ATG initiation codon begins two nucleotides downstream of the end of the splice leader sequence, resulting in a total of 23 untranslated nucleotides at the 5′ end of the Ac-mtp-1 cDNA. A TAA terminator is located at nucleotides 1666 to 1668, followed by a 35 bp 3'UTR containing the AATAAA polyadenylation signal (Blumenthal and Steward, 1997), which is 12 bp upstream of the polyA tail (bases 1687- 1692). Amino acids 1 to 16 of the deduced protein sequence were predicted to represent a hydrophobic signal peptide, with a possible cleavage site between Ala at position 6 and Gly at position 7 (Nielson et al., 1995). The deduced sequence contains 2 possible N-linked glycosylation sites (N-X-S/T) at Asn39 and Asn159.

A BLAST search using the predicted amino acid sequence of Ac-MTP-1 (Altschul et al., 1997) GenBank revealed significant homology to members of the family of zinc metalloproteases called astaxanthin (Bond and Benyon, 1995), Shrimp The zinc metalloprotease of red pigment is named after the digestive protease astaxanthin of the crayfish crawfish (Astacus astacu). A search of protein structure databases (Apweiler et al., 2000) with the deduced amino acid sequence of Ac-MTP-1 revealed the presence of a characteristic astaxanthin fingerprint, including an extended zinc-binding domain as well as a conserved Met turn located 37° downstream. amino acids. The catalytic domain containing the zinc binding site is followed by a domain homologous to epidermal growth factor (EGF), from amino acids 334 to 368. Amino acids 374 to 484 are domains with poor homology to the CUB domain, named because of its presence in complement subcomponents C1r/C1s, embryonic echinoprotein U egf and B MP-1. The EGF and CUB domains are identical in astaxanthin metalloproteases and are believed to be involved in protein-protein interactions (Bond and Benyon, 1995).

The N-signal peptide is followed by a 119 amino acid helix-rich pre-peptide domain. The C-terminus of the propeptide domain contains four basic amino acids (R-E-K-R) from amino acid 132 to amino acid 135, which are possible recognition sites for furin or other trypsin-like processing enzymes (Bond and Benyon, 1995). Proteolysis at this site will activate Ac-MTP-I to a putative 412 amino acid processed form with a calculated MW of 46419 and a pi of 8.04.

Stage-specific RT-PCR analysis : The stage-specificity of Ac-mtp-1 expression was investigated by qualitative RT-PCR of cDNA from different developmental stages of H. caninum. Ac-mtp-1 specific primers were designed to amplify a 434 bp portion of the Ac-mtp-1 cDNA corresponding to nucleotides 985 to 1419 of the complete sequence. Products of the predicted size were amplified from unactivated and activated _L3 cDNA, but not from H. caninum eggs or _L1 / _L2 mixed stage cDNA. A less intense band was observed in the mature cDNA. A longer fragment was amplified from genomic DNA, showing that the primer spanned an intron, and confirming that the amplicon from cDNA was derived from amplification of cDNA rather than contaminating genomic DNA. Control primers amplified a partially constitutively expressed catalytic subunit of L. canis protein kinase A (Hawdon et al., 1995), which successfully amplified products from all DNA samples, indicating the presence of an amplifiable template.

Expression and immunoblotting of recombinant MTP : Recombinant MTP-1 was produced in Escherichia coli, purified by Ni column chromatography, and used to immunize BALB/c mice to generate specific antiserum. Antisera against lysates of E. coli were adsorbed and used to determine whether Ac-MTP-1 was secreted by A. caninum _L3 in vitro. ES products obtained from 10,000 unactivated (unfed) and activated (fed) _L3 were analyzed by Western blot using rMTP-1 antiserum. The antiserum recognized the full-length and processed (ie, without the propeptide domain) form of rMTP-1 expressed in E. coli BL21(DE3) cells, but failed to recognize any band in lysates of induced cells containing only the vector.

rMTP antiserum recognizes bands at MW 47.5 and 44.5 in the ES product of 10,000 A. caninum L3s that have been activated to reinitiate feeding in vitro . The antiserum did not recognize any bands only in the ES of 10,000 unactivated _L3 in culture or in mature Ancylostoma canium ES products or hookworm lysates (not shown). The slower migrating band in activated ES had a similar MW to the processed form of rMTP (47.5 vs. 46.5), suggesting that A. caninum _L3 releases processed MTP-1 during in vitro activation. The lower MW band was also recognized by pre-immune mouse serum (not shown), showing that the antiserum recognized a protein not related to Ac-MTP-1. To confirm that this recognition is non-specific, mouse antiserum directed against BL21(DE3) cells expressing full-length MTP-1 was adsorbed and used to probe Western blots. Adsorbed antiserum failed to recognize any rMTP-1, but recognized a band at MWr = 44.5 in the activated ES product, showing that recognition of low molecular weight bands by antisera was non-specific.

While recombinant MTP-1 was recognized by pooled sera used to screen the library, sera from individuals living in non-infectious areas (Shanghai) did not recognize rMTP-1 (not shown).

Isolation and identification of embodiment 3B.MTP-1 cDNA

Sera from hookworm-infected patients in China were used as probes to isolate and characterize cDNA from an A. caninum L3 expression library encoding an astaxanthin family zinc metalloprotease (Ac- mtp-1). According to the manufacturer's instructions, the L3 cDNA expression library of A. caninum (Baltimore strain) (Hawdon et al. , 1995), Ancystoma duodenale (A.Duodenale) is the main hookworm species in Anhui Province, China. Using sera from patients with low fecal egg counts and high titers of circulating antibodies to the L3 whole lysate antigen of A. caninum, it was suggested that these patients may be resistant to hookworm infection. Six identical truncated clones were recovered after plaque purification. Isolation of the 5′ end from A. caninum L3 cDNA by nested PCR using the nematode splicing leader sequence together with two gene-specific primers (Hawdon et al., 1995) and sequencing of two independent 5′ ends clone.

Results of Example 3B

The amplified sequence was considered to represent the entire 5′ end of the transcript because the predicted ATG start codon is followed by the first methionine of the splicing leader sequence and the first 16 putative amino acids encode a characteristic of the secreted protein The signal peptide (Nielson et al., 1997), and alignment with similar metalloproteases shows that this is the complete amino acid sequence. The full-length cDNA without a polyA tail is 1703bp and encodes an open reading frame of 547 amino acids, with a calculated molecular weight of 61,730 and a pI of 8.72. Amino acids 1 to 16 in the deduced protein sequence were predicted to represent a hydrophobic signal peptide, with a possible cleavage site between Ala16 and Gly17 (Nielson et al. 1997). The protein sequence contains two possible N-linked glycosylation sites (NX-S/T) at Asn39 and Asn159. A BLAST search (Altschul et al., 1997) of GenBank using the predicted amino acid sequence of Ac-MTP-1 revealed significant homology to members of the zinc metalloprotease family known as astaxanthin (Bond and Beynon, 1995) , the astaxanthin is named after the degrading protease of the crayfish Astacus astacus. Members of this family are characterized by a short-terminal signal peptide that targets them for secretion, followed by a propeptide, and a catalytic domain containing a characteristic zinc-binding domain and "methionine turn". Unlike astaxanthin, most of the other family members contain a C-terminal domain, including varying numbers of EGF and CUB domains (Bond and Beynon, 1995). Searches of protein structure databases with the deduced amino acid sequence of Ac-MTP-1 (Apweiler et al., 2000) revealed the presence of characteristic astaxanthin fingerprints, including an extended zinc-binding domain, and a conserved 37 amino acids downstream of Methionine turn. The catalytic domain containing the zinc-binding site is followed by a domain homologous to epidermal growth factor (EGF), from amino acids 334 to 368. Amino acids 374 to 484 are a domain with less homology to the CUB domain, named for its role in complement subcomponents C1r/C1s, embryonic sea urchin protein Uegf and BMP-1 exist (Bork and Beckman, 1993).

Astaxanthin metalloprotease is synthesized as an inactive proenzyme. Removal of the propeptide by the processing enzyme activates the enzyme (Bond and Beynon, 1995). Ac-MTP-1 contains a 119-amino acid N-terminal domain in which four amino acid recognition sites (R ₁₃₂ E ₁₃₃ K ₁₃₄ R ₁₃₅ ) predicted for trypsin- or furin-processing enzymes are located. C-terminus (Bond and Benyon, 1995). Proteolysis at this site will activate Ac-MTP-1 to a putative 412 amino acid processed form with a calculated MW of 46419 and a pi of 8.04. The propeptide is also predicted to contain four amphipathic α-helices separated by a short linker region (from amino acid 23 to amino acid 86) (Kelley et al., 2000).

Stage-specific expression of Ac-mtp-1 was investigated by qualitative RT-PCR of cDNA from various developmental stages of A. caninum. Specific primers were designed to amplify a 434 bp portion of Ac-mtp-1 cDNA corresponding to nucleotides 985-1419 of the full sequence. Presumed sized products were amplified from inactive and activated L3 cDNA, but not from A. caninum eggs or L1/L2 mixed stage cDNA, showing that Ac-mtp-1 mainly Expressed at the L3 stage. Bands of weaker intensity were observed in the mature cDNA. Although this band is faint, it is not possible to draw conclusions about the amount of gene expression since RT-PCR is only used for qualitative analysis. However, Western blot of mature lysates using mouse anti-rMTP serum failed to recognize mature ES or any of the proteins in the lysates (not shown). This suggests that the expression of Ac-MTP-1 is restricted to the L3 stage and that the message present in the mature stage is not translated or possibly partially degraded.

Recombinant MTP-1 was produced in E. coli, purified by Ni column chromatography, and used to immunize BALB/c mice to generate specific antisera. Antisera against E. coli lysates were adsorbed and used to determine whether Ac-MTP-1 was secreted by A. caninum L3 in vitro. ES products collected from 10,000 unactivated (unfed) and activated (fed) L3 (Hawdon and Schad, 1993) were analyzed by Western blot using rMTP-1 antiserum. The antiserum recognizes the full-length and processed (i.e., without the propeptide domain) form of rMTP-1 expressed in E. coli BL21(DE3) cells, but not any band in induced cell lysates containing vector alone . A smaller molecular weight band was observed and was similar in size to the processed rMTP (ie, lacking the prosequence), suggesting that some rMTPs expressed in E. coli underwent in vitro cleavage at the C-terminus of the propeptide. This is likely an autocatalyzed cleavage, although nonspecific cleavage by bacterial proteases is also possible. Autocatalysis may also represent a mechanism for the physiological activation of Ac-MTP-1 in vivo.

The rMTP antiserum recognized bands with Mr 47.5 and 44.5 in the ES product of 10,000 Ancylostoma canium L3 that had been activated for re-feeding in vitro. Antisera did not recognize any specific bands in ES from non-activated L3, only in culture medium, or in mature A. aninum ES products or hookworm lysates (not shown). A slowly migrating band in activated ES had a similar molecular weight to the processed form of rMTP (47.5 vs. 46.5), indicating that Ancylostoma canium L3 releases processed MTP-1 during in vitro activation. Furthermore, MTP-1 is only released in response to the stimulus of activated L3 to resume feeding and, therefore, most likely plays a role at some stage of the infectious process (Hawdon et al., 1996). The previously described metalloproteolytic activity is also specifically released during activation and has a similar molecular size (Hawdon et al., 1995), suggesting that Ac-MTP-1 may be responsible for at least part of this activity.

A lower molecular weight band (44.5 kDa) in the activated ES product was also recognized by pre-immune mouse serum (not shown), indicating that the antiserum recognized a protein unrelated to Ac-MTP-1. To confirm that this recognition is non-specific, mouse antiserum directed against E. coli cells expressing full-length MTP-1 was adsorbed and used to probe Western blots. The adsorbed antiserum did not recognize any rMTP-1, but recognized a band with a molecular weight of 44.5 in the activated ES product, indicating that the antisera recognized low molecular weight bands non-specifically. Recombinant MTP-1 was recognized by the pooled sera used to screen the library, but sera from individuals living in a disease-free area (Shanghai) did not recognize rMTP-1 (not shown).

Although the exact function of Ac-MTP-1 is unknown, the phase specificity of expression and specific release during activation suggest a decisive role in the infectious process. Intervention of the function of Ac-MTP-1 in vivo therefore provides a useful strategy for the development of vaccines for the control of hookworm disease.

This example demonstrates that MTP-1 is an important enzyme for invasion by hookworm parasites and that the protein is an immunodominant antigen as it was detected in sera from patients with low hookworm numbers despite repeated exposure to hookworms. recognized. MTP is therefore an attractive candidate for use as a vaccine antigen.

Example 3C. Canine Vaccine Trials Using Ac-MTP-1 Antigen

To test whether Ac-MTP-1 is an effective vaccine, two groups of 5 purpose-bred male beagles aged 8+1 weeks were treated with recombinant (expressed and isolated from E. coli) fusion protein with AS02A adjuvant preparations, or only adjuvants for immunization. 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). Specific animal management and housing conditions have been previously reported (Hotez et al., 2002a). Recombinant fusion proteins containing a polyhistidine tag were purified from washed E. coli inclusion bodies in 10 mM Tris HCl, pH 8.0, in 6M HCl guanidine. The dissolved inclusion bodies were subjected to gel filtration chromatography (Saphacryl S-300, 26/60 gel filtration column [AmershamPharmacia] pre-equilibrated in a buffer containing 0.1NaH2PO4, 10mM Tris-HCl and 6M guanidine) at room temperature (2ml flow rate per minute) in batches of 5-10ml. Selected fractions containing Ac-MTP-1 (as determined by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) were pooled according to the method of Singh et al. (2001), and then Folded, then loaded onto the Hi-Trap IMAC column (Amersham Pharmacia) of 5ml, the Hi-TrapIMAC column was charged with ZnCl _and equilibrated at 50mM sodium phosphate (pH7.2), 1M urea, and 0.5M of NaCl. The column was then washed with 15 column volumes of equilibration buffer, and bound proteins were eluted with 50 mM sodium phosphate (pH 7.2), 1 M urea, 0.5 M NaCl, and 50 mM ethylenediaminetetraacetic acid (EDTA). . Eluted protein-containing samples were pooled and dialyzed against 10 mM Tris-HCl (pH 8.0), 5% glycerol, 1 mM dithiothreitol, and 2 mM EDTA. Purified recombinant Ac-MTP-1 showed no enzymatic activity (data not shown).

Recombinant Ac-MTP-1 fusion protein was mixed with SBAS2 adjuvant and administered to each of 5 dogs by 4 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 injected intramuscularly with AS02A according to the same administration procedure. After immunization, blood was collected weekly by venipuncture and serum was separated and stored at -20°C. Antigen-specific canine IgG2 and IgE antibodies were determined by indirect enzyme-linked immunosorbent assay (ELISA) as previously described (Hotez et al., 2002a). Immunoblotting of secreted products from non-activated L3 and L3 activated under host-stimulated conditions was performed as previously described (Zhan et al., 2002) using pooled sera obtained from Ac-MTP-1 -vaccinated dogs. Each study dog was infected subcutaneously with 500 Ancylostoma canium L3 14 days after the last immunization. The source of hookworm strains used for the study is described elsewhere (Hotez et al., 2002c). Identification of the hookworm species used for the study was confirmed by polymerase chain reaction followed by restriction fragment length polymorphism (Hawdon, 1996). Following infection, dogs were bled weekly by venipuncture for complete blood counts (CBC). Chemical characterization of serum was also obtained at the end of the vaccination protocol and prior to autopsy. Quantitative hookworm egg counts (McMaster technique) for each dog were obtained 3 days per week starting on day 12 post-infection (PI). At week 5 post-infection, dogs were sacrificed by intravenous barbiturate injection, and mature hookworms were recovered and counted from the small and large intestines during necropsy (Hotez et al., 2002c). Statistically significant differences between the numbers of mature hookworms were determined using an ANOVA test (Anovtest), as were differences in hematological parameters and quantified hookworm egg numbers. Comparison of hookworm numbers and egg numbers to antibody titers was determined using a Pierman rank (non-parametric) correlation.

SDS-PAGE analysis of the Ac-MTP-1 recombinant fusion protein followed by Coomassie blue staining revealed that the migrated protein had an apparent molecular weight of 61 kDa predicted by the zymogen. Also shown is a triplet of bands migrating with a low apparent molecular weight, likely corresponding to partially processed Ac-MTP-1. After immunization, each vaccinated dog showed high titers of IgG2 anti-Ac-MTP-1-specific antibodies ranging between 1:40,500 and 1:364,500; between 1:500 and 1:500 Anti-Ac-MTP-1-specific IgE antibody responses ranged between 1,500. Serum from vaccinated dogs recognized a tightly migrating trio of proteins with the predicted molecular weight of the zymogen and Ac-MTP-1 processed in the secreted product of host-activated L3, whereas in non-inactivated L3 Secreted products are not. Other bands may also correspond to other related metalloproteases secreted by Ancylostoma canium L3; at least 3 closely related expressed sequence tags from Ancylostoma canium L3 were found in the dbEST database (ncbi.nim.nih.gov/dbEST/index.html).

Overall, the number of mature hookworms recovered from vaccinated dogs (154 + 34 hookworms) (mean + standard deviation) was not statistically compared to the number of mature hookworms recovered from control dogs (143 + 30 hookworms) significant difference in the. However, as shown in Figure 33A, there was a statistically significant reduction in the number of mature hookworms recovered from the gut of vaccinated dogs with high anti-H. canis IgG2 antibody titers. The Peelman's correlation coefficient between antibody titers and number of mature hookworms was -0.89 (P=0.04). The number of hookworms recovered from dogs with the highest antibody titers (98 hookworms) was equivalent to a 50% reduction in the number of enteroworms compared to the number of mature hookworms recovered from dogs with the lowest antibody titers (189 hookworms). The same relationship was shown between IgG2 antibody titers and median egg counts (Fig. 33B).

These studies suggest that Ac-MTP-1 may provide downstream predictors as an anti-hookworm vaccine antigen.

Example 4. Canine Vaccine Trials Using Ac-TMP, Ac-AP, and Ac-APR-1 Antigens

In order to evaluate whether antibodies directed against parasite enzymes and enzyme inhibitors have a therapeutic effect on hookworm disease, a canine immunization trial was performed using an encoding mature A. caninum protease or a protease inhibitor recombinant fusion protein. Because only small amounts of protein are available from live hookworms, testing these molecules as vaccine candidates requires expression of recombinant vectors in prokaryotic or eukaryotic host systems, followed by immunization of dogs with purified recombinant fusion proteins.

Materials and methods used in Example 4.

Dog and Husbandry Studies: After protocol approval by the Institutional Institutional Care and Use Committee (IACUC) established by The George Washington University, specially fed, parasite-naive 8±1-week-old male beagles were purchased by ear The tattoos were identified and maintained at the AALAC (Association for Assessment and Accreditation of Laboratory Animal Care) authorized by the George Washington University Animal Research Institute. Dogs were housed for the study in a room at 70+4°F with 10-15 air changes per hour, consisting of 100% fresh air, with 12-hour light cycles repeated with 12-hour dark cycles alternately. Monitor airflow and timer function daily. Dogs were fed on the diet of Teklad Certified Dog Food #8727 supplemented with canned soft food if anorexic. Drinking water was supplied from a water filtration plant and passed through an automated water supply system; water quality analysis was performed by the USArmy Corps of Engineers. Water from the facility's automated systems is cultured annually for bacteria and fungi. The pens are washed daily and sanitized every two weeks. Dogs in a given study group were allowed to live and move together before hookworm larval challenge, but were housed separately after infection. All dogs were quarantined for about a week before starting the vaccine trial. Prior to vaccination, a complete blood count (CBC), serum chemistry profile, and pre-vaccination serum samples were obtained.

Vaccine study protocol and sample size : The vaccine trial was designed to test three different antigens, each formulated with alum, and an alum adjuvant control. A total of 24 dogs were randomly assigned to four groups, each consisting of 6 dogs. The number of dog samples was selected based on the ability to detect a 40-50% reduction in the number of mature hookworms in the small intestine of the vaccinated group relative to control dogs, with 80% statistical power (α = 0.05, two-tailed). The data are from the mean and standard deviation of mature hookworms previously recovered from 400 age-matched dogs infected with Ancylostoma canium _L3 (Hotez et al., 2002).

Recombinant antigens : 6 dogs per group were immunized with recombinant hookworm proteins expressed as fusion proteins in E. coli or insect cell lines harboring 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 the baculovirus pBacPAK6 vector (Clontech) modified to contain a polyhistidine-encoding sequence and other restriction enzyme sites ( Brindley et al., 2001). The recombinant Ac-AP and Ac-TMP fusion proteins were then purified by nickel affinity chromatography followed by a second purification step. 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). ) was purified by gel filtration chromatography. Ac-APR-1 (Harrop et al., 1996) was purified by substrate affinity chromatography using pepstatin sepharose (Brindley et al., 2001). Antigen stock protein concentration was determined by Pierce Micro BCA analysis (Pierce Chemicals) or by the absorbance of the sample at 289 nm using the extinction coefficient calculated from the deduced amino acid composition of the fusion protein. The amount of protein adsorbed by alum in each dose of antigen was determined by Pierce Micro BCA assay using bovine serum albumin standards. The relative purity of each antigen relative to contaminating E. coli or insect cell proteins was determined by analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Adjuvant formulations: Recombinant Ac-TMP and Ac-APR fusion proteins were subjected to alum precipitation with a mixture of potassium aluminum sulfate dodecahydrate and sodium bicarbonate as previously described (Ghosh et al., 1996). The method requires precipitation of an aqueous protein solution with aluminum salts under basic 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, two initial doses of Ac-AP were administered without adjuvant. However, the last two doses of Ac-AP were adsorbed to the amorphous non-crystalline calcium phosphate gel.

Canine Immunization : Select a 4-dose immunization schedule (Table II). Each dog was vaccinated by subcutaneous immunization at two sites at the shoulder with a 22-gauge needle. Injected volumes were between 0.5 and 1.0 ml. Four doses of each antigen were administered over a 38-day period. The first two injections (primary immunizations) were administered on days 1 and 4, while the last two immunizations (boosts) were administered on days 34 and 38. Dogs in the control group were injected with the same amount of alum.

Table II. Amount of antigen and adjuvant used for each dog immunization. Ac-AP Ac-TMP Ac-APR-1 alum Dose 1 (Day 1) 100μg 71μg 12.5 μg- - Adjuvant none alum alum alum Dose 1 (Day 1) 100μg 71μg 12.5 μg- - Adjuvant none alum alum alum Dose 1 (Day 1) 180μg 61μg 95μg- - Adjuvant calcium phosphate alum alum alum Dose 1 (Day 1) 273μg 69μg 86μg- - Adjuvant calcium phosphate alum alum alum

Canine Antibody Assay : Blood was collected weekly by venipuncture and serum was separated and refrigerated at -20°C. Antigen-specific canine IgG1 antibodies were determined by an indirect enzyme-linked immunosorbent assay (ELISA). Other IgG subclasses were not assayed due to unavailability of suitable high quality canine-specific reagents. Optimal concentrations of sample serum and enzyme-linked detection antibody were determined by assay plate titration. Optimal antigen concentration is determined using a saturation technique. NUNC Maxisorp F96 certified plates (Rosklide, Denmark; lot number 045638) were coated with 0.1 ml antigen per well 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) by DYNEX Opsys ^™ plate washer (Chantilly, VA). The plate was treated with 0.25 ml per well of 0.5% Tween 20 in 0.15 MPBS (PBS-Tween 20) (pH 7.2) for 1.5 hours at room temperature (RT), decanted and blotted dry with paper towels. Various serial dilutions of test sera were prepared in 0.1 ml of PBS-Tween 20 and incubated overnight at 4°C. After washing, 0.1 ml of anti-canine IgGl conjugated to alkaline phosphatase (Bethyl Laboratories, Montgomery, TX) was added to each well at a dilution of 1:1000. After 1.5 hours at room temperature, the plates were treated with 0.1 ml of 2.5 mM p-nitrophenyl phosphate (Sigma. St. Louis, MO) in 10 mM diethanolamine (Sigma, St. Louis, MO) and 0.5 mM magnesium chloride (Sigma, St. Louis, MO) (pH 9.5) solution was added to each well and washed 10 times with PBS-Tween20. Plates were incubated in the dark for 30 minutes and read at a wavelength of 405 nm by 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 final serum dilution greater than 3 times the above reference was regarded as the end point of the titration. The geometric mean of these endpoints was calculated for six dogs from each group.

Ancylostoma canium infection and parasite recovery : 14 days after the last immunization, each dog studied was orally infected with 400 Ancylostoma canium _L3 contained in capsules. The origin of the hookworm strains used for the study is described elsewhere (Hotez et al., 2002). Identification of the hookworm species used in the study was confirmed by polymerase chain reaction followed by restriction fragment length polymorphism (Hawdon, 1996). Following infection, dogs were bled weekly by venipuncture for complete blood counts (CBC). Serum chemistry profiles were also obtained at the end of vaccination and before autopsy. Quantitative hookworm egg counts (McMaster technique) per dog were obtained on three days per week beginning 12 days after infection. Five weeks after infection, dogs were sacrificed by intravenous barbiturate injection and mature hookworms were recovered from the small and large intestines at necropsy and enumerated (Hotez et al., 2002). The sex of each mature hookworm was determined by visual inspection. Necropsies were performed over a three day period when 8 dogs (two dogs from each of the four groups) were euthanized per day. Small intestines of approximately 1-2 cm were isolated and placed in formalin for future histopathological analysis.

Statistical method : The percentage reduction or increase in the number of mature hookworms in the vaccinated group relative to the control group is expressed by the following formula:

Statistical significance of differences in the number of mature hookworms was determined using nonparametric tests; the Kruskal-Wallis and Dunn methods, and the Mann-Whitney U test. Differences in hematological parameters and quantitative hookworm egg counts between groups were evaluated by ANOVA tests. When more than two trials are calculated with the same variable, the level of significance should be adjusted for the number of trials. Sex differences in recovered mature hookworms were compared statistically by the Wilcoxon-Signed Ranks test for two related 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).

The results of Example 4.

Mature Ancylostoma canium antigens : Three recombinant Ancylostoma canium antigens were selected for canine immunization. Two of them, Ac-AP and Ac-TMP, are only protease inhibitors secreted by adult hookworms. Ac-AP is a 91 amino acid factor Xa inhibitor anticoagulant (Cappello et al., 1995; 1996), while Ac-TMP is a 140 amino acid putative tissue inhibitor of metalloproteases, and was produced by H. canis (Ancylostoma canium) secreted most abundant protein. A third antigen of choice is Ac-APR-1, a 422 amino acid aspartic cathepsin (Harrop et al., 1996). Coomassie blue staining was performed after SDS-PAGE analysis of the recombinant fusion protein. As expected, the apparent molecular weights of the recombinant fusion proteins Ac-APR-1 and Ac-TMP migrated on SDS-PAGE were 45,000 and 18,000, respectively. The predicted molecular weight of Ac-AP expressed as a pET28 fusion protein with an N-terminal polyhistidine tag is 12,191 Da (Cappello, 1996). By SDS-PAGE, the recombinant Ac-AP fusion protein was observed as a major band with a molecular weight of 22,000 and a fainter band migrating at approximately 15,000 Da. This observation may correspond to the organization of polypeptide oligomers. This phenomenon has previously appeared during the purification of Ac-AP natural products (Cappello et al., 1995). Factor Xa inhibitory activity, DNA sequence analysis of the pET28 plasmid encoding the recombinant Ac-AP fusion protein, and amino-terminal peptide sequence analysis of the 22 kDa band by Edman degradation confirmed the identity of the gene product (data not shown).

Canine Antibody Response. The selected canine immunization schedule was a primary immunization with two subcutaneous doses (Day 1 and Day 4) administered during the initial 4-day period, followed by two subsequent subcutaneous booster immunizations in the primary immunization Started 30 days later (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, Ac-AP was administered subcutaneously without adjuvant for the first two doses. However, during the 30-day period between the second and third immunizations, the protocol using calcium phosphate gel was shown to efficiently precipitate Ac-AP (data not shown). For this reason, calcium phosphate was chosen as the adjuvant for the last two doses of Ac-AP immunization.

The geometric means of IgGl antibody titers for the 3 vaccine antigens are shown in Figure 34A-C. In dogs vaccinated against Ac-APR-1 (FIG. 34A), antigen-specific IgGl increased approximately 6 weeks after the primary immunization followed by the final two booster immunizations. In contrast, anti-Ac-TMP IgG1 antibody responses were further boosted ( FIG. 34B ) and started to increase 2 weeks after the primary immunization, before the third and fourth doses. Anti-Ac-TMP antibody titers increased a second time after the last two boosts, with titers exceeding 1:10,000. Five of six dogs vaccinated against Ac-AP did not mount an immune response to the antigen. As shown in Figure 34C, individual dogs that responded to Ac-AP immunization displayed an antigen-specific antibody response after the last two doses.

Recovery of Mature Ancylostoma canium from the Small Intestine The number of mature Ancylostoma canium recovered from the small intestine of immunized dogs is shown in Table III. Reductions in hookworm counts in vaccinated dogs relative to dogs injected with alum alone ranged from 4.5% to 18%. The aforementioned reductions were insufficient to demonstrate statistical significance between groups (Kruskal-Wallis test, P=0.19). However, the probability of an 18% reduction in the number of hookworms recovered from the small intestine of dogs vaccinated with Ac-APR-1 (the largest reduction in a group) was less than 0.05 by Dunn's method and less than 0.03 by the Mann-Whitney U one-sided test . Dogs vaccinated against Ac-TMP also showed a reduction in the number of mature hookworms (10.8%), although these were not statistically significant. The five dogs that did not show an anti-Ac-AP antibody response also showed an insignificant reduction in hookworm numbers. Individual dogs, however, with a significant anti-Ac-AP antibody response, showed a 34.7% reduction in the number of mature hookworms. As shown in Table III, the data do not provide sufficient evidence for a statistically significant reduction in quantitative hookworm egg counts between vaccinated and control dogs. Similarly, immunization did not affect the dogs on hematological parameters, including hematocrit, hemoglobin, white blood cell count, and eosinophils (data not shown). As expected, the hookworm challenge dose used in this study did not induce anemia in control alum-injected dogs (data not shown).

Mature Ancylostoma canium was recovered from the colon.

Table III. Reduction of mature hookworms in the small intestine of immunized dogs versus alum-injected dogs test group dog number Intestinal worms reduce% average SD median value control 6 176 twenty two 180 Ac-AP 5 168 36 170 4.5 Ac-AP ^* 1 115 115 34.7 Ac-TMP 6 157 26 161 10.8 Ac-APR-1 6 144 31 138 18 ^**

^* Positive immune response

^** P＜0.05(Dunn's method)

Conversely, the number of mature hookworms recovered from the small intestine of vaccinated dogs was reduced, and the number of mature hookworms recovered from the colon was correspondingly increased (Table IV).

Table IV. Increase of mature H. canis in the colon of immunized versus alum-injected dogs Experiment 1 group dog number Intestinal worms Increase% average standard deviation median value control 4 6 8 4 Ac-AP 5 17 17 14 183 Ac-AP ^* 1 71 71 1083 Ac-TMP 4 36 11 32 500 ^** Ac-APR-1 5 twenty four 11 27 300 ^**

^* Positive immune response

^** P＜0.05(Dunn's method)

The increase in the number of mature hookworms recovered from the large intestine was statistically significant (Kruskal-Wallis test, P=0.07). Dogs immunized with either Ac-TMP (500% increase) or Ac-APR-1 (300% increase) showed a statistically significant increase relative to alum-injected dogs (Dunn's method, P<0.05). Dogs vaccinated with Ac-AP that did not exhibit antigen-specific antibody responses did not have a statistically significant increase in the number of mature hookworms recovered from the colon. However, a single dog with a significant anti-Ac-AP antibody response showed a 1083% increase in the number of mature hookworms in its colon.

Overall, there were no statistically significant differences between vaccinated and control dogs for the total number of mature hookworm combinations recovered from the small and large intestines (data not shown). In contrast, antibody responses to recombinant hookworm antigens resulted in a marked migration of mature hookworms out of the small intestine and into the colon. The ratio of mature hookworms in the small intestine relative to the colon was reduced from 43.9 in the alum-injected dogs to a ratio between 6.6 and 7.3 in Ac-TMP and Ac-APR-1 vaccinated dogs, respectively. The ratio of hookworm populations in the small intestine to colon in the individual dog showing an anti-Ac-AP antibody response was 1.6, indicating that almost half of the hookworm population in this dog had migrated to the colon.

Sex-dependent differences. The number of male and female hookworms that migrate away from the small intestine and into the colon is unequal. As shown in Figure 35, mature female hookworms recovered from the colon were more prevalent than male hookworms recovered. A higher number of female hookworms colonizing 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 females to be recovered from the small intestine, although the difference was not statistically significant. Sexing was not performed on hookworms attached to 1-2 cm segments of the small intestine that were preserved for histopathological analysis. The average number of hookworms in these segments ranged between 5 and 6 intestinal worms. These small numbers of intestinal worms did not contribute significantly to the scores for sex-dependent differences (data not shown).

These examples demonstrate that it is feasible to vaccinate mammals with recombinant fusion proteins to elicit an antigen-specific response, and that the antibody response correlates with a reduction in the number of hookworms in the intestine or a transfer of intestinal parasitic hookworms.

Example 5. Canine vaccine trials of Ac-MTP-1 and Ac-TTR

Example 5A. Reduction of Antibody Titers and Hookworms

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

As shown in Figures 36A and B, there was a statistically significant reduction in the number of mature hookworms recovered from the intestines of vaccinated dogs with high anti-Ancylostomacanium IgG2 anti-MTP-1 antibody titers. The Peelman's correlation coefficient between antibody titer and number of mature hookworms 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% reduction in the number of intestinal worms compared to the number of mature hookworms recovered from the dog with the lowest antibody titer (189 hookworms). The same relationship was shown between IgG2 antibody titers and median quantitative egg counts.

SDS-PAGE analysis of the Ac-MTP-1 recombinant fusion protein followed by Coomassie blue staining revealed that the migrating protein had an apparent molecular weight of 61 kDa, the predicted molecular weight of the zymogen. A triplet of bands is also shown, migrating at a lower apparent molecular weight, likely corresponding to partially processed Ac-MTP-1. After immunization, each vaccinated dog developed high titers of IgG2 anti-Ac-MTP-1-specific antibodies ranging between 1:40,500 and 1:364,500; anti-Ac-MTP-1-specific IgE antibodies The response range is between 1:500 and 1:1,500. Serum from immunized dogs recognizes trios of tightly migrating proteins with predicted molecular weights of the proenzyme and processed form Ac-MTP-1 at L3 of host activation but not in the secreted product of unactivated L3.

Regarding the use of TTR antigen, see Figure 37A and B, one dog with high levels of IgE and IgGl antibodies to TTR showed a reduction in the number of hookworms (6%).

This example demonstrates that vaccination of mammals with MTP or TTR elicited a protective antibody response and that reductions in hookworm numbers were observed in mammals with high antibody titers.

Example 5B. Prevention of blood loss and reduction in hookworm size due to immunization with hookworm antigens

Animals were also tested to determine if vaccination with hookworm antigens prevented blood loss. Vaccination with Ac-TTR showed significant protection against blood loss (Fig. 38A and B). Differences in both hemoglobin (38B) concentrations (P=0.036) and hematicrit (38A) concentrations (P=0.009) between TTR and adjuvant controls were statistically significant using the Mann-Whitney test.

Furthermore, the difference in hemoglobin concentration was explained by a statistically significant reduction in worm size. Data were collected using an imaging system based on scans of intestinal worms recovered from the host. Intestinal worms were photographed with a CoolSnapPro digital camera (Media Cybernetics), and the image was measured using Macro's ImagePro software to determine the length (mm) of intestinal worms compared between treatments. As shown in Figure 39, the TTR vaccinated group had a statistically significant reduction in worm size (between 1 and 2 mm) relative to the adjuvanted control group.

This example shows that vaccination with TTR, in addition to reducing the number of hookworms in the body, also reduces blood loss.

Example 6. Chimeric hookworm antigens

This example investigates the protective effect of two different hepatitis B core particles expressing a peptide epitope corresponding to amino acids 291-303 of Na-ASP-1 (also found in Ac-ASP-1). Previously, by studying the relative hydrophilicity (hydrophobicity and hydrophilicity) of the amino acid sequence of Na-ASP-1 and Ac-ASP-1, it was found that both molecules showed a hydrophilic sequence, and the The model predicts a loop-out region that could represent the molecule. Covalent adsorption of peptides to KLH (keyhole limpet hemocyanin) demonstrated that the chimeric molecule protects mice from challenge infection.

Two different chimeric molecules expressing ASP-1 were constructed. ICC-1546 expresses ASP-1 amino acids 291-303 as the "loop-out" tether structure, while ICC-1564 expresses the same peptide as the N-terminal structure. Previous studies have demonstrated that mouse anti-L3 antibodies recognize ICC-1546, but not ICC-1564.

Antigen chimeras were administered as described above with algel as adjuvant. DSM (detergent-dissolved membrane extract of mature Ancylostoma canium) was used as a negative control. Larval challenge was performed by subcutaneous injection of L3 stage larvae.

The results showed that dogs immunized with either particle produced high levels of anti-particle antibodies. Most antibodies are directed against hepatitis core antigen components. However, in one dog vaccinated with ICC-1546, there were high levels of anti-ASP-1 (and anti-peptide) antibodies. This dog showed a marked reduction in hookworm populations (Table V).

Table V. Comparison of Anti-ASP-1 Antibodies and Hookworm Counts ICC 1546 total hookworm Anti-ASP-1 IgG1 IgG2 A1 139 1:800 0 A2 181 1:800 0 A3 170 1:200 0 A4 180 1:800 0 A5 118 1:1,600 1:1,600 A mean 158 ICC 1564 total hookworm total hookworm IgG2 B1 135 1:100 0 B2 143 1:100 0 B3 206 1;200 0 B4 195 1:800 0 B5 217 1:400 0 B mean 179 alum total hookworm total hookworm total hookworm D1 176 0 0 D2 150 0 0 D3 161 0 0 D4 241 0 0 D5 255 0 0 D mean 191

This example demonstrates that high antibody titers against specific epitopes associated with ASP-1 lead to reduced numbers of hookworms in vivo.

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

Expression of hookworm antigens in eukaryotic expression systems such as baculovirus/insect cells and Pichia pastoris offers the greatest potential for obtaining soluble and biologically active recombinant proteins.

A. Expression in Pichia pastoris

The antigens Na-ASP-1, Ac-TTR, Ac-API, and Ay-ASP-2 have been successfully expressed in a Pichia pastoris fermentation system. Antigens are isolated by tagging with polyhistidine for ease of isolation.

B. Expression in the Baculovirus/Insect Cell System

Antigens Na-CTL, Ac-MEP-1, Ac-ASP-2, and Ac-MTP-1 have been successfully expressed in the baculovirus/insect cell expression system. Antigens are isolated using a polyhistidine tag for easy isolation.

Embodiment 8. Cloning of the cDNAs of the orthologous antigen Ay-ASP-1, Ay-ASP-2 and Ay-MTP of Ancylostoma elegans

Orthologous antigens from the hamster-parasitic hookworm A. ceylancium were successfully cloned following the construction of a cDNA library from the larvae of A. ceylancium.

The N. ceylonostomum orthologue of ASP-1 was cloned by screening the N. ceylonis L3 cDNA library using a 900 bp ³² P-labeled Ac-ASP1 cDNA fragment as a probe. Screening of approximately 500,000 clones resulted in 85 positive clones. Among them, 21 clones were sequenced, and 19 of them encoded the same cDNA. No other ASP-1 orthologous genes were found. This clone showed 85% identity and 92% similarity to Na-ASP-1.

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

By using a 590 bp ³² P-labeled Ac-MTP cDNA fragment as a probe to screen about 500,000 L3 cDNA libraries of N. ceylonis, 700 positive clones were obtained, and 8 of them were sequenced. A further 7 of 8 encoded Ay-MTP-1, while one additional encoded a putative isoform (Ay-MTP-2).

This example demonstrates a high degree of similarity between hookworm antigens from Ancylostoma canium and Ancylostoma canium species, and the data show a high degree of identity (>80% ).

Example 9. Immunolocalization

Immunolocalization of some major vaccine antigens was performed in mature hookworm sections. Immunolocalization was determined as follows: Ac-103 as hookworm surface antigen, Ac-FAR-1 and Ac-API as components of pseudocoelomic fluid, (Ac-API is also a pharyngeal protein), and Ac-CP-1 as Amphidial gland proteins, Ac-TMP in secretory glands, and ASP-3 as amphidial and esophageal proteins. Furthermore, the total protein of this hookworm ES product localized to the double organ and secretory glands, as well as the brush boundary membrane of the hookworm digestive tract.

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

Example 10. Human Studies in Minas Gerais State, Brazil

As previously reported in China and elsewhere, human hookworm infections display unique epidemiological features compared with other soil-transmitted hookworm diseases (e.g., ascariasis and whipworm disease) and schistosomiasis (Gandhi et al., 2001 ). The prevalence and intensity of these other helminth infections peak during childhood and adolescence and then decline in adulthood, whereas the prevalence and intensity of hookworm infections increase with age. Middle-aged and even older residents showed the most severe infections in many Chinese and Brazilian Villajes villages (and presumably elsewhere).

The underlying immunological mechanisms explaining this observation have been investigated. As shown in Figures 40 and 41, CD-4+ lymphocytes were gated from the whole blood of hookworm-infected residents and gated with L3 soluble hookworm antigen (Figure 40) or Pichia-expressed recombinant Na-ASP-1 (Figure 40). 41) Stimulate. Host cytokine production was measured by intracellular cytokine staining techniques. Both antigens stimulated high levels of IL-10 and IL-5 but not IL-4. IL-10 is a strong immunomodulator with down-regulated, anti-inflammatory properties, while IL-4 is associated with antibody production and TH-2 type immunity. These findings suggest that hookworm-infected individuals may be unresponsive to hookworm and possibly other antigenic challenges.

In contrast, the study found that individuals treated with hookworms produced IL-4. These observations suggest that removing hookworms from the gut helps reconstitute the patient's immunity. This is an important observation because it suggests that treatment lacking recombinant hookworm vaccine may not function as a therapeutic vaccine in actively infected patients, and that anthelmintic chemotherapy may be necessary prior to immunization.

Furthermore, these observations also suggest that hookworm infection may hinder successful immunization against pathogens such as HIV and malaria. In some regions of Subsaharan Africa, where hookworm overlaps with HIV and malaria, monitoring of study participants for hookworm status prior to HIV or malaria immunization and management of those participants found to be actively infected prior to immunization may be necessary .

While the invention has been described in terms of 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. Therefore, the present invention should not be limited by the described embodiments, but should further include all changes and equivalents coming within the spirit and scope of the description provided herein.

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Hawdon JM, Jones BF, Hotez PJ.1995b. Cloning and characterization of a cDNA encoding the catalytic subunit of cAMP-dependent protein kinase from the third stage infectious larva of H. canisostomum (Cloning and characterization of a cDNA encoding the catalytic subunit of a cAMP-dependent protein kinase from Ancylostoma caninum third-stage infectious larvae). Mol Biochem Parasitol 69: 127-30.

Hawdon JM, Jones BF, Hoffinan DR, Hotez PJ. Cloning and characterization of Ancylostoma secreted protein: a novel protein associated with the transformation of infectious hookworm larvae into parasites (Cloning and characterization of Ancylostoma secreted protein: a novel protein associated with the transition toparasitism by infectious hookworm larvae). J Biol Chem 1996; 271: 6672-8.

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Claims (97)

1. compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, and,

Pharmaceutically suitable carrier.

2. the compositions of claim 1, wherein said reorganization or synthetic antigen show and are selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL are arranged, 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 compositions of claim 2, wherein said antigen is Ac-TMP.

4. the compositions of claim 2, wherein said antigen is Ac-MEP-1.

5. the compositions of claim 2, wherein said antigen is Ac-MTP-1.

6. the compositions of claim 1, the kind of wherein said ancylostome is selected from Necator americanus (Nector americanus), ancylostoma caninum (Ancylostoma canium), Ancylostoma ceylonicum (Ancylostoma ceylancium), and Ancylostoma duodenale (Ancylostomaduodenale).

7. one kind is caused the interior method to the ancylostome immunne response of mammalian body, and described method comprises the steps:

Give the compositions of described administration effective dose, described compositions comprises from the reorganization of ancylostome or synthetic antigen or its fragment, and

Pharmaceutically suitable carrier.

8. the method for claim 7, wherein said reorganization or synthetic antigen show and are selected from following antigen about at least 80% homogeneity: Na-ASP-1, Na-ACE, Na-CTL are arranged, 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 for claim 8, wherein said antigen is Ac-TMP.

10. the method for claim 8, wherein said antigen is Ac-MEP-1.

11. the method for claim 8, wherein said antigen is Ac-MTP-1.

12. the method for claim 7, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

13. the method for the anti-ancylostome of immunity inoculation mammal, described method comprises the steps:

Give the compositions of described administration effective dose, described compositions comprises from the reorganization of ancylostome or synthetic antigen or its fragment, and

Pharmaceutically suitable carrier.

14. the method for claim 13, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: 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 for claim 14, wherein said antigen is Ac-TMP.

16. the method for claim 14, wherein said antigen is Ac-MEP-1.

17. the method for claim 14, wherein said antigen is Ac-MTP-1.

18. the method for claim 13, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

19. a compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: 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

Pharmaceutically suitable carrier.

20. the method for claim 19, wherein said antigen is Ac-TMP.

21. the method for claim 19, wherein said antigen is Ac-MEP-1.

22. the method for claim 19, wherein said antigen is Ac-MTP-1.

23. the method for claim 19, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

24. a vaccine comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: 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

Pharmaceutically suitable carrier.

25. the method for claim 24, wherein said antigen is Ac-TMP.

26. the method for claim 24, wherein said antigen is Ac-MEP-1.

27. the method for claim 24, wherein said antigen is Ac-MTP-1.

28. the method for claim 24, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

29. a compositions that causes immunne response, described compositions comprises:

From reorganization or synthetic antigen or its fragment of ancylostome, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: 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

Pharmaceutically suitable carrier.

30. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-TMP.

31. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-MEP-1.

32. the method for claim 29, wherein said antigen protein, polypeptide or its fragment are Ac-MTP-1.

33. the method for claim 29, the kind of wherein said ancylostome is selected from Necator americanus, ancylostoma caninum, Ancylostoma ceylonicum, and Ancylostoma duodenale.

34. one kind can the anti-infection method of immunity inoculation patient, described method comprises the steps:

A) the treatment hookworm infection is to being enough to increase lymphopoietic degree; And

B) the anti-described infectious disease of the described patient of immunity inoculation.

35. the method for claim 34, wherein said infectious disease is selected from HIV, tuberculosis, malaria, measles, tetanus, diphtheria, pertussis, and poliomyelitis.

36. a method that is used for the ancylostome immunity inoculation, described method comprises the steps:

A) patient of chemotherapy hookworm infection improves hookworm infection; And

B) after improving hookworm infection, use reorganization or synthetic antigen or the described patient of its fragment immunity inoculation from ancylostome.

37. the method for claim 36, wherein said reorganization or synthetic antigen show and is selected from following antigen about at least 80% homogeneity: 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. one kind is reduced the method that the hookworm infection patient loses blood, described comprising the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and,

Pharmaceutically suitable carrier.

39. a method that reduces ancylostome size in the hookworm infection patient body, described method comprises the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and

Pharmaceutically suitable carrier.

40. one kind is reduced the method that infects ancylostome quantity in the ancylostome patient body, described method comprises the steps:

Use a kind of compositions for described patient, described compositions comprises

From reorganization or synthetic antigen or its fragment of ancylostome, and

Pharmaceutically suitable carrier.

41.SEQ?ID?NO.11。

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.11。

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. Ac-APR-2 antigen.

96. Ay-TTR antigen from nematicide.

97. the Ay-TTR antigen of claim 96, wherein said nematicide is an ancylostome.

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