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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/20—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
  • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Ascaris lumbricoides Trichuris trichiura. Enterobius vermicularus. Strongyloides stercoralis or Wuchereria bancrofti. and particularly Trichostrongylus colubriformis or Haemonchus contortus.
• the expression product comprises a first polypeptide sequence homologous to the host and a second polypeptide sequence which is an amino acid sequence coding for an antigen of the first embodiment.
• the first amino acid sequence is part or all of ⁇ -galactosidase or Tra-T and the host cell is £_ * _ colj.
• a process for the biosynthesis of a proteinaceous product comprising an antigen of the first embodiment which process comprises: transforming a host with a recombinant DNA molecule of the fourth embodiment so that the host is capable of expressing a proteinaceous product which includes an antigen of the first embodiment; culturing the host to obtain expression; and collecting the proteinaceous product.
• a process for the preparation of an anti-idiotype antibody of the thirteenth embodiment which process comprises immunizing an immunoresponsive host with an antibody of the twelfth embodiment.
• the amount of antigen, expression product, epitope and/or anti-idiotype antibody that may be combined with carrier, excipient, diluent and/or adjuvant to produce a single vaccine dosage form will vary depending upon the infection being treated or prevented, the host to be treated and the particular mode of administration.
• pharmaceutically acceptable adjuvant can mean either the standard compositions which are suitable for human administration or the typical adjuvants employed in animal vaccinations.
• An appropriate adjuvant can be selected using ordinary skill in the art.
• Suitable adjuvants for the vaccination of animals and humans include but are not limited to aluminium hydroxide and oil emulsions such as Marcol 52: Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC, Paris.).
• Other adjuvants suitable for use in the present invention include conjugates comprising the expression product together with an integral membrane protein of prokaryotic or eukaryotic origin, such as TraT.
• Figure 4 illustrates the structure of a Tra T-Tc Ad ESA1 fusion.
• nucleotide sequences, fused genes, recombinant DNA molecules and transformed hosts of the invention are prepared using standard techniques of molecular biology such as those described in Maniatis et ⁇ (1982).
• the vaccine may be administered orally or parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants as desired.
• Antibodies are raised using standard vaccination regimes in appropriate hosts.
• the host is vaccinated with an antigen, expression product, epitope, anti-idiotype antibody and/or vaccine of the invention.
• the compounds acting in a similar manner to the antibodies of the invention may be purified naturally occuring compounds or synthetically prepared using standard techniques including standard chemical or biosynthetic techniques.
• the antibody composition is prepared by mixing, preferably homogeneously mixing, antibody with a pharmaceutically acceptable carrier, diluent and/or excipient using standard methods of pharmaceutical preparation.
• the amount of antibody required to produce a single dosage form will vary depending upon the infection to be treated or prevented, host to be treated and the particular mode of administration.
• the specific dose level for any particular host will depend upon a variety of factors including the activity of the antibody employed, the age, body weight, general health, sex and diet of the host, time of administration, route. of administration, rate of excretion, drug combination and the severity of the infection undergoing treatment.
• the antibody composition may be administered orally or parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents and/or excipients as desired.
• ESA excretory/secretory antigens
• Excretory/secretory antigens were prepared from L4 and adult T. colubriformis as described in Example 1. This material was used to vaccinate guinea pigs intraperitoneally using the procedure described by O'Donnell et al (1985). It can be seen (Tables 1 and 2) that the ESA from L4 or young adult nematodes gave highly significant protection in each experiment (62-92% reduction in parasitism) .
• the components described here that were recovered from the LL fraction were Tc Ad ESAl, with an apparent molecular weight of 30kD; Tc Ad ESA2 with an apparent molecular weight of 37kD and Tc Ad ESA5 with an apparent molecular weight of 81kD (Fig.2).
• the components described here that were recovered from the LL ⁇ fraction were Tc Ad ESA3 with an apparent molecular weight of 17kD and Tc Ad ESA4 with an apparent molecular weight of llkD (Fig.2).
• the LL + 32kD component and the LL ⁇ 28-30kD components Fig.
• Tc Ad ESAl LL 30kD antigen
• Vaccinates were injected intraperitioneally with the relevant antigen. Animals were challenged with 2000 larvae 28 days later and killed for worm counts 13 days post challenge (n indicates the number of animals in each group) . Table 4
• Vaccinates were injected intraperitoneally with antigens isolated from the adult T. colubriformis ESA preparations. Animals were challenged with 2000 infective larvae 28 days later and killed for worm counts 13 days post challenge.
• polypeptides isolated as described in Example 5 were analysed for N-terminal amino acid sequence on an Applied Biosystems gas phase amino acid sequencer. To obtain internal sequences, purified protein was digested with proteinase [37°C, overnight, in 0.1M NH.HCO- pH 7.8 at 5% w/w enzyme/substrate ratio] . Peptides were separated by HPLC using a 30 x 2.1 mm Aquapore RP-300 column with a gradient of 0.1% TFA to 0.1% TFA/70% acetonitrile. Some of the amino acid sequences obtained are shown in Table 5: the underlined sequences were found to be particularly useful in providing information to design oligonucleotide probes suitable for isolation of cDNA clones.
• RNA was isolated from the L4 stage of T. colubriformis by grinding the larvae in a buffer containing guanidine hydrochloride (6M) sodium acetate (0.2M pH 5.2), and 2-mercaptoethanol (50mM), precipitation with ethanol and fractionation on an oligo(dT)-cellulose column.
• the L4 PolyA mRNA was used as the template for synthesis of double-stranded cDNA using the Amersham ribonuclease H/DNA polymerase I kit (Amersham cDNA synthesis system, #RPN.1256) as recommended by the manufacturers.
• amino acid sequence D I E L M P was used to design a degenerate oligonucleotide probe 5' G G C A T A A G T T G T T C A A T A T C 3 * G C C G G C
• amino acid sequence N P P I K D T P was used to design a degenerate oligonucleotide probe using deoxyinosine in positions of 4-fold degeneracy
• amino acid sequence D E E I I K D A was used to design a degenerate oligonucleotide probe
• the DNA sequence of the partial clone coding for Tc Ad ESA3 is shown in Table 8.
• the DNA contains an open reading frame which codes for a peptide of 43 amino acids.
• the sequence corresponding to the N-terminal amino acid sequence from the natural protein is underlined.
• An E_. coli strain DH5 ⁇ F (BRL) transformed with plasmid pT_T 3 (Pharmacia) containing the Tc Ad ESA3 gene fragment shown in Table 8 has been given the inhouse reference number BTA 1691.
• TraT-Tc Ad ESAl fusion protein of apparent molecular weight 22kD at up to 50mgs per litre per OD g _ Q .
• the signal sequence may be cleaved from the fusion product (as is normally the case when TraT is produced in E. coli) if the level of"expression does not exceed the processing capacity of the cell and the terminal cysteine may be further modified.
• this modification may be advantageous as it may confer a self adjuvanting character to the protein (International Patent Application PCT/AU87/00107 Title: Immunopotentiation) .
• the cDNA fragment encoding Tc Ad ESAl was inserted into a yeast expression vector, pYEUC114 (Fig 5), developed in the CSIRO Division of Biotechnology.
• This vector employs the Cup 1 gene (encoding metallothionine) of Saccharomyces cerevisiae.
• the accompanying promoter is inducible with copper when contained in yeast cells.
• the Cup 1 gene casette containing the copper-inducible Cup 1 promoter and a multi-cloning site is described in Australian Patent Application No. 15845/88 and in Macreadie et al, Plasmid 2_, 147-150.
• Tc Ad ESAl To induce expression of Tc Ad ESAl, copper sulphate was added to the culture medium to 0.5mM. * After 2 hours in the presence of copper, the cells were harvested, treated with Zymolyase to remove the yeast outer cell wall and then examined by SDS-PAGE and western blotting.
• the recombinant plasmid containing Tc Ad ESAl encoding DNA was named pYEUC30B4E
• the antigens expressed by recombinant IL. coli cells can be purified for vaccination trials.
• the following is an illustration of how the Tc Ad ESAl is isolated.
• Bacterial cells containing the recombinant plasmid described in Example 9 are grown in a suitable medium at 28 C and the expression of Tc Ad EAS1 is induced by increasing the temperature to 42°C and incubation the cultures at that temperature for 4-6 hours. Cells are recovered from cultures by centrifugation at 10,000 xg for 10 mins at 4°C. The pellet is then resuspended in a suitable buffer such as 50 mM Tris-HCl, lOmM EDTA, 50 mM NaCl, pH 8.0 and cells pelleted by centrifugation as before.
• a suitable buffer such as 50 mM Tris-HCl, lOmM EDTA, 50 mM NaCl, pH 8.0 and cells pelleted by centrifugation as before.
• the retenate can then be adjusted to pH 3.0 by addition of phosphoric acid, diluted 1:1 with 8M Urea to reduce the Na concentration to 50 mM and passed over a column of S-sepharose "Fast Flow" equilibrated with 8 M Urea, 50 mM NaPi, 5 mM EDTA, 5 mM DTT, pH 3.0.
• the recombinant antigen is eluted off the column with a 50 - 400 mM NaPi gradient. Fractions containing the 21 kD recombinant Tc Ad ESA antigen are pooled and concentrated on a "Diaflo" Amicon YM10 membrane.
• This concentrate is then made 0.1% with respect to SDS and dialysed in a 1000 D cut off dialysis sac against 8 M Urea, 50 mM NaPi, 2mM DTT, 0.1% SDS, pH 8.5 to reduce the Na + concentration to 50 mM and increase the pH to 8.5.
• the antigen can then be dialysed against a solution containing 150 mM NaCl, 10 mM Tris-HCl, 0.006 mM Oxidised Glutathione, 0.06 mM Reduced Glutathione, 0.1% SDS, pH 8.5, at room temperature for 24 hours and finally against a solution containing 150 mM NaCl, 10 mM Tris-HCl, 0.1% SDS, pH 7.4, at room temperature for 24 hours.
• the antigen recovered from the dialysis sac can be sterilised by filtration through a 0.22 ⁇ m filter prior to formulation in a suitable adjuvant prior to vaccination of host animals.
• Guinea pigs were vaccinated with a preparation of recombinant Tc Ad ESAl and challenged with T. colubriformis as described above. It should be noted that the worm numbers in the control animals were uncharacteristically low and scattered in this particular experiment. In previous instances where this has occurred (see e.g. Table 3, Experiment 257) repeat experiments have resulted in increased levels of protection being observed (see e.g. Table 3, Experiment 236).
• the only means by which the related antigens can be tested is to use recombinant DNA methods to isolate the gene coding for the related proteins and to express the related proteins in recombinant organisms, purify the related proteins from those recombinant organisms and vaccinate animals and challenge them with the other species of parasitic nematodes.
• the above approach using molecular biology to clone genes coding for protective antigens related to the Tc Ad ESA antigen genes is a preferable approach to developing vaccines.
• the digested DNA together with appropriate size markers was electrophoresed in 0.4% agarose gels to separate the DNA fragments according to their size. After staining the gels in ethidium bromide and photographing the DNA, the DNA in the gels was transferred to positively charged nylon membranes by alkaline transfer and the membranes prepared for hybridisation as recommended by the manufacturers (Amersham) . Fragments of DNA coding for Tc Ad ESAl and 4 were labeled with 32P by the random labeling method described in the kit sold by
• the production and purification techniques so far described are carried out at laboratory scale.
• large scale fermentation of transformed hosts is required.
• the large scale fermentations are performed according to standard techniques, the particular techniques selected being appropriate to the transformed host used for production of the antigen.
• the invention provides antigens which can be used as an effective vaccine for protection against parasitism of animals by parasitic nematodes, particularly
• Trichostrongylus colubriformis and Haemonchus contortus Trichostrongylus colubriformis and Haemonchus contortus.
• Antibodies raised against the purified antigens isolated from Trichostrongylus colubriformis and the DNA sequences coding for these proteins can be used to identify the related polypeptides and genes coding for the antigens from species of parasitic nematode other than Trichostrongylus colubriformis.
• the same DNA sequences and antibodies can be used to identify related antigens and genes coding for those proteins in a range of other species of nematode which are parasitic to man and domestic animals and it is anticipated that these proteins will provide effective vaccines against parasitism by those species of nematode whether isolated from the parasite itself or produced by recombinant DNA technology.
• Species of parasites and hosts they may infect include for example:
• Trichinella spiralis or Ancylostoma caninum infections of man Strongylus vulgaris infections of horses, Trichostrongylus colubriformis infections of sheep, Haemonchus contortus infections of goats, Ostertagia ostertagi infections of cattle, Ascaris suum or Trichinella spiralis infections of pigs, Toxascaris leonina or Uncinaria stenocephala infections of cats and Ancylostoma caninum or Trichuris vulpis infections of dogs as well as infections of the circulatory system of man by larvae of Toxocara spp and of the circulatory system of dogs by Dirofilaria immitis as well as infections of the circulatory system, urogenital system, respiratory system, skin and subcutaneous tissues of these and other species of animal. It should be noted that this list is by no means complete.

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