AU629249B2 - Vaccine - Google Patents

Description

:1 OPI DATE 18/04/90 4 PC AOJP DATE 24/05/90 APPLN. ID 43360 89 PCT NUMBER PCT/AU89/00416 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4: (11) International Publication Number: WO 90/03433 C12N 15/00, 1/16, 1/20 C07K 13/00, 7/10, 3/28 CO7K 15/08, 15/12, C12P 21/02 Al C07H 21/04, C12Q 1/68 A A61K 39/00, 37/02 C12N 1/20 C12R 1/19, C12N 1/16 (43) International Publication Date: 5 April 1990 (05.04.90) C12R 1/865__ (21) International Application Number: PCT/AU89/00416 (72) Inventors; and Inventors/Applicants (for US only): DOPHEIDE, Theodo- (22) International Filing Date: 26 September 1989 (26.09.89) rus, Antonius, Aloisius [AU/AU]; 124 Brougham Street, Eltham, VIC 3095 FRENKEL, Maurice, Joseph [AU/AU]; 23 Ellington Street, South Caulfield, VIC Priority data: 3162 GRANT, Warwick, Norman [AU/AU]; 156 PJ 0621 26 September 1988 (26.09.88) AU Mann Street, Armidale, NSW 2350 SAVIN, PJ 0622 26 September 1988 (26.09.88) AU Keith, William [AU/AU]; 3 Mercury Street, South Caul- PJ 0623 26 September 1988 (26.09.88) AU field, VIC 3162 WAGLAND, Barry, Maxwell PJ 0624 26 September 1988 (26.09.88) AU [AU/AU]; 31 Norwood Avenue, Carlingford, NSW 2118 (AU).

(71) Applicants (for all designated States except US): BIOTECH- (74) Agent: GRIFFITH HACK CO.; 71 York Street, Syd- NOLOGY AUSTRALIA PTY. LTD. [AU/AU]; 28 Bar- ney, NSW 2000 (AU).

coo Street, Roseville, NSW 2069 COMMON- WEALTH SCIENTIFIC AND INDUSTRIAL RE- SEARCH ORGANISATION [AU/AU]; 1 Limestone (81) Designated States: AT (European patent), AU, BE (Euro- Avenue, Campbell, ACT 2600 pean patent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT S(European patent), JP, LU (European patent), NL (European patent), SE (European patent), US.

Published With international search report With amended claims (54)Title: VACCINE (57) Abstract The invention provides excretory/secretory antigens derived from parasitic nematode species which are capable of inducing protective immunity against infection by parasitic nematode species, and related antigenic molecules. The invention also provides nucleotide sequences encoding the antigens and related molecules of the invention, recombinant DNA molecules comprising the nucleotide sequences, and transformed hosts carrying the recombinant DNA molecules. The invention further provides antibodies against the antigens and related molecules, and antibody compositions comprising the antibodies, vaccines comprising the antigens and/or related molecules and methods of treating or preventing nematode infections using the antigens and related molecules, vaccines, antibodies and/or antibody compositions of the invention.... I ii:i di erit co ll iiS the alnendmnunts allowed under Section 83 by the Supervising Examiner on and is correct for Printing t ag

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WO 90/03433 PC/AU89/00416 -1-

VACCINE

TECHNICAL FIELD The invention relates to the identification of antigens which induce protective immunity in a host against infection by parasitic nematode species, such as species of the genera TrichineIa-, Ancyclostoma, Strony.ilus, Trichostronavius, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Negator, Enterobius, Stronavloides and Wuchereria, especially the genera Trichostronavylus and Haemonchus. Examples of such species include Trichinella spiralis, Anclostoma caninum, Stronaylus vulaaris, Trichostronivlus colubriformis, Haemonchus contortus, Ostertaaia ostertagi, Ascaris suum, Toxascaris leonina, Uncinaria stenocephala, Trichuris vulvis, Dirofilaria immitis, the larvae of Toxocara spp.; Necator americanus, Ancylostome duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Stronavloides stercoralis and Wuchereri bancrofti, particularly Trichostronavlus colubriformis and Haemonchus contortus.

The invention also relates to nucleotide sequences encoding these antigens, as well as to recombinant DNA molecules containing such nucleotide sequences and host cells expressing these nucleotide sequences.

The invention further relates to methods for the production of the antigens, nucleotide sequences, recombinant DNA molecules and hosts of the invention.

The invention relates to antibodies raised against the antigens of the invention and to compounds which act in a manner similar to those antibodies.

Additionally, the invention relates to vaccines which induce protective immunity against infection by parasitic nematodes such as species of the genera Trichinella, Ancyclostoma, Stronylus§, Trichostronylus, Haemonchu, ostertagia, Ascarisj Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara,, Necato, .Enterobius, Strongyloides, and Wuchereria, especially the genera Trichostronylus and

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WO 90/03433 PCr/AU89/004 16 2 Haemonchus. Examples of such species include Trichinella spiralis or Ancyostoma caninum in man, StronQYlu vulgaris in horses, Trichostronvlus colubriformis in sheep and goats, Haemonchu contortus in sheep and goats, lOstertagi atertSagi in cattle, Ascaris suu or Trichinella spiralis in pigs, Toxascaris leonina or Uncinaria stenocephala in cats, Ancvlostoma cninum or Trichuris vulvis in dogs, Dirofilaria immitis in dogs, or the larvae of Toxocara spp in man, or infection by Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiur, Enterobiu$ vermicularus, Stronvipyides stercoralis or Wuhereribancrofti, and particularly Trichostrgnaylus colubriformis or Haomonchus contortus.

BACKGROUND ART Nematodes (nema thread; oides resembling), which are unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle, are virtually ubiquitous in nature, inhabiting soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.

The roundworm parasites of mammals belong to the phylum Nemathelminthes. The roundworms include the hookworm (e.g.

Necator americanus and Ancvlostoma duodenale), roundworm the common roundworm Asgaris lumbricoides), whipworm Trichuris trichiura), and the pinworm or threadworm Enterobiu5 vermicularus), as well as Strongvloides stercoralis, Trichinella sniralis and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancvlostoma caninum (infections of man), StrOngylus xvugari (infections of horses), Trichostrongylus colubriformis, Ostertagia circumcincte (infections of sheep and goats), Haemonhu conto2tua (infections of sheep and goats), Ostertagii ostertagi, Haemonchus Placei (infections of cattle), AScarii sum (infections of pigs), Toxascaris J.oin a or Uiinarj stenocePhala ,(infections of" dogs), Toxocara spp (circulatory infections of man) and Dirofilaria Li i. WO 90/03433 PCT/AU89/00416 -3immitis (circulatory infections of cats and dogs).

Even when symptom-free, parasitic worm infections are harmful to the host animal for a number of reasons; e.g.

they deprive the host of food, injure organs or obstruct ducts, may elaborate substances toxic to the host, and provide a port of entry for other organisms. In other cases, the host may be a species raised for food and the parasite may be transmitted upon eating to infect the ingesting animal. It is highly desirable to eliminate such parasites as soon as they have been discovered.

More commonly, such infections are not symptom-free.

Helminth infections of mammals, particularly by parasitic nematodes, are a source of great economic loss, especially of livestock and pets, e.g. sheep, cattle, horses, pigs, goats, dogs, cats, and birds, especially poultry (see CSIRO/BAE Report "Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research"). These animals must be regularly treated with anthelminthic chemicals in order to keep such infections under control, or else the disease may result in anaemia, diarrhoea, dehydration, loss of appetite, and even death.

The only currently available means for controlling helminth infections is with the use of anthelminthic chemicals, but these are only effective against resident worms present at the time of treatment. Therefore, treatment must be continuous since the animals are constantly exposed to infection; e.g. anthelminthic treatment with diethylcarbamazine is required every day or every other day most of the year to control Dirofilaria immitis or the dog heartworm. This is an expensive and labour intensive procedure. Due to the widespread use of anthelminthic chemicals, the worms may develop resistance and so new and more potent classes of chemicals must be developed. An alternative approach is clearly desirable.

The development of a vaccine against parasitic nematodes would overcome many of the drawbacks inherent in chemical treatment for the prevention and curing of helminthic i WO 90/03433 PCT/AU89/00416 o 4 infections. The protection would certainly last longer, only the vaccinated animal would be affected, and the problems of toxicity and persistence of residues would be minimized or avoided. Accordingly, there have been several reported attempts to develop such vaccines using parasitic nematodes; unfortunately, they have met with limited success and factors such as material availability and vaccine stability have precluded their large scale use.

One such attempt described by J.K. Dineen, (1977) involves the use of irradiated larval vaccines. As with other such attempts, the utility of this method is restricted by the requirement to maintain viable nematodes for prolonged periods.

The failure of killed vaccine preparations to afford good anthelminthic protection has been thought to be due to a number of factors. For example, it has been considered by J.T.M. Neilson (1975) that parasitic nematodes may have evolved mechanisms by which they can secrete products which immunosuppress or immunomodulate the host's immune system, thereby both preventing the development of an effective immune response and rendering the host susceptible to other infections. It is believed by Dineen and Wagland.(1982), that immunosuppressants or immunomodulators may be present in the crude preparations of parasitic nematodes which are used in the killed vaccines. A second problem suggested by this review article is that parasitic nematodes may have altered their antigen profile to one which resembles that of the host so that, in a natural infection, vigorous immunlogical reactions are not provoked by protective parasitic antigens. Such a phenomenon would also occur following vaccination with impure preparations of killed nematodes or extracts thereof.

Some workers have shown accelerated explusion of worms from host animals using whole homogenates of worms and impure subfractions see for example Rothwell and co-workers (1974, 1977, 1979), O'Donnell et at (1985), Neilson and Van de Walle (1987), Silverman: U.S. Patent 894603, Australian 1 1 1 1 1 1 1 SWO 90/03433 PCT/AU89/00416 5 Patent 247 354, Adams (1989), East et al (1989), 'Munn and Greenwood (1987) (Australian Patent Application No. 77590/87), Connan (1965), Savin ett a (1988) and McGillivery et al (1988).

In all of these studies, crude extracts of nematodes have been used to vaccinate animals, and no defined antigen or individual components of the extracts have been identified as being responsible for protection.

There have been some reports attempting to identify purified protective components, see for example Silberstein and Despommier (1985), Hoetz et al (1985), Grandea et al (1989), Lucius _t al (1988), Donelson et al (1988), Nilsen et al (1988). However, protection has either not been shown or not substantiated for the components described.

In only one natural host/parasitic nematode system has a purified cloned subunit been shown to be protective. In Australian Patent Application No. 19998/88, it was demonstrated that a recombinant DNA derived antigen shown to be nematode tropomyosin, gave 50% protection in sheep against Haemonchus contortus challenge. For reasons which will become clear later in this specification, this antigen is different to those identified in the current specification: the current antigens being found in the excretory/secretory fluids of nematodes following incubation in vitro.

The CSIRO/BAE working paper "Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research" cited intestinal parasites as one of the three most urgent health problems in the Australian sheep industry and indicated that the development of vaccines holds great promise for better control of these infections.

It is well established that animals which are infected with parasitic nematodes develop an immunity which renders them less susceptible to subsequent infection (see Rothwell 1989 for review).

Although it has been demonstrated O'Donnell at al 1985) that many parasite proteins are recognised by the

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-6immune system of infected host animals during parasitic infection, many of the immune responses will have no functional significance in terms of resistance to re-infection. The major step is to identify, from the many thousands of proteins present in the parasitic organism, the individual proteins which can induce immune responses in the host animal that protect it from re-infection.

Recent advances in biotechnology and in particular recombinant DNA technology, realistically offer the opportunity to produce commercially-viable vaccines against a range of economically-important parasites of man and domestic animals. This approach would overcome many of the problems proposed to account for the lack of efficacy of killed vaccines using crude parasite preparations. For example, the vaccines produced by recombinant DNA techniques would not contain immunosuppressants or immunomodulators which may be found in crude extracts of parasitic nematode species. But it is necessary to first identify the antigens. Once identified and characterised, recombinant DNA technology could be used to construct microorganisms which synthesize those proteins or portions of the proteins containing protective epitopes and use the products 25 synthesized by the recombinant organism in vaccines to protect animals from infection with the parasites.

The present inventors have studied in detail the excretory/secretory products from adult T. colubriformis .and components from the mixture which are capable of 30 giving protection following vaccination of target animals have been purified and characterised at the molecular e level.

*g Definitions The term adjuvant" as used throughout the specification refers to an agent used in immunising compositions to enhance the immune response of an S20998-A1 0.07.2 20998-AP/10.07.92 6a immunised host to the administered immunising composition.

The term "parenteral" as used herein includes subcutaneous injections, intraperitoneal or intramuscular injections, or infusion techniques.

The term "homologue" refers to proteinaceous molecules or to DNA sequences coding for those proteinaceous molecules which are related in structure to a first proteinaceous molecule or DNA sequence to such an extent that it is clear that the proteinaceous molecules themselves, or as encoded by the DNA, are related.

Related DNA sequences are referred to as homologous genes and the related proteins are referred to as homologous antigens. The homology is expected to be at least over 20 amino acids at the amino acid sequence level and at least 50% over 60 nucleotides at the DNA level.

It is recognised that the nematode population worldwide is genetically diverse as is the case for all organisms which reproduce sexually. Each individual of a population differs subtly from the others in the population and these differences are a consequence of differences in the sequence of the DNA which each individual inherits from its parents.

Further, random mutational events which can occur in 25 either sexually or asexually reproducing organisms are a further source of genetic variation.

Thus, for each gene encoding a particular protein, there are likely to be differences in the sequence among S.the population of individuals.

30 Such related molecules are referred to herein as homologues.

h omlFurther homologous antigens may be defined as antigens related by evolution but not necessarily by function. Similar but not necessarily identical DNA or protein sequences may be provided. It should be noted however that function in this sense relates to the natural in vivo function of the protein.

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i8-AP/10.07.92 r1-- r 1_ S iil 1:

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6b Illustration of this point is provided by considering: 1. Tc Ad ESA 1-5 from Trichostrongylus colubriformis and other nematode species.

2. Tc Ad ESA 1-5 from variants or different individuals of the T. colubriformis population.

3. Tc Ad ESA 1-5 and related proteins from nematodes, which are homologues of Tc Ad ESA 1-5 as defined herein.

It is stressed that for the purposes of this invention, the homologues of antigens encompassed include only those molecules which share the immunological function of the antigens as defined herein.

Such homologous molecules may exist in the nematode population worldwide and will be capable, when incorporated into a vaccine either alone or in combination with other antigens, of eliciting in animals vaccinated with those molecules protective immune response.

In the context of this invention, the DNA from T.

colubriformis which codes for an antigen of the invention can be used in DNA hybridisation experiments to identify specific DNA sequences in other species of parasitic nematodes. The conditions used for the hybridisation experiments will indicate the approximate homology of the related DNA sequences to the DNA isolated from T. colubriformis. Typically, the conditions will be such that the related DNA sequences hybridising to the DNA isolated from T. colubriformis are at least homologous in nucleotide sequence. These related DNA segments code for antigens in those other species of parasitic nematodes which are also related in amino acid sequence to the protective antigens isolated from T. colubriformis. It is contended that the related proteins will act as effective immunogens to protect animals from parasitism by the other species of parasitic nematodes with the possibility also of cross-species Sprotection. These related DNA sequences are referred to 1 ;i

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j 4 0.07.92 ii.; i -I i 6c as homologous genes and the related proteins are referred to as homologous antigens. Homologues of the invention may also be generated in vitro as herein described.

The term "derived" in the context of the antigens of the invention as used herein is intended to encompass antigens obtained by isolation from a nematode life stage expressing the antigen, as well as antigens obtained by manipulation of and expression from nucleotide sequences prepared from nematodes, including genomic DNA, mRNA, cDNA synthesized from mRNA and synthetic nucleotides prepared to have sequences corresponding to the antigen encoding sequences.

It is also intended to encompass synthetic peptide antigens prepared on the basis of the known amino acid sequences of the antigens as expressed by nematodes or cell lines expressing recombinant forms of the antigens.

Further, it should be recognised that it is possible to generate molecules which are not related to the Tc Ad ESA 1-5 antigens by evolution or necessarily by structure but which may serve as immunogens to generate an immune response against protective epitopes on the Tc Ad ESA antigens and thereby act as effective vaccines. These molecules are referred to herein as "analogues" and, to the extent that they fulfil the functions of immunogens as defined herein, they are included within the scope of the invention. Such analogues include chemically synthesized oligopeptide molecules with sequences .corresponding to portions of the amino acid backbone of the Tc Ad ESA 1-5 molecules, oligopeptides which when used as immunogens elicit an immune response which recognises native Tc Ad ESA 1-5 antigens in nematodes, and anti-idiotype antibodies raised against the variable region of antibodies which recognise the epitope(s) of the Tc Ad ESA 1-5 antigens.

Derivatives of anticrens of the invention are molecules made from the antigens or molecules which are related to the antigens in a manner which suggests their preparation from the antigens. 20998-AP/10.07.92 -iii

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6d DESCRIPTION OF INVENTION The present inventors have found that protective immunity against infection by parasitic nematodes can be induced by immunization with excretory/secretory products of a parasitic nematode species. Five molecules termed Tc Ad ESA1, Tc Ad ESA2, Tc Ad ESA3, Tc Ad ESA4 and Tc Ad are described which have been purified from the 9 9 9 9 9 .9 9 9 9 9* 20998-AP/10.07.92 Ile Val Cys Phe Phe Gin Lys Asn Tyr Asp Trp Met Lys Sly Gln Trp Gin Ash ./3 SWO 90/03433 PCF/AU89/00416 -7excretory-secretory fluids of mature adults of T. colubriformis and characterized. The present inventors have found that on vaccination, these proteins induce protective responses in guinea pigs against infection with T. colubriformis.

Adult worms were recovered from sheep 21 days after infection, washed and maintained in RPMI 1640 culture medium, containing antibiotics at 37 0 C for 16 hours. This Sculture medium which contains the excretory/secretory fluids from T. colubriformis, was concentrated over Diaflo membranes, and fractionated by adsorption to a lentil lectin-Sepharose 4B column.

The unbound fraction and the bound fraction (LL eluted with methylmannoside) each contained only a few protein bands and were fractionated further by polyacrylamide gel electrophoresis and electroelution.

Three proteins, designated Tc Ad ESA1, Tc Ad ESA2 and Tc Ad have been isolated from the lentil lectin bound fraction and a further two proteins designated Tc Ad ES3 and Tc Ad ES4 were isolated from the unbound fraction. All five proteins confer immunity to T. colubriformis infection following intraperitoneal injection of guinea pigs, a laboratory model for sheep.

Examples of the antigens of the invention are the purified proteins Tc Ad ESA1, Tc Ad ESA2, Tc Ad ESA3, Tc Ad ESA4 and Tc Ad ESA5 having molecular weights of 30, 37, 17, 11 and 81kD respectively as estimated by SDS-PAGE.

According to a first embodiment of this invention there is provided an antigen comprising: an excretory/secretory protein derived from a first parasitic nematode species and capable of inducing protective immunity against infection of a host by a second parasitic nematode species, which may be the same as or different from the first nematode species; or a protein molecule comprising all, part, an analogue, homologue, derivative or combination thereof of the excretory/secretory protein, which protein molecule is capable of inducing protective immunity in a host against I or amino acids 13 to 219 inclusive of said amino acid sequence.

p- WO 90/03433 PCT/AU89/00416 -8infection by a parasitic nematode.

Preferably, the excreto-ry/secretory protein has an approximate molecular weight of 11, 17, 30, 37 or 81 kD as estimated by SDS-PAGE.

Typically, the first parasitic nematode species is selected from species of the genera Trichinella, Anylostoma, trnyu., Trichostronlvlus, ajemonc.hua, Ostet.agia., Asaris, Tox ascar is, Uncinaria, 2drbcnxris, Dirofilaria, ToxgcaLr., NgCr Enterobius, Stroncivloides and Wuhrra Examples of such species include Trichinella spiralis., Ancylostona caninum, StrQonaylia vulgaria, Trichostrongylus colubriformis, lliemonchu§L ctort1L, Osetai os t er ta ci, Asarais siium, Toxacaia leonin.a, Uncinaria stenocephala, Trichuris vulpis, Dirofilaia imrnitis, Toxocara s.p, Necator americanus, trichurD., Enterobi-us vermicularus, Stro~nayloides stercoralis and W hrei bancofti.

Typically, the second parasitic nematode species is selected from species of the genera Trichinell-a, Ancy&lostoma, Stxr.ngvlus., Trichostronivlus, Jiaemncnhus, Ostta.±i, Asarais, Toxa scar is, Uncinaria., Trichrisa, Dirofilaria, Txoara, Ne-c-tor, Enterobius, St-ronayloides and Wuhrra Examples of such species include Trichinella spiralis, AncyQoatQni caninu~m, Strongvyiu§ vugari.s, Trichostroncivlus colubriformis, Hae onchuIm contrtu, Osteragia- osetai Asari-. Toxa scar i ileonina, Uncinaria stenoceiphala, Trichur. v.ulpis, AncyJ.o±.Qma dudnale, Asarxis lumbricoides, Trichuis~ trichiura, Enterkbius vermicularus, Stronayloides stercralis and Wucherxia bancrofti.

Preferably, the first parasitic nematode species is T. colubriformis.

Preferably, the second parasitic nematode species is I colubriformis or H. contortus.

According to a second embodiment of this invention there rW0 90/03433 PCT/AU89/00416 9 -9is provided: a first nucleotide sequence encoding the amino acid sequence of an antigen of the first embodiment; a nucleotide sequence which hybridizes to the first nucleotide sequence; or a nucleotide related by mutation including single or multiple base substitutions, insertions or deletions to the first nucleotide sequence.

Preferred nucleotide sequences of the invention are those encoding the excretory/secretory proteins of the first embodiment having approximate molecular weights of 11, 17, 30, 37 and 81kD as estimated by SDS-PAGE.

Preferably, the nucleotide sequence is a DNA sequence.

The DNA sequences embraced by the present invention can be prepared, for example, from T. colubriformis cells by extracting total DNA therefrom and isolating the sequences by standard techniques. Alternatively, the DNA may be prepared in vitro, synthetically or biosynthetically, such as by the use of an mRNA template.

According to a third embodiment of this invention there is provided a process for selecting a DNA or RNA sequence coding for an antigen according to the first embodiment which process comprises providing one or more DNA or RNA sequences and determining which of the sequences hybridizes with a DNA or RNA sequence known to code for an antigen of the first embodiment or providing an antiserum to the antigen and identifying host-vector combinations that express the antigen.

The sequences may be from natural sources, may be RNA sequences, synthetic sequences, DNA sequences from recombinant DNA molecules or combinations of such sequences.

Preferably, the process used to identify and characterize DNA coding for the antigen involves the extraction of mRNA species from cells producing the antigen, their conversion to double stranded DNA (cDNA) and the insertion of these into an autonomously replicating factor, such as a plasmid or phage vector. This is followed by transformation of a host cell such as a bacterial strain with the factor and screening of the library produced with i :i 1 1 1 1 1 1 WO 90/03433 PCT/AU89/00416, :t 10 synthetic DNA probes which are complementary to the antigen encoding mRNA or DNA sequences in order to detect those clones which contain DNA coding for the antigen as opposed to any other cell proteinaceous components.

According to a fourth embodiment of this invention, there is provided a recombinant DNA molecule comprising a DNA sequence of the third embodiment and vector DNA.

The DNA sequence may be a natural, synthetic or biosynthetic DNA sequence.

Preferred recombinant DNA molecules of the invention include an expression control sequence operatively linked to the DNA sequence.

In one preferred form of the invention, the DNA sequence is operatively linked to the P-galactosidase gene of E. gcli. Other preferred control systems include those of the tryptophan (Trp) operon, the Tra-T gene of E. 9li, the leftward promoter of bacteriophage lambda, the Cup 1 promoter and hybrid promoters such as tac or viral promoters such as the SV40 early promoter.

Preferably, the vector DNA is plasmid DNA. Suitable plasmid vectors include pUR290, pUC18, pYEUC114 and derivatives thereof.

Alternatively, the vector DNA may be bacteriophage DNA such as bacteriophage lambda and derivatives thereof, such as lambda gtll and lambda gtlO.

According to a fifth embodiment of this invention there is provided a fused gene comprising a promoter, a translation start signal and a DNA sequence of the third embodiment.

According to a sixth embodiment of this invention there is provided a process for the preparation of a recombinant DNA molecule of the fourth embodiment which process comprises providing a DNA insert comprising a DNA sequence of the third embodiment and introducing the DNA insert into a cloning vector.

Preferably, the DNA insert is introduced into the cloning vector in correct spacing and correct reading frame and Wuchereria, especially the genera Trichostronavlus and SO 90/03433 PCT/AU89/00416 11 with respect to an expression control sequence.

According to a seventh embodiment of this invention there is provided a host transformed with at least one recombinant DNA molecule of the fourth embodiment.

Preferably, the transformed host is capable of expressing an antigen of the first embodiment.

Suitable hosts include bacterial cells, yeasts such as Saccharomvces cerevisiae strain CL13-ABSY86 other fungi, vertebrate cells, insects cells, plant cells, human cells, human tissue cells live viruses such as vaccinia and baculovirus and whole eukaryotic organisms.

Suitable bacterial hosts include E. coli and other enteric organisms, Pseudomonas, and Bacillus species.

Preferred hosts are E. coli K12 derivatives; in particular JM109 and Y1090.

According to an eighth embodiment of this invention there is provided a process for transforming a host to provide a transformed host of the seventh embodiment which process comprises providing a host, making the host competent for transformation, and introducing into the host a recombinant DNA molecule of the fourth embodiment.

According to a ninth embodiment of this invention there is provided an expression product of a transformed host of the seventh embodiment which product comprises an antigen of the first embodiment.

Preferably, the expression product is provided in substantially pure form.

Preferably, 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.

More preferably, the first amino acid sequence is part or all of P-galactosidase or Tra-T and the host cell is E coli.

According to a tenth embodiment of this invention there is provided a process for the biosynthesis of a proteinaceous product comprising an antigen of the first i Toxocara spp (circulatory infections of man) and Dirofilaria i 113~~ WO 90/03433 PCT/AU89/00416, 12 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.

According to an eleventh embodiment of this invention there is provided an epitope of an antigen of the first embodiment which is responsible for the protective immune response. The epitope may be created artificially by the synthetic production of oligopeptides which contain sequences of portions of the antigen which can be predicted from the results of immunochemical tests on fragments of the proteins produced in bacteria or generated as a result of chemical or enzymatic cleavage of the native or recombinant peptides.

According to a twelfth embodiment of this invention there is provided an antibody generated against an epitope of the eleventh embodiment. These antibodies or idiotypes can be used for passive protection of animals.

According to a thirteenth embodiment of this invention there is provided an antibody generated against the variable region of an antibody of the twelfth embodiment, a so called anti-idiotype antibody, which mimics a protective epitope of the antigen and may be used as an effective vaccine in active immunization of animals.

According to a fourteenth embodiment of this invention there is provided a vaccine comprising an effective amount of one or more antigens of the first embodiment, expression products of the ninth embodiment, epitopes of the eleventh embodiment and/or anti-idiotype antibodies of the thirteenth embodiment, together with a pharmaceutically acceptable excipient, carrier, adjuvant and/or diluent.

Preferred vaccines include those suitable for injectable or oral administration. Preferably, injectable vaccines i include a pharmaceutically acceptable adjuvant, i I .I *o treatment for the prevention and curing of helminthic 1 WO 90/03433 PC/AU89/00416 13 According to a fifteenth embodiment of this invention there is provided an antibody prepared as a result of vaccination of a host by administration of one or more antigens, expression products, epitopes, anti-idiotype antibodies and/or vaccines of the present invention to the host. Such antibodies include polyclonal and monoclonal antibodies.

It is recognised that there are compounds which act in a manner similar to the antibodies of the fifteenth embodiment. Although these compounds are not antibodies their presence in the host can produce a similar protective effect to the antibodies. Throughout the specification and claims, reference to antibodies of the fifteenth embodiment should be construed as extending to these compounds.

According to a sixteenth embodiment of this invention there is provided: an antibody composition comprising at least one antibody of the twelfth and/or fifteenth embodiment together with a pharmaceutically acceptable carrier, diluent and/or excipient.

According to a seventeeth embodiment of this invention, there is provided a process for the preparation of an antigen of the first embodiment which process comprises: collecting excretory-secretory fluids from a parasitic nematode species; fractionating the fluid by lentil lectin chromatography with methylmannoside as eluent; collecting the bound and unbound fractions; further fractionating by SDS-gel electrophoresis; and electroeluting the antigen.

According to an eighteenth embodiment of this invention there is provided a process for the preparation of a fused gene of the fifth embodiment which process comprises providing a promoter, a translation start signal and a DNA sequence of the third embodiment and operatively linking the promoter, translation start signal and DNA sequence.

According to a nineteenth embodiment of this invention there is provided a process for the preparation of a vaccine of the fourteenth embodiment which process comprises admixing an effective amount of at least one antigen of the Un bliverman: U.S. Patent 894603, Australian WO 90/03433 PCT/AU89/00416' 14 first embodiment and/or expression product of the ninth embodiment and/or epitope of the eleventh embodiment and/or anti-idiotype antibody of the thirteenth embodiment with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

According to a twentieth embodiment of this invention there is provided a process for the preparation of an antibody of the fifteenth embodiment which process comprises immunizing an immunoresponsive host with an antigen of the first embodiment and/or expression product of the ninth embodiment and/or epitope of the eleventh embodiment and/or anti-idiotype antibody of the thirteenth embodiment and/or a vaccine of the fourteenth embodiment.

According to a twenty-first embodiment of this invention there is provided 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.

According to a twenty-second embodiment of this invention there is provided a process for the preparation of an antibody composition of the sixteenth embodiment which process compries: admixing an effective amount of at least one antibody of the twelfth and/or fifteenth embodiment with a pharmaceutically acceptable carrier, diluent and/or excipient.

According to a twenty-third embodiment of this invention there is provided a method of protecting a host in need of such treatment from infection by a parasitic nematode species which method comprises vaccinating the host with an antigen, expression product, vaccine, epitope and/or anti-idiotype antibody of the invention.

According to a twenty-fourth embodiment of this invention there is provided a method of passively protecting a host in need of such treatment against infection by a parasitic nematode species which method comprises passively vaccinating the host with at least one antibody of the twelfth and/or fifteenth embodiment and/or antibody

.I

ivs) tnat many parasite proteins are recognised by the r 4.

I:

15 composition of the sixteenth embodiment.

It is recognised that variation in amino acid and nucleotide sequences can occur between different allelic forms of a particular protein and the gene(s) encoding the protein. Further, once the sequence of a particular gene or protein is known, a skilled addressee, using available techniques, would be able to manipulate those sequences in order to alter them from the specific sequences obtained to provide a gene or protein which still functions in the same way as the gene or protein to which it is related. These molecules are referred to herein as "homologues" and are intended also to be encompassed by the present invention.

In this regard, a "homologue" is a polypeptide that retains the basic functional attribute, namely, the protective activity of an antigen of the invention, and that is homologous to an antigen of the invention. For purposes of this description, "homology" between two sequences connotes a likeness short of identity indicative of a derivation of the first sequence from the second. In particular, a polypeptide is "homologous" to an antigen of the invention if a comparison of amino acid sequences between the polypeptide and the antigen, reveals an identity of greater than about 70% over amino acids. Such a sequence comparison can be performed via known algorithms, such as the one described by Lipman and Pearson (1985), which are readily implemented by computer.

Homologues can be produced in accordance with the present invention, by conventional site-directed mutagenesis, which is one avenue for routinely identifying residues of the molecule that can be modified without rendering the resulting polypeptide biologically inactive. Oligonucleotide-directed mutagenesis, comprising synthesis of an oligonucleotide with a sequence that contains the desired nucleotide r substitution (mutation), [ii] hybridizing the oligonucleotide to a template comprising a structural 20998-AP/10.07 .92 i h 1 20998-AP/10.07.92 z7T~--ir-- I 1' II .C.i :*ICL. LIC i 15a sequence coding for an antigen of the invention and [iii] using T4 DNA polymerase to extend the oligonucleotide as a primer, is preferred because of its ready utility in determining the effects of particular changes to the antigen sequence.

Also exemplary of antigen homologues within the present invention are molecules that comprise a portion of the antigen without being coincident with the natural molecule, and that display the protective activity of an antigen of the invention.

Also, encompassed by the present invention are synthetic polypeptides that correspond to a portion of the antigen amino-acid sequence and (ii) retain protective activity characteristic of the antigen. Such synthetic polypeptides would preferably be between 6 and amino residues in length.

Whether a synthetic polypeptide meeting criterion also satisfies criterion (ii) can be routinely determined by assaying for protective activity, in an appropriate host.

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 25 infection being treated or prevented, the host to be treated and the particular mode of administration.

It will be understood, also, that the specific dose level for any particular host will depend upon a variety of factors including the activity of the specific 30 antigen, expression product, epitope, anti-idiotype antibody and/or vaccine employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, the particular infection to be treated or prevented and the severity of the particular infection undergoing treatment or prevention.

The vaccine of the present invention may be administered orally or parenterally, in unit dosage 20998-AP/10.07.92 ii: L 15b formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, adjuvants and/or excipients as desired.

Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to known arts using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.

In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The term "pharmaceutically acceptable adjuvant" can mean 20998-AP/10.07.92 <Ie related DNA sequences are referred to 1 I 2098-AP/10.07.92

P

WO 90/03433 PCr/AU89/00416 -16 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.

Routes of administration, dosages to be administered as well as frequency of injections are all factors which can be optimized using ordinary skill in the art. Typically, the initial vaccination is followed some weeks later by one or more "booster" vaccinations, the net effect of which is the production of vigorous immunological responses such as high titres of antibodies against the antigen epitope, anti-idiotype antibody or expression product.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, antigens, epitopes, anti-idiotype antibodies and/or expression products may be admixed with at least one inert diluent such as sucrose, lactose or starch.

Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include nanoparticles, microcapsules, LTB conjugates, cholera or its B subunit as a conjugate, in pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as R water. Such compositions may also comprise adjuvants, such i or -I

A

preparation trom tne antigens.

20998-AP/10.07.92 r o

I

-17 as wetting agents, emulsifying and suspending agents or TraT as a conjugate, and sweetening, flavouring, and perfuming agents including sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol, propylene glycol etc, oils such as sesame oil, olive oil, soybean oil etc., antiseptics such as alkylparahydroxybenzoate etc, and flavours such as strawberry flavour, peppermint etc.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates SDS-PAGE analysis of the LL+ and LL fractions.

Figure 2 illustrates SDS-PAGE analysis of Tc Ad ESA 1,2,3,4 and Figure 3 illustrates SDS-PAGE analysis of Tc Ad ESA1 before and after deglycosylation.

Figure 4 illustrates the structure of a Tra T Tc Ad ESA 1 fusion.

Figure 5 illustrates the yeast expression vector pYEUC114 used to express Tc Ad ESA1 in Saccharomyces cerevisiae.

Figure 6 illustrates the detection of ESA encoding sequences in H. contortus and Dirofilaria immitis.

Figure 7 illustrates the detection of ESA2 encoding sequences in H. contortus, Ostertacia ostertagi,Ostertacia circumcincta and D. immitis.

25 BEST MODE AND OTHER MODES OF CARRYING OUT THE INVENTION The nucleotide sequences, fused genes, recombinant I DNA molecules and transformed hosts of the invention are prepared using standard techniques of molecular biology such as those described in Maniatis et al (1982).

In preparing the nucleotide sequences of the invention, it is recognised that the genes of interest, and also cDNA copies made from the genes may be provided in low yield. PCR (polymerase chain reaction) techniques can be used to amplify the relevant DNA to facilitate detection and cloning.

SR 20998AP/10.07.92 20998-AP/10.07.92 20998-AP/1O.07.92 17a Expression products of the invention are obtained by culturing the transformed hosts of the invention under standard conditions as appropriate to the particular host and separating the expression product from the culture by standard techniques. The expression product may be used I MA 20998-AP/10.07.92 r -e i, capable of inducing protective immunity in a host against appropriate to the expression product being produced and the product, anti-idiotype antibody and/or epitope with a WO 90/03433 PCT/AU89/004'l6 pharmaceutically acceptable carrier, diluent, excipient 18 impure form ormay be purified by standard methods of ptechniques as appropriate to the expression product being produced and the particular host.

The vaccines of the invention are prepared by mixing, preferably homogeneously mixing, antigen, expression product, anti-idiotype antibody and/or epitope with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant using standard methods of pharmaceutical preparation.

ac0 The amount of antigen, expression product, anti-idiotype antibody and/or epitope required to produce a single dsage form will vary depending upon the infection to be treated or prevented, the 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 antigen, expression product, anti-idiotype antibody and/or epitope employed, the age, body weight, general health, sex, and diet of the host, time of administration, route of adminstration, rate of excretion, drug combination and the severity of the infection undergoing treatment.

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 L i According to a second embodiment of this invention there WO 90/03433 PCT/AU89/00416 19 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.

The invention is further described with reference to the following Examples.

Example 1 Preparation of excretory/secretory antiaens (ESA) from adult T. colubriformis.

Young merino Border Leicester cross bred lambs 12 months old and reared worm free were infected with 60,000 infective larvae of T. colubriformis. Twenty one days post-infection, the sheep were slaughtered and the nematodes were recovered from the intestine by Baermanization. The worms were washed in RPMI 1640 culture medium containing penicillin (100 units/ml) and streptomycin (100g/ml) and incubated in the same medium (approximately 1000 worms/ml) for 16h at 37 0 C in an incubator with 5% CO 2 The viability of the worms was monitored by visual inspection and routinely more than 95% were alive and motile.

The worms and large debris were removed from the culture media by filtration or centrifugation. The supernatant or filtrate thus obtained is referred to as adult ESA (Tc Ad

ESA).

1

I

WO 90/03433 PCT/AU89/00416' 20 Similar preparations referred to as Tc L4 ESA have been made from T. colubriformis fourth stage larvae recovered from sheep after 7-8 days infection. The subsequent analysis of the components of the extracts by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (SDS-PAGE) showed that L4 and adult extracts contained similar antigens but the extracts from the adults have been used in preference as they yielded more material than L4 extracts.

Example 2 Vaccination of guinea pigs with L4 ESA and Adult ESA.

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).

Table 1 Protection of Guinea Pigs with L4 ESA and Fractions derived from it by Lentil Lectin Affinity Chromatography Expt. Group Antigen Injected n Worm Prote- No. (Pg) Numbers ction (mean SD) 110 Controls 7 556+152 Vaccinates L4 ESA 60 5 94±122 83 112 Controls 5 655+463 Vaccinates L4 ESA 100 5 249±547 62 121 Controls 10 1103±336 Vaccinates L4 ESA 100 5 82+127 93 (collected 0-24 h) Vaccinates L4 ESA 100 8 190±159 83 (collected 48-72 h) f .unjuLy vector in correct spacing and correct reading frame 11 ,:rll, l* i:biir.l~ -i WO 90/03433 PCU/AU89/00416 21 120 Controls Vaccinates Vaccinates Vaccinates Total ESA(L4) LL+(L4) LL-(L4) Total ESA(L4) LL+(L4) LL-(L4) 821±442 103±144 193±141 51± 51 1103±314 83± 57 158±142 191±149 123 Controls Vaccinates Vaccinates Vaccinates 54 22 200 Vaccinates were injected intraperitoneally with the relevant antigen (n indicates the number of guinea pigs in each group). Animals were challenged with 2000 larvae 28 days later and killed for worm counts 13 days post challenge. LL is material bound and eluted from the lentil lectin column. LL is the unbound, run through material.

Table 2 Protection of Guinea Pigs with Adult ESA and Fractions Derived from it by Lentil Lectin Affinity Chromatography Expt. Group Antigen Inje- n Worm Prote- No. cted Numbers ction (Rg) 126 Controls Vaccinates Ad ESA 164 Controls Vaccinates Ad ESA Vaccinates Ad ESA LL+ Vaccinates Ad ESA LL+ 280 Controls Vaccinates Ad ESA 100 1069±343 300±239 910±243 140±211 219±277 544±348 1484 375 252±435 11 10 9 Vaccinates were injected intraperitoneally with the relevant antigen (n indicates the number of guinea pigs in each group). Animals were challenged with 2000 larvae 28 days later and killed for worm counts 13 days post challenge. The material bound by the lentil lectin column is LL that unbound is LL'.

proteinaceous product comprising an antigen or tne rirst WO 90/03433 PC'/AU89/004161 22 Example 3 Fractionation of adult ESA The culture supernatant was concentrated 40 fold on a "Diaflo" (Amicon) YM10 membrane. The concentrated fluid was absorbed onto a lentil lectin Sepharose-4B (Pharmacia) column (5 x Icm) equilibrated with Tris-buffered saline (TBS; 10mM Tris, 150mM NaC1, pH7.4). The column was washed with 100ml of TBS at a flow rate of Iml/min and fractions containing unabsorbed material (measured by absorbance at 280nm) were collected. The specifically bound glycopeptides were eluted from the column using a solution of 2% methylmannoside in TBS. Fractions containing material absorbing at 280nm were pooled. Both the lentil lectin bound (LL and unbound components were recipitated from solution by the addition of 10 volumes of methanol, chilling the mixture at -20 0 C for 16 hours and centrifugation at 12,000 xg for 15 min.

When analysed by SDS-PAGE (Fig 1) the LL+ fraction contained Coomassie staining bands with apparent molecular weights 81, 37, 32 and 30 kilodaltons (kD) together with some smaller molecular weight material when compared with molecular weight standards. The LL- fraction contained several components including predominant bands at about 28-32, 17 and 10-12 kilodaltons.

Example 4.

Vaccination of guinea vies with Lentil Lectin fractionated L4 and adult ESA.

The material prepared from L4 or adult ESA by lentil lectin chromatography was used to vaccinate guinea pigs intraperitoneally (O'Donnell et al 1985). Both the bound material and the unbound fraction gave highly significant degrees of protection to subsequent challenge of those guinea pigs with T. colubriformis (Tables 1 and It is thus clear that there are components in both the bound and flow through fractions which are capable of eliciting a protective immune response following vaccination.

I: r I I SWO 90/03433 PCT/AU89/00416 23 Example Further fractionation of lentil lectin bound and unbound components of adult ESA and recovery of individual antigens from preparative SDS gels.

Samples of the LL+ and LL fractions from adult ESA (100-500mg protein) were suspended in Laemmli buffer (Laemmli, 1970) and subjected to electrophoretic separation on preparative 12.5% SDS-polyacrylamide gels. Proteins were visualised with Coomassie R-250 and electroeluted (Stearne et al, 1985).

*The components described here that were recovered from the LL fraction were Tc Ad ESA1, 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. 1) are believed to be related to the LL antigen (Tc Ad ESAI), as western transfers resolved with antibodies raised against the purified Tc Ad ESA1 show cross-reaction with these components. These differences are likely to be due at least in part to differential degrees of glycosylation of the Tc Ad ESA1 as analysis of the cloned DNA sequence predicts that this component is extensively glycosylated.

Example 6 Vaccination of guinea pias with purified antigens The individual antigens electroeluted from SDS gels were used to vaccinate guinea pigs as described in Example 2. The guinea pigs were challenged with T. colubriformis and shown to be significantly protected from parasitism (Tables 3 and 4).

_I i i i dULI.LLiY CIL =IL:_Lt:L..LY O&IIJ -w 1 I I WO 90/03433 PCI7/AU89/00416' 24 Table 3 Protection of Guinea Pigs by Vaccination with Purified Antigens, Tc Ad ESA1, Tc Ad ESA2 and Tc Ad from the lentil lectin bound fraction Group Antigen Inje- n Worm Protected Numbers ction (jg) Experiment 200 Controls 12 1135+263 Vaccinates 1 Total LL+ 60 5 354+341 69 Vaccinates 2 Tc Ad ESA1 21 5 458+534 Vaccinates 3 Tc Ad ESA2 24 5 482+683 57 Vaccinates 4 Tc Ad ESA5 8.5 5 530±361 53 Experiment 218 Controls 8 1389±773 Vaccinates Total LL+ 10 3 149±226 89 Vaccinates Tc Ad ESA1 20 4 563±828 59 Vaccinates Tc Ad ESA2 10 3 999±339 28 Vaccinates Tc Ad ESA5 10 3 706+283 49 Experiment 257 Controls 10 482+200 Vaccinates Tc Ad ESA1 12.5 5 254±170 47 (deglycosylated) Experiment 236 Controls 11 1223±236 Vaccinates Tc Ad ESA1 17 5 121±126 (deglycosylated) Experiment 267 Controls 7 652±281 Vaccinates Tc Ad ESA1 10 5 238±178 63 (in Alhydrogel) 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).

j :3 1 1~ JL.L.L L ==11L11 tauwuuimenr ana/or antibocy ii iii.

SWO 90/03433 PCT/AU89/00416 25 Table 4 Protection of Guinea Pigs by Vaccination with Purified Antigens Tc Ad ESA3 and Tc Ad ESA4 from the lentil lectinunbound fraction Group Antigen Injec- n Worm Proteted Numbers ction (Rg) Experiment 236 Controls 5 1103+186 Vaccinates 1 Tc Ad ESA3 40 5 182+230 83 Vaccinates 2 Tc Ad ESA4 20 5 266+248 76 Experiment 241 Controls 10 773+609 Vaccinates 1 Tc Ad ESA3 20 5 310+418 Vaccinates 2 Tc Ad ESA4 20 5 338+487 56 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.

It is clear from the results in Tables 3 and 4 that antigens electroeluted from SDS-PAGE of both the LL+ and LL fractions were capable of conferring substantial protection to guinea pigs against challenge infection by T.

colubriformis. Of particular relevance in this work are the Tc Ad ESA1, Tc Ad ESA2 and Tc Ad ESA5 components of the LL fraction and the Tc Ad ESA3 and Tc Ad ESA4 components of the LL fraction. Other Tc Ad ESA components also had effects and are of relevance.

Vaccination of guinea pigs with Tc Ad ESA1 adjuvanted in Alhydrogel resulted in 63% protection being obtained.

Deglycosylation of Tc Ad ESA1 did not result in a decrease in the extent of protection obtained (in experiment 257, the worm numbers in the controls were abnormally low) indicating that the protein portion of the molecule was capable of giving protection: the carbohydrate was apparently not the 'S ~L ~I "f 20998-AP/10.07.92 26 protective component.

Example 7 Amino acid sequence analysis of isolated peptides The polypeptides isolated as described in Example 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 0 C, overnight, in 0.1M NH 4 HC03 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% 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.

Table Some N-terminal and Internal Amino Acid Sequences from Tc Ad 20 Tc Ad ESA1 Amino Terminal sequence AN N K XX DIEQLM PKY Armillaria proteinase peptides K EQ Y S

KLIXD

Tc Ad ESA2 25 Armillaria proteinase peptides SSL

KVIPXNPPIKDTP

Tc Ad ESA3 Amino Terminal sequence KSDEEIIK DALSA L Armillaria proteinase peptide: K DALSALDVVPLG

S

(overlap with N-terminal sequence) Tc Ad ESA4 Tryptic peptides RLADDSDFG

NYDWMKGQWQN

Tc Ad Amino Terminal sequence: SXSLKD

TA

20998-AP/15.07.92 1 1 1 1 1 L 1 20998-AP/10.07.92 -27 For Tc Ad ESA1, amino acid analysis after reduction and carboxymethylation (O'Donnell et al., 1973) indicated the presence of 2 residues of half-cystine. Deglycosylation of Tc Ad ESA1 with N-glycanase (Genzyme), which removes asparagine-linked carbohydrate, reduced the apparent molecular weight from 30kD to 15kD (Fig. This is in close accordance with information provided by the cDNA sequence (see below).

Deglycosylation of Tc Ad ESA2 by the same treatment reduced the apparent molecular weight as analysed by SDS-PAGE from 37kD to approximately 30kD. A tryptic peptide from digestion of deglycosylated Tc Ad ESA 2 gave the sequence E-

I-A-D-D/S-S-K-R.

Example 8.

Isolation of recombinant organisms containing the genes coding for the Tc Ad ESA components A. Construction of cDNA Libraries.

Messenger 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 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. Following the addition of EcoRI linkers, the double-stranded cDNA was ligated to lambda gtll and packaged into viable bacteriophage which were used to infect E. coli Y1090 cells, essentially as described 30 by Huynh et al (1985). Using the above methods, a cDNA S""library was established consisting of 2 x 105 independent recombinants. A similar technique was used to establish an adult cDNA library in lambda gt 10 containing 1.5 x 105 independent recombinants.

20998-AP/10.07.92 20998-AP/10.07.92

II

PcTIAU89O4 1W WO 90/03433 28 B. Oligonucleotide probes i) Tc Ad ESA1 The amino acid sequence D I E Q L M P was used to design a degenerate oligonucleotide probe G G C A T A AG T T G T T C A A T A T C 3' G C C G G

C

ii) Tc Ad ESA2 The 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 A T A G G I G T G T C C T T I A T I G G I G G G TT 3' iii) Tc Ad ESA3 The amino acid sequence D E E I I K D A was used to design a degenerate oligonucleotide probe A T T T A GCGTCCTTTATTATCTCCT CGTC iv) Tc Ad ESA4 The amino acid sequence W M K G Q W Q N was used to design an oligonucleotide probe T T T T G C C A T T G I C C T T T C A T C C A 3' v) Tc Ad G T G A T C C T T I A A I G A I I I I G A 3' All oligonucleotides are the reverse complement of the DNA sequence coding for the amino acid sequences selected.

C. Selection of Recombinants from cDNA Libraries The L4 and young adult cDNA libraries in lambda gtll and gtlO respectively were amplified and aliquots were ff, "I i i

B

i II I WO 90/03433 PC'/AU89/00416 29 screened using the above synthetic oligonucleotides to probe duplicate filter lifts as described by Wallace et. al.

[1985] and Benton and Davis [1977].

D. Sequence of cDNA clones A number of the selected clones contained an insert which could be resected with EcoRI and subcloned into M13mpl8 digested with the same enzyme. The DNA sequence of the subcloned inserts were determined using the method of Sanger et. al. [1980] The DNA sequence of several clones of the Tc Ad ESAl cDNA was determined and is summarised in Table 6. The DNA sequence contains an open reading frame which codes for a protein of 130 amino acids. The N-terminal amino acid sequence corresponds to the sequence obtained by gas phase sequence analysis of the antigen isolated from Ad ESA1 (underlined in Table 6) and the two internal peptide sequences obtained from Armillaria mellea digests of Tc Ad ESA1 can also be identified. An E. coli stain TG1 transformed with plasmid vector pTTQ18 containing the Tc Ad ESAl gene has been given the inhouse reference number BTA 1689.

Sequence of the DNA from several isolates has shown some variation in the translated amino acid sequence. The amino acids which have varied are doubly underlined in Table 6. The sequence corresponding to the mature protein has been determined. The sequence of the presumed N-terminal leader sequence has yet to be established.

The amino acid sequence shows four sites of potential N-linked glycosylation (consensus sequence AsnXSer/Thr) which is consistent with the lentil lectin binding properties of this antigen and with the altered mobility of the antigen in SDS-PAGE following treatment with N-glycanase. Finally, the molecular weight calculated from the amino acid sequence shown (15,300 daltons) is in close agreement with that obtained for the N-glycanase treated antigen (Fig. 3).

20998-AP/1 0.07.92 WO 90/03433 PCT'/AU89/00416, 30 DNA sequence of the cDNA coding for Tc Ad ESAl and the translated amino acid sequence Coding for the complete mature protein

GGC

20 AAC ACT TAC AGT 30 GCA AAC AAT AAaLAnAs AAG CAA CAG ACC Lvs Gln Gln.Thr GAA TTC GGG GAC ATA GAA CAA CTC ATG CCC AAA Asp Ile Glu Gln Leu Met Pro Lys

ATG

Met

GTA

Val

GCT

Ala

AAC

Asn

GTC

Val1

CAC

His AAT GGA Asn Gly 140 TCT GAT 5pxr Asp ACC TTC Thr Phe 230 GCA ACG Ala Thr TTG AGA Leu Arg 320 TAC GCA Tyr Ala 100

AAC

Asn

GCG

Ala 190

AAG

Lys

ATG

Met 280

GCC

Ala

TGC

Cys 110 TAT AAC TCG ACG Tyr. Asn 5_e Thr 120 CTG ATC TGG GAT TLpt T1P Tn A~Rn

TTC

Phe GCG AAG Ala Lys 130 TAT AGT Tyr Ser 150 CTG CAA Leu G2ln ATC CGT Ile Arg 240 GAG GAA Glu Glu GAT AAA Asp 10M~ 330 AAT GGA Asn Gly TAT AAG Tyr Ly GAC AGC 160

AAG

Lvs 170 GAA GCA GJlu Ala 200 CGG AGA Arg Arg AAA GTG Lys Val 290 TTT CTT Phe Leu

AAG

Lys 250

GAG

G lu

CGC

Arg 340

GAT

Asp GAG CAA 210 GTG TTC Val Phe GGA GCT Gly Ala 300 CGT CTT Arg Leu TAC AGT Tvr Spr ATA AAG Ile Lys 260 CTG AAG Leu Lys CTC TGG Leu Trp

ACG

Thr 220

GGC

G ly

TAC

Tyr 310

TTC

Phe

ATG

ae t 180

AAT

Asn

GAT

Asp 270

CCC

Pro

ACA

Thr 350 TAT TAC I~Tyr ACG AAA Thr Lys GGT GGA Gly Gly 360 CAC GAT Hi s .P

,I

3 31 370 380 390 400 GTC CTG ACT GTC GCG TGT CTC TAC AGA GAG ATC GAT TAC AAA AAT Val Leu Thr Val Ala Cys Leu Tyr Arg Glu lie Asp Tyr Lys Asn 410 420 430 440 450 TCT CAC TAT TAG AAA GCA GTC AAC AAA AAC AGC AGA GTA AAC TGA Ser His Tyr 460 470 480 490 CTG CAC ATT TCC GCA GTT TTT GAA TAA ATA CTT GAT GCA ACT CAA 500 AAA AAA AAA AAA The DNA sequence of the clone coding for Tc Ad ESA4 is shown in Table 7. The DNA sequence contains an open reading frame which codes for a protein of 95 amino 20 acids, and contains only a single potential glycosylation site. E coli strain TG1 transformed with an inhouse pBR322 based vector, pBTA503, containing the Tc Ad ESA4 gene has been given the inhouse reference number BTA 1690. The lack of binding to the lentil lectin column i 25 and the close agreement between the estimated molecular weight on SDS-PAGE and the predicted molecular weight i based on the sequence suggests the protein is not i glycosylated.

SI20998 5.07.92 20998-AP/15.07.92

-L,

1.J iii.iY adli .ouay wirn a pharmaceutically acceptable carrier, diluent and/or 32 Table7 DNA sequence of the cDNA coding for Tc Ad ESA4 and the translated amino acid sequence TPICPIPGPCCC CCAIPTTSTPIC PICSPIPITTCT TCIPCSPGAI PIPIPCPGCCTPI AAPTCTSPGPIT GGAAPCCCACAP T PITS TCG OPIS CAT GCT CTPI CAAP GPIP PITT GAGS PIPI CCPI 555 Met 8cr Gin His Alac LCe Gin Glu Ilie Siu Lys Pro Sly 1 3 PIPIP TTT TOG CAAP AAAIP GAT TCPI GCP TAIT TTC PIPI CTC SPA PIPC AAPG PISS Lye Phe Set- Sin Lys Asep Ser A~la Tyr Phe Lys LCLL GiLU PAn Lys PAtg 20 SPA CTS SiU Leu PIPG GGI SPIC PAT CTPI COCi GTS GAG GPIG AAAIP GTPI 050 CAA ACT Lys Sly Asep PAen LeUt Pro Val Silu SiL Lys Val PArg Sin Thr 3540. PITT SPA PIPIA TTC APIS SPIT SGIT ETA AGO GAPI PTC AGAP CST CTC GOT SPIT Ilie Siu Lys Phe Lys Asp Asp Val Se- Sli Ilie PArg Arg Leu Ala PAsp 55 SPIT TOG SPIT TTT GOPI TSO PAOC 50 PIAAP PIA ACOC GAG SST GOPI ITS OCAC Asp Ser Asp Phe Sly Cys Asn Sly Lys SiLu Thr Sil Sly Ala Met His 70 PITT STS TOT TTO TTC OPIG APIS PIPT TPIT GAO TOO PITS PAAA GSA CAAI TOG Ile Vali Cys Phe Phs Sin Lys PAen Tyr PAsp Trp Met Lys Sly Sin Trp BO 85 CPIA PIAC Sin Aen TOPITTTTTCT GPIPITPIOTTG TTSSPITTCTT CGTPISPATCG PITGCACAPIPI 0000 0 0 @000 *000 0 0 0 0 00 00 @0 0

S

0000 00 0 *0 0* 0@ 0@ @0 @0@ TPCCTTTTTT SSSPISPCAAC TTCOCITAAI PIOTTCTOSPT SPAAAPIPIA PIAPIA 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 DH5uF (BRL) transformed with plasmid pT 7

T

3 (Pharmacia) containing the Tc Ad ESA3 gene fragment shown in Table 8 has been given the inhouse reference number BTA 1691.

'0 00 0 0 Z"v LA 20998-AP/1O.07.92

ESA).

r .1 33 Table 8 DNA sequence of partial cDNA coding for Tc Ad ESA3 and the translated amino acid sequence CGG TTC CTT CTT CTA GCA GCG TTC GTC GCC TAT GCG TAT GCA AAG Arg Phe Leu Leu Leu Ala Ala Phe Val Ala Tyr Ala Tyr Ala Lys TCA GAT GAA GAA ATC CGA AAA GAT GCA CTA TCT GCT CTG GAT GTA Ser Asp Glu Glu Ile Arg Lys Asp Ala Leu Ser Ala Leu Asp Val 100 110 120 GTT CCA CTG GGT TCG ACT CCC GAA AAA CTG GAA AAT GGC Val Pro Leu Gly Ser Thr Pro Glu Lys Leu Glu Asn Gly

S

S

S

S

S

SI S

S.

0 555

S.

S S .5

SI

S

S 55 .5,

S.

55 5

S

The inosine-containing oligonucleotide: 5' GG IGT ATC TTT IAT IGG IGG ATT 3' G C G was used to screen the young adult lambda gtlO cDNA library.

20 The cDNA library was screened with the radiolabeled oligonucleotide as described previously [Sambrook et al., 1989; Wallace et al., 1980] and positively hybridizing plaques were purified through several rounds of screening.

Filters were hybridized at 37°C and washed to a stringency 25 of 6XSSC-0.1%SDS (1XSSC is 15 mM sodium citrate, 150 mM NaCI, pH7.0), at 40 0 C. DNA from recombinant lambda clones was prepared using DE52 and CTAB [Manfioletti and Schneider, 1988] Polvmerase Chain Reaction. Uncut, recombinant lambda DNA 30 was subjected to the polymerase chain reaction (PCR) using S 25pmol of lambda gtl0 forward and reverse primers (based on the sequences surrounding the EcoRI site) in the presence of 100 AM dNTP's, 16.6 mM ammonium sulphate, 67 mM Tris-HCl 20998-AP/10.07.92 i .i i L. -FI I (collected 48-72 h) i 4

I

33a pH8.8, 6.7 mM magnesium chloride and 2.5 units of Taq polymerase (Biotech International). The reaction conditions used were 20 cycles of 95 0 C for 1 min, 55 0 C for 1 min and 72 0 C for 2 min. Following PCR, amplified DNA was phenol:chloroform extracted, ethanol precipitated and then digested with EcoRI. The insert DNA was purified by electroelution from 0.5% agarose and precipitated in the presence of glycogen.

Cloning into pT7T3/18U and DNA sequencing. EcoRI-digested lambda DNA and PCR-amplified lambda insert DNA were subcloned into the plasmid vector pT7T3/18U (Pharmacia).

The subclones were transformed [King and Blakesley, 1986] into Escherichia coli TG1 cells. Single-stranded DNA was prepared using the method supplied with pT7T3/18U (Pharmacia) using M13K07 helper phage. DNA sequencing was performed with Taq polymerase according to the manufacturer's instructions (Promega) using the universal M13 primer or synthetic oligonucleotides based on previously determined DNA sequences.

Database search. The following databases were searched for protein sequences with homology to Tc Ad ESA2: EMBL Nucleic Acid Sequence Data Bank, Release 28.0, September 1991; Ooi- Nakashima Protein Sequence Data Bank, March 1986; Protein Identification Resource (PIR), Release 29.0, June 1991; 25 National Biomedical Research Foundation, (NBRF), Release 36.0, March 1991; PRF Protein Sequence Database, Release 91/09 (Peptide Institute, Protein Research Foundation, Osaka, Japan); Genetic Sequence Data Bank (Genbank Release 69.0, September 1991); GBTrans Protein Data Base, Release 13.0, October 1991 (Compiled from Genbank Release 68.0 by The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia); Swiss-Prot Protein Sequence Databank, Release, 19.0, August 1991 (European Molecular Biology Laboratory, FRG); and DNA Data Bank of Japan, Release 8, January 1991. These databases were searched with the whole Tc Ad ESA2 protein sequence or the sequence lacking the S proline rich region (residues 133-169 deleted), using the il

\);TR

V1.

20998-AP/15.07.92 challenge. The material bound by the lentil lectin column is LL that unbound is LL_.

33b FASTA program (Pearson/Lipman algorithm) [Pearson Lipman, 1988] From 250,000 recombinants screened, four positive clones were purified through rounds of screening and were found to contain inserts of approximately 700-800 bp in size.

Complete DNA sequence data was obtained from the recombinants isolated from the lambda library.

The DNA sequence for the gene is shown in the attached Table (Table The DNA sequence has an uninterrupted reading frame which extends for 701 nucleotides followed by 63 bases of non-coding sequence which contains a polyadenylation addition sequence (AATAAA) but no poly A tail. The predicted amino acid sequence codes for a protein of 220 amino acids, with a molecular weight of 26.4 kilodaltons. The initiating methionine is followed by an eleven amino acid sequence of hydrophobic amino acids which are likely to be a leader sequence which is posttranslationally cleaved from the molecule.

The peptide sequence from which the oligonucleotide probe was designed can be seen starting at amino acid 161.

The only difference between the peptide sequence and that predicted from the DNA sequence is that Ile 173 is a Thr in the cloned sequence. This is likely to be either a sequencing artefact or represent the variation in the amino 25 acid sequences of various genes within the outbred population (homologues). The predicted amino acid sequence also contains the two other peptide sequences which were obtained from the native polypeptide with some small variation. It can be assured that the cDNA sequence codes 30 for Tc Ad ESA2. Database searches have not shown any significant homology between the DNA sequence for Tc Ad ESA2 and genes coding for any other protein.

o..

0 ~L 20998-AP/10.07.92 33c Table 9 DNA sequence of cDNA coding for Tc Ad ESA2 and the translated amino acid sequence CCTGGTTGTT CCSCACTTTC ACTCGGCGCA GCTCTTCGAC SATO ATG CTS ATC CTT Met Le I le Leu 1

CTG

Leu

AST

Ser

CAG

Gin

TTG

Leu

CAA

Gin

SAC

Asp

ACT

Th r

TTC

Ph e

CAA

Gin

CCS

Pro

TCC

Ser 165

ACT

Thr

SCA

Al a

CTC

GCC

Al a

SAC

Asp

CAA

Sin

AGO

Arg

GTS

Vali 71)

CST

Arg

STG

Val

AAA

Lys

CTS

LeLt

GTC

Val 1501

GAT

Asp

CCG

Pro

OTT

Va 1

AAT

ATT

Ile

TAT

Tyr

CCG

Pr o

AAA

Lys

SG

Si Lu

GAG

Si LU

GTT

Val

SAC

Asp

CCA

Pro 17.5

AST

Ser

TCC

Ser

CAC

His

SCC

Ala

TGT

TTS

LSLI

ASS

Ar g

TTT

Ph e

CTS

LeL

TTC

Ph e

ACA

Thr

SGT

Sily

TAT

Tyr 120

CCT

Pro

CCT

Proa

CTC

Leu

ACA

Thr

AAT

Asn 2001

TTC

STC

Vai

STT

Val1

AAA

Lys

AGA

Arg

AAC

Asn

ABA

Arg

SSC

Sily 1015

CTT

Leu

STT

Val

CCS

Pr o

AAT

Asn

CCT

Pro 185

SG

Si Lt

ACT

SSC

Sly 11:Q

CAC

His

SAA

51 u 656 Sly

AAC

Aen

TTT

Phe 90

SAC

Asp

CTA

LSLI

CTS

Leu

AAT

Asn

AAS

Lys 170

CCA

Pro

AAA

Lys

TGT

ACC GTS CCT Thr Val Pro AAT SAC CAC Asn Asp His ATC GCC AAC Ile Ala Asn 45 ATT 555 SAT Ile Sly Asp 60 SAC AGC AAT Asp Ser Aen 75 TCA CSS AAC Ssr Arg Aen TTC ATS TSS Phe Met Trp AAS TST GAS Lys Cys Siu 125 TCA TSC SAC Ser Cys Asp 140 SAG SAC SCT Slu Asp Ala 155 GTC ACT CCT Val Thr Pro CCS CSS SAT Pro Arg Asp TCC ACC ACT Ser Thr Thr 205 TTT TSATAAC

TCC

Ser

TST

Cys

TCS

Ser

ACT

Thr

CCS

Pro

TAT

Tyr

AAS

Lys 1

ASC

Ser

CSS

Ar g

CCS

Pro

CCC

Pro

TTC

Ph e 190

AAA

SAG TCG GlI uSer 15 AAA TAC Lys Tyr TCA CTA Ser LSLI SAC TSC Asp Cys TTT TAT P" Tyr 8o ACC STC Thr Val 975 TCA TST Ser Cys SAA GAG SilU SiLt ACC CCT Thr Pro CCC ACC Pro Thr 160 AAT CCT Asn Pro 175 ACT ACT Thr Thr AAA 555 TCS CTC Ser Leu AST. SAG Ser Siu CSA TCA Arg Ser STS CAS Val Gin STC TTT Vai Ph e TSC SST Cys Sly SAT AAA Asp Lye AAC AGA Asn Arg 130 AAT CCT Asn Pro 145 CTC CCT Leu Pro CCC ATT Pro Ile ATC CCT Ile Pro ,TTC CTA

AAC

Asn

AAS

Lys

CTT

LeuL

TAC

Tyr

ATC

Ile

STT

Val1 I

AAA

Lys

CCA

Pro

TCS

Ser

ASS

Arg

SAC

Asp 180)

CGA

Arg

AAG

Lys Lye Sly Phe Leu Ser Lys 210 )TT 6TGCTGGCAC CAAAATTSAA Leu Asn Cys 215 Phe Thr Cys Phe CCTSTTACAT TATTSASAAT AAASGTTTSC ATG 20998-AP/1 0.07.92 33d Example 9 Expression of Tc Ad ESA1 as a Tra T Fusion in E. coli TraT is an outer membrane lipoprotein of certain strains of E. coli. We have cloned the gene coding for TraT obtained from the antibiotic resistance plasmid R100 (Ogata R.T. et al., 1982, J. Bact. 151 819-827) and have transferred this gene to a plasmid in which the expression of TraT is under the control of the leftward promoter (PL) of the bacteriophage lambda. High levels of TraT can be obtained when the cells harbour the thermolabile repressor of PL, XcI857 (Remaut E et al 1980 Gene 15 81-93) are incubated at 38-42 0

C.

The gene coding for Tc Ad ESA1 has been fused to the position of the coding region of TraT in such a way that the new gene codes for the first 30 amino acids of TraT (including the 20 amino acid long signal sequence) followed by some amino acids generated by restriction sites used for the DNA manipulations followed by the gene coding for Tc Ad ESA1 (Fig Insertion into this position of the Tra-T gene was made possible by the creation of a PvuII Aii I 20998-AP/10.07.92 L U AI .LO C.J 6.1 LD challenge (n indicates the number of animals in each group).

1 WO 90/03433 34 PC/AU89/004161 restriction site at codons 31 and 32 of the TraT gene by site directed mutagenesis. The Tc Ad ESAl gene was obtained as a 570bp XmnI (generated by cutting an EcoRI site, and filling with DNA polymerase I Klenow fragment and religating) to Hind III fragment.

In a suitable E. coli host, raising the temperature of a culture leads to the production of TraT-Tc Ad ESAl fusion protein of apparent molecular weight 22kD at up to per litre per OD 600 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. When producing this TraT-immunogen fusion this modification may be advantageous as it may confer a self adjuvanting character to the protein (International Patent Application PCT/AU87/00107 Title: Immunopotentiation).

Expression of Tc Ad ESA4 as a TraT Fusion The gene coding for Tc Ad ESA4 has been fused to the portion of the coding region of TraT in a manner identical to the Tc Ad ESAl-TraT construct. The whole of the coding region of Tc Ad ESA4 (95 amino acids) is expressed as a TraT Tc Ad ESA4 fusion under the control of the Lambda leftward promoter.

Cloning into DYEUC114 and expression in Yeast The cDNA fragment encoding Tc Ad ESA1 was inserted into a yeast expression vector, pYEUC114 (Fig 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 a -ta s 1 1 1 1 11 i| 1 1 1 /r *j 1

I

p f S 1 v 1 1 1 1 V 1 \r 1 1 1 1 11 1 ,i74 tnat the protein portion of the molecule was capanle or Sgiving protection: the carbohydrate was apparently not the WO 90/03433 PC/AU89/00416 1 35 e.t aj, Plasmid 2, 147-150. The EcoRl fragment containing the (previously described) Tc Ad ESA1 cDNA was inserted into pYEUC114 replacing most of the Cup 1 coding sequence.

This results in the synthesis of a fusion protein consisting of 4 amino acids from the N-terminus of metallothionine followed by the sequence shown in Table 6.

Saccharomvces cerevisiae cells (strain CL13-ABSY86, [a, AUra3 leu2 his pral prbl prcl cpsl]) carrying the recombinant plasmid (pYEUC30B4E) were grown in minimal medium containing histidine and leucine. To induce expression of Tc Ad ESA1, 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 ESA1 encoding DNA was named pYEUC30B4E Example Purification of recombinant antigens from bacteria and yeast The antigens expressed by recombinant EL sli cells can be purified for vaccination trials. By means of example 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 280C 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, 10mM EDTA, 50 mM NaC1, pH 8.0 and cells pelleted by centrifugation as before. The washed pellet is resuspended in a buffer such as 50 mM Tris-HCl, 1 mM EDTA, 5 mM DTT, 0.1 mM PMSF, pH and homogenised in Marton-Gaulin Homogeniser, 6 passes at 9000 psi. The cell homogenate is then centrifuged at 20,000 xg for 20 min at 4°C to collect the dense j 20998-AP/15.07.92 WO 90/03433 PCT/AU89/00416' 36 inclusion bodies which contain the recombinant antigen.

The supernatant is decanted off and discarded and the pellet is resuspended in a solution suitable for solubilising the proteins in the inclusion bodies such as 8 M Urea, 100 mM NaPi, 1 mM EDTA, 40 mM DTT, pH 8.5 and incubated at 37 0 C for 4 hours with stirring. The solubilised antigen can be recovered by passing the solution through a "Diaflo" Amicon YM30 membrane followed by concentration of the eluant on a "Diaflo" Amicon membrane. 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 NaC1, 10 mM Tris-HC1, 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 NaCI, 10 mM Tris-HC1, 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 pm filter prior to formulation in a suitable adjuvant prior to vaccination of host animals.

A similar approach can be taken to purify the other recombinant antigens according to this specification although the details of the purification protocols will differ with each antigen.

20998-AP/10.07.92 WO 90/03433 PCT/AU89/00416 37 Preparation of Recombinant Tc Ad ESA1 from Yeast Yeast cells carrying pYEUC30B4E were grown for 2 days in minimal medium containing histidine and leucine. The cells were then placed in fresh medium, incubated for a further 2hrs and then copper sulphate was added to Incubations was continued for a further 2hrs whereupon the cells were harvested by centrifugation and lysed using the Braun cell homogenizer according to the manufacturer's instructions. Briefly the cells are broken by shaking with glass beads at high speed. The glass beads are allowed to settle out under gravity and the cell lysate collected.

The crude lysate was centrifuged at 15,000 rpm and the resulting supernatant and pellet examined for the presence of Tc Ad ESA1 protein. The latter was found exclusively in the 15krpm pellet. This pellet was subsequently dissolved in 50mM ammonium bicarbonate solution containing 8M urea, 2% SDS, 10mM EDTA and 2% mercaptoethanol. This crude material was then fractionated using a Sephadex G75 column run in 50mM ammonium bicarbonate solution containing imM EDTA, 0.1% SDS and 0.1% mercaptoethanol. Fractions containing material with the molecular weight expected of Tc Ad ESA1 (non-glycosylated) and reacting with an anti-serum (R45) raised in rabbits against Tc Ad ESAl from adult parasites, were pooled and used in subsequent vaccination trials.

Example 11 Host-Protection Using Recombinant Tc Ad ESAl produced by yeast #295 Worm numbers ±SD Protection Controls 413±299 Vaccinates 275±166 33 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 L. _i i; 38 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).

Recombinant bacteria were constructed which synthesize each of Tc Ad ESA 1-4. Standard molecular biology techniques were used in all cases. The details of the constructs varied slightly with each antigen but were similar in principle to that described in Example for Tc Ad ESA1.

The recombinant antigens were purified from the recombinant bacteria essentially as described in Example 9 and used in vaccination and challenge trials against T colubriformis as described for the native antigens in Example 2. Both sheep and guinea pigs have been used in these challenge experiments. All of the recombinant antigens have protected the vaccinated animals as measured by decreased worm burdens at slaughter and/or decreased egg output by vaccinated sheep as compared with non-vaccinated controls. Table 11 summarises the protection data obtained in these initial trials. Sheep were vaccinated intraperitoneally with the recombinant antigens in the presence of an immune stimulatory adjuvant. Two weeks after the second vaccination the sheep were challenged with T. colubriformis larvae. Post patency, faecal samples were taken each week to determine the number of eggs per g. Approximately 50 days after infection, the sheep were slaughtered, adult parasites recovered from the intestines and counted. protection is compared with non-vaccinated control animals (the decrease in worm counts were very similar to the decreased faecal egg counts).

35 It is to be appreciated that the degree of protection can be reasonably expected to be improved by such parameters as optimising the dose of antigen, the adjuvant used, the vaccination route, the conformation of 20998-AP/10.07.92 38a the antigen and/or by vaccinating animals with 1 combinations of the antigens.

TABLE 11 Recombinant TcAdESAl TcAdESA2 TcAdESA3 TcAdESA4 antigen Protection in 53 74 0 53 guinea pigs Protection in 23 46 35 23 sheep Example 12 Extension to other Parasites The Tc Ad ESA antigens produced by recombinant DNA technology are capable of inducing a protective immune response against T. colubriformis infestation in vaccinated animals. It is possible that this immune response may also provide protection against other species of parasitic nematodes such as those cited elsewhere in this specification, but it is more likely that the other species of parasitic nematodes express proteins which are related but not identical to the Tc Ad ESA antigens. For most species of parasitic nematodes, it is not practical to obtain sufficient parasite material to purify these components and identify their structure in preparation for cloning the gene from those parasites and testing the protective potential of the components. In these cases, 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 i nematodes. Even in the cases where it is possible to obtain sufficient parasite material to purify antigens, the above approach using molecular biology to clone genes i coding for protective antigens related to the Tc Ad ESA antigen genes is a preferable approach to developing 20998-AP/10.07.92

SS

S*

55 _i i- -I i His Tyr Ala Cys Asn Gly Tyr Tyr Asp Thr Lys Gly Gly His A

I

39vaccines. To demonstrate that this approach is feasible, the following example demonstrates that there are genes that are related to Tc Ad ESA1 and 4 in two species of parasitic nematodes other than T. colubriformis, namely Haemonchus contortus which is an abomasal parasite of sheep and goats in particular and Dirofilaria immitis which is a parasitise of dogs and cats. In addition, it is demonstrated that genes related to Tc Ad ESA2 are present in H. contortus, Ostertagia ostertagi, Ostertagia circumcincta and D. immitis.

Genomic DNA was purified from the various species of parasites by the method described by Herrmann and Frischauf 1987. l1g of each DNA preparation from T.

colubriformis, H. contortus, and D. immitis was digested with each of the restriction enzymes BamHI, PstI and HindIII (Promega). In a separate experiment, 8 pg of each DNA preparation from T. colubriformis, H. contortus, Ostertagia ostertagi, Ostertaqia circumcincta and D.

immitis was digested with the restriction enzyme HinfI (Promega). The digested DNA together with appropriate size markers was electrophoresed in 0.4% agarose gels (Fig 6) and 1.0% agarose gels (Fig 7) 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 ESA1, 2 and 4 were labeled with 32 p by the random 30 labeling method described in the kit sold by SBoehringer-Manheim. The filters were then incubated for hours at 42 0 C (Fig 6) and 16 hours at 55 0 C (Fig 7) in a solution of 5xSSPE, 5x Denhardts solution, 0.5% SDS and Ag/ml of denatured Herring sperm DNA (see Maniatis et 35 al 1982) containing 106 cpm/ml of the radioactive Tc Ad ESA DNA. The filters were then washed to remove any non-specifically bound DNA and exposed to X-ray film.

J Fig. 6 demonstrates that there are specific DNA sequences 20998-AP/10.07.92 *I 39a in both H. contortus and D. immitis DNA which hybridise to the Tc Ad ESA1 and 4 DNA fragments. Fig 7 demonstrates that there are specific DNA sequences in H.

contortus. Ostertagia ostertai. Ostertagia circumcincta and D. immitis which hybridise to the Tc Ad ESA2 DNA fragment. This demonstrates that these DNA sequences could be cloned from genomic DNA libraries or from cDNA libraries or prepared by other recombinant DNA techniques such as the polymerase chain reaction from these species of parasite and by extension from any other species of parasitic nematode and recombinant organisms could be constructed which synthesise these related genes for use in vaccines against the other species of parasitic nematodes.

20998-AP/10.07.92 C AT. 20998-AP/10.07.92 Exmple 3 Scale up of Manufacturing for Commercial Vaccines The production and purification techniques so far described are carried out at laboratory scale. For commercial production of the antigens of the invention, 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.

INDUSTRIAL APPLICATIONS 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.

Antibodies raised against the purified antigens isolated from Trichostronavlus 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 Trichostroncylus 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 iali or Ancvlostoma caninum infections of man, Stronqylus vulagris infections of horses, Trichostronavlus colubriformis infections of sheep, Haemonchus contortu infections of goats, Ostertaa D ostertaai infections of cattle, Ascaris suu or Trichinella sliralis infections of pigs, Toxascaris leoia or Uncinaria steocephala. infections of cats and Ancvlostoma i i 1 6 -ii: ir .4, hr.nt G LL J.jLl.Lt:2.a :Lasea on the sequences surrounding the EcoRI site) in the presence of 100 (M dNTP's, 16.6 mM ammonium sulphate, 67 mM Tris-HCl 20998-AP/10.07.92 SWO 90/03433 r PCT/AU89/00416 41 caninum or Trichuris vulpis infections of dogs as well as infections of the circulatory system of man by larvae of Toxocara Msy 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.

proline rich region (residues 133-169 deleted), using the 20998-AP/15.07.92 41a Deposition of Microoranisms E coli strain BTA 1691 was deposited with the American Type Culture Collection of 12301 Parklawn Drive, Rockville, MD, 20852, United States of America on 26 September 1989 under accession number ATCC 68098.

E coli strain BTA 1689 was deposited with the American Type Culture Collection of 12301 Parklawn Drive, Rockville, MD, 20852, United States of America on 26 September 1989 under accession number ATCC 68099.

E coli BTA 1690 was deposited with the American Type Culture Collection of 12301 Parklawn Drive, Rockville, MD, 20852, United States of America on 26 September 1989 under accession number ATCC 68100.

53S/SD ii I 51 20998-AP/10.07.92 -42

REFERENCES

Adams, D B and Cobon, G S (1984) in Reviews in Rural Science Vol. 6 (Leng et al eds) National Library of Australia pp 67-74 Adams, D B (1989). Int. J. Parasitol, 19, 169-175.

Connan, R M (1965) Parasitology 55, p Dineen, J K and Wagland B M (1982) In "Biology and Control of Endoparasites" (Eds. L E A Symons, A D Donald and J K Dineen), Academic Press, pp 297-323.

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7 209 98-AP/1 0.07.92

Claims (6)

1. An antigen comprising: an excretory/secretory protein, derived from a parasitic stage of a first parasitic nematode species and capable of inducing protective immunity against infection of a host by a second parasitic nematode species, which may be the same as or different from the first nematode species; or a protein molecule comprising all, part, an analogue, homologue, derivative or combination thereof of the excretory/secretory protein, which protein molecule is capable of conferring protective immunity on a host against infection by a parasitic nematode.

2. An antigen comprising an excretory/secretory protein according to claim 1, having an approximate molecular weight of 11, 17, 30, 37 or 81 kD as estimated by SDS-PAGE.

3. An antigen comprising an excretory/secretory protein according to Claim 1 or Claim 2 having an approximate molecular weight of 11 kD as estimated by SDS-PAGE. S 20 4. An a-tigen comprising an excretory/secretory protein according to Claim 1 or Claim 2 having an approximate molecular weight of 17kD as estimated by SDS-PAGE. An antigen comprising an excretory/secretory protein :according to Claim 1 or Claim 2 having an approximate 25 molecular weight of 30kD as estimated by SDS-PAGE.

6. An antigen comprising an excretory/secretory protein according to Claim 1 or Claim 2 having an approximate molecular weight of 37kD as estimated by SDS-PAGE.

7. An antigen comprising an excretory/secretory protein according to Claim 1 or Claim 2 having an approximate ¢e molecular weight of 81kD as estimated by SDS-PAGE.

20998-AP/10.07.92 8. An antigen according to one of claims 1 to 7 wherein the first nematode species is selected from the genera Trichinella, Ancylostoma, IStroncwlus, Trichostroniylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Stroncwloides, and Wuchereria. 9. An antigen according to claim 8 wherein the first parasitic nematode species is selected from Trichinella s-piralis, Ancylostoma caninum, Stronqvlus vulcraris, Trichostrongrylus colubriformis, Haemonchus contortus, Ostertagia ostertagi, Ascaris suum, Toxascaris leonina, Uncinaria stenocelphala, Trichuris vullpis, Dirofilaria immitis, Toxocara spp, Necator ainericanus, Ancylostoma duodenale, Ascaris lunibricoides, Trichuris trichiura, Enterobius vermicularus, Stronayloides stercoralis and Wuchereria bancrofti. An antigen according to any one of claims 1 to 9 wherein the second nematode species is selected from the genera Trichinella, Ancylostoma, Stronciylus, Trichostronqylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Stroncryloides, and Wuchereria. 11. An antigen according to claim 10 wherein the second parasitic nematode species is selected f rom Trichinella sipiralis, Ancylostoma caninum Stroncrylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostertagfia ostertacri, Ascaris suum, Toxascaris leonina, Unciariastenocephala, Trichuris vulpois, Dirofilaria immitis, Toxocara species, Necator americanus, Ancylostoma duodenale, Ascaris liubricoides, Trichuris trichiura, Enterobius vermicularus, Stroncryloides stercoralis and Wuchereria bancrofti. 201-98-API1 5.07.92 46 12. An antigen according to any one of claims 1 to 11 wherein the first parasitic nematode species is Trichostrongrylus colubriformis. 13. An antigen according to any one of claims 1 to 12 wherein the second nematode species is Trichostroncrylus colubriformis. 14. An antigen according to claim 1 or 2 comprising the amino acid sequence: Al a Lys Tyr Gin Ile Met Arg Tyr Val Asn Tyr Lys Giu Arg Giu Ala Ala Leu Asn Asn Leu Ala Arg Giu Asp Cys Thr Lys Ser Ile Lys Arg Lys Lys Asn Val Gin Thr Trp Giu Liys Val Phe Gly Ala Gin Phe Asp Gin Val Giu Leu Tyr Cys Thr Ala Asp Tyr Phe Gly Arg Tyr Leu Asp Lys Ser Ser Ile Ala Arg Asp Tyr Ile Met Met Thr Lys Leu Leu Thr Arg Giu As n Vai Asn Gly Lys Leu Lys Glu Gin Gly Ser Ala Asp Tyr Trp Gly Ile Leu Asn Asp Thr Asn Pro Phe Gly Asp Met Tyr Ala Phe Aia Vai Thr His Tyr Pro Ser Leu Lys Thr Leu His Asp Lys Asn Ser His Tyr An antigen according to any 20 comprising the amino acid sequence: one of claim 1 or Met Set- Sin His Ala Lau Sin Siu Ile Glu Lys Pro Sly Lys Phe Set- Gin Lys Asp Set- Ala Tyr Phe Lys Lau Giu Asn Lys Ar-a Glu Laeu Lys Sly Asp Asn Lau Pro Val Giu Glu Lys Val Ara Sin Thr Ile Siu Lys Phe Lys Asp Asp Val Set- Su Ile Arc -Arg Laeu Ala Asp Asp Set- Asp Phe Sly Cys Asn Sly Lys Slu Thr Slu Sly Ala Met Hi s Ilie Val Cys Phe Phe Sin Lys Asn Tyr Asp Trp Met Lys Sly Sin Trp Sin Asn /NO 20998-AP/110.07.92 X 1, 47 16. An antigen according to claim 1 or 2 amino acid sequence: Arg Phe Leu Leu Leu Ala Ala Phe Val Ala Ty Lys Ser Asp Glu Glu Ile Arg Lys Asp Ala Lei Asp Val Val Pro Leu Gly Ser Thr Pro Glu Ly Gly or Lys Ser Asp Glu Glu Ile Arg Lys As] Ala Leu Asp Val Val Pro Leu Gly Ser Thr Pr Glu Asn Giy. comprising the 01 Ala Ser Leu Ala Glu Tyr Ala Ala Leu Glu Asn Leu Ser Lys Leu 17. An antigen according to claim1 comprising the amino acid sequence: Me or claim Leu Ilie LeU LaLL Ala Ile LeUt Val Sly Thr Vali Pr-o Ser GILL Ser Ser Leu Val Aen 1C) 152) Ser Asp Tyr Arg Val Hi s Asn Asp His Cys Lys Tyr Ser GILL Val Lys Sin Gin Pro Phe Lys GILL Ile Ala Aen Ser Ser Leu Arg Ser Phe Leu 45 Leu Ar-g Lye LSLL Arg Sly lie Sly Asp Thr Asp Cys Val 554 Sin SE r Tyr 4 0 Sin Vaa GILL Phe Aen Asn Asp 8cr gsn Fro Fhe Tyr Val Fhe r Ile 75 SO Asp Arg SILL Thr Ai-g Phe Ser Arg Aen Tyr Thr Val Cys Sly Val Val 9C0 9510C Thr Val Val Sly Sly Asp Phe Met Trp Lys Ser Cys Asp Lys Ser Lye 105 110 115 Phe Lye Asp Tyr Leu Leu Lye Cye Slu Ser Slut Siu Aen Ar-a His Pro 12C0 125 13Z"0 Sin Leu Pro Pro Val Leut Ser Cys Asp Arg Thr Pro Aen Pro Val Ser 135 140. 145 Pro Val Ser Pro Pro Asn GILL Asp Ala Pro Pro Thr Leu Pro Pro Arg 150 155 160 Ser Asp Ser Leu Aen Lye Val Thr Pro Pro Aen Pro Pro Ile Lye Asp 165 170: 175 180 Thr Pro His Thr Pro Pro Pro Arg Asp Phe Thr Thr Ile Pro Pro Arg 185 19o 195 Ala Val Ala Aen SILL Lye Ser Thr Thr Lye Lye Sly Phe Leu 6cr Lye 200 205 210 Leu Asn Cye Phe Thr Cys Phe 215 or amino acids 13 to 219 inclusive of said amino acid sequence. 2.0998-AP/1 5.07.92 re ~rc 48 18. Tc Ad ESA1 as hereinbefore defined, or non-glycosylated form. 19. Tc Ad ESA2 as hereinbefore defined, or non-glycosylated form. 20. Tc AD ESA3 as hereinbefore defined. 21. Tc Ad ESA4 as hereinbefore defined. 22. Tc Ad ESA5 as hereinbefore defined, or non-glycosylated form. in glycosylated in glycosylated in glycosylated 23. A first nucleotide sequence encoding the amino acid sequence of an antigen according to any one of claims 1 to 22, a nucleotide sequence which hybridizes to the first nucleotide sequence, or a nucleotide sequence related by mutation including single or multiple base substitutions, insertions or deletions, to the first nucleotide sequence. 24. A nucleotide sequence according to claim 23 wherein the nucleotide sequence encodes the amino acid sequence of an antigen according to any one of claims 2 to 7 or any one of claims 14 to 22. 25. A nucleotide sequence according to claim 23 or claim 24 wherein the nucleotide sequence is a DNA sequence. 20998-AP/15.07.92 .,iL L. i I. 49 26. GAA ACC GCG GAC TAC A DNA sequence according to claim 25 TTC GGG GGC AAC ACT TAC AGT GCA AAC GAC ATA OAA CAA CTC ATO CCC AAA TAT AAG ATG AAT GGA AAC TAT AOT TAT AAG AOC ATO GTA TCT OAT GCG CTO CAA GAA AGT ACO AAT GCT ACC TTC AAG ATC CGT TTC ATA AAG GGC GAT AAC OCA ACG ATO OGA CGC TAC CTC GCA GCA AAA ATO TAT OCG TTC GCA GTC ACA CAC GCT COT OAT TAC OTC OTT or CCC AOT CTG AAG ACG TTO CAC GAT CTG AAO TAC CTT CTC TG ACG A~AA GOT AGA GAG ATC AAC AAA AAC CCC TTC GGA GAT AGC OTC TTG AGA ACA CAC TAC CAC OAT GTC TAC AAA AAT AGA OTA AAC TTT OCA AAA TAT CAA ATC ATG AGA TAC GTC OAA TAA AAC AAT TAT AAC AAG CTO GAA GCA COT CG GAG GAA GCC GAT OCA TGC CTG ACT ATA CTT GAT GCA AAG CAA CAG ACC TCG ACG TTC GCO ATC TGG OAT GAC AAG GAG CAA TAC AGA AAG OTO TTC AAA OTO GAG OGA AAA TTT CTT COC AAT OGA TAT TAC OTC OCO TOT CTC GAO 0CC GCA CTG TCT TOA ACT GAC AAG AGC AOT ATA OCT COT OAT TAC comprisingt AAT AAG CAA AAC TCO ACO CTO ATC TOO GCA AAG GAO COG AGA AAO OAA AAA GTO OAT AAA TTT TOC AAT OGA ACT OTC GCG CAC TAT TAO CTO CAC ATT CAA AAA AAA ATA GAA CAA ATO AAT OGA ATO OTA TCT ACO AAT OCT AAO GOC OAT CTO AAO TAC CTT CTC TOO ACO AAA OOT AGA GAO ATC CAG TTC OAT CAA OTO GAO CTT TAT TOT APA TCC AAA CTC AAC OAT ACC AAC CCC TTC OGA OAT TAC AAA AAT TCT CAC TAT. 20998-AP/1 5.07.92 J V 50 27. A DNA sequence according to claim 25 comprising: TACAAGACCC CCAATTSTAC ACGAAATTCT TCAACGAAGA AAACAGCCTM- AATCTGAGAI GGAACCCACA T ATG TCS CAG CAT GCT CTA CAA SAA ATT S MAS CCA GGS. AAA TTT TCS CAA AAA SAT TCA SCA TAT TTC AAG CTC GAA AAC AAS AGS GAA CTS AAG GGA SAC AAT CTA CCA GTG SAG GAG AAA GTA CSC CAA ACT ATT GAA AAA TTC AAG EAT SAT ETA AGC SAA ATC AGA CST CTC GCT SAT E AT ATT CAA TOG SAT TT T GSA TSC AAC EEC AAA GAA ACC GAG GST GOA ATG CAC GE TGT TTC TMTC CAG AAG AAT TAT SAC TGG ATE AAA GSA CAA TSG AAC TGATTT -iTCT GAAGTAC -ai TTSSATTCTT1 CSTAC-AiTCG ATGCACAAAA TACCTTT or AAA TrT SAA CTG ATT -APA EAT TOG TTT SSGASACAAC TTCSCA-LAAA.ACTTCTCOAT SAAAAAAAAA AAAAA, ATe TCG CAS CAT GCT CTA CAA SAA ATT SAG AAS CCA SGa TOG CAA AAA SAT TCA SCA TAT TT C AAS CTC SAA AAC AAG AGE AAS SEA SAC AAT CTA CCA STS SAG SAG AAA STA CEC CAA ACT AAA TTC AAS SAT SAT ETA ASC SAA ATC AGA CST CTC GOT SAT SAT TTT GSA TSC AAC SSC AAA GAA ACC SAG 557 SCA ATG CAC 757 TTC TTC CAS AAS AAT TAT SAC TGG ATE AAA GSA CAA TGS ATT ST a CAA AAC 20998-AP/1 5.07.92 ij I 28. CGG AAG GAT GGC GCT GAA 29. 51 A DNA sequence according to claim 25 comprising: TTC CTT CTT CTA GCA GCG TTC GTC GCC TAT GCG TAT GCA TCA GAT GAA GAA ATC CGA AAA GAT GCA CTA TCT GCT CTG GTA GTT CCA CTG GGT TCG ACT CCC GAA AAA CTG GAA AAT or AAG TCA GAT GAA GAA ATC CGA APIA GAT GCA CTA TCT CTG GAT GTA GTT CCA CTG GGT TCG ACT CCC GAA AAA CTG AAT GGC. A DNA sequence according to claim 25 comprising: OCT TCC GA TCG TCG CTC GTA AAC AST SAC TAT ASS GTT CAC AAT SAC S S 5* S S. S S S. 9 SS S. CAG TTG CAAC ACT TTC CAA1 CCG TCC ACT SCA CAA1 AGA GTG CST GTS AAA CTS GTC GAT CCG GTT CCG GAS GAG GTT SAC CCA AGT TCC CAC GCC TTT CTG TTC ACA GGT TAT CCT CCT CTC ACA AAT AAA AGA AiAC AGAl GGC CTT GTT CC AIAT CCT GAG AA GGG AAC TTT SAC CTA CTG AAT AAG CCA AAA ATC ATT SAC TCA TTC AAG TCA SAG GTC CC TCC 6CC GGG ABC C66 ATG TST TGC SAC ACT CGS ACC CAC TGT AAC TC6 GAT ACT AAT CC AAC TAT TGG AAG GAG ABC GAC CGG GCT CC CCT CCC GAT TTC ACT AAA AAA TCA SAC TTT ACC TCA GAA ACC CCC AAT ACT AAA TAC CTA TSC TAT STC TST SAG CCT ACC CCT ACT G6G AST. BAG CGA TCA GTG CAB 6TC TTT TGC GGT GAT AAA AAC AGA AAT CCT CTC CCT CCC ATT ATC CCT TTC CTA GTT rTC TCT CGC GTG TC CAT GTC CCG AAA CCC ABC AAG CTT TAC CTC GTT AAA CCA TC ASS SAC CGAI AASG CTC 20998-AP/1 5.07.92 1, L AAT TGT TTC ACT TST TTT, OI i i) -52- CCTGGTT6TT CCGCACTTTC ACTCGGCGCA GCTCTTCGAC GFATG ATG CTG ATC CTT CTG GCC ATT TTG GTC GEC ACC GTE CCT TCC BGE TCG TC6 CTC ETiA AC ASET SA~C TAT ASSG6TT CAC AAT SAC CAC TET AA TAC AGTGA G~ TT AA6 CAG CAA CCG TTT AA GAA ATC GCC AAC TCG TCA CTA C6Gt TCA TTC CTT TT6 AG AA CTG AG 666 tATT 666 GAiT ACT GiAC TEC 6T6 CAG TCT TAC CAAg GTE GAG TTC AC AAC SAC AGC AAT CC6 TTT TAT ETC TTT CGC ATC SA~C CST GAG6 AC AG TTT TCPA CGG AAC TAT ACC ETC TGC GET GTE 6TT ACT 6TG GTT GET EEC GAtC TTC ATG TEG AP t E TCA TET GAT AAA~ TC6 AAA TTC AA SAC TAT CTT CTA AAG T6T GAG AGC SAA GAG AiC AG CAT CCA CAA CTE CCA CCT ETT CTG TCA TEC SAC CEE ACC CCT AAT CCT ETC TCG CC6 ETC AST CCT CCE AAT GAG SAC ECT CC6 CCC ACC CTC CCT CCE ASS *s *TCC GAT TCC CTC AAT AAS ETC A~CT CCT CCC AAT CCT CCC ATT AA SAC C @6*ACT CCE CAC AC CCT CCA CC6 GE GAT TTC ACT ACT ATC CCT CCC CGA 6CA GTT 6CC AAT GAG AA TCC ACC ACT AA AAA 666 TTC CTA AGC AA6 CCTC VVAT TET TTC ACT TET TTT TGATAACATT ETGCTG6CAC CAATTGAA T CCTGTTACAT TATTGAGAAT AAA6GTTT6C ATG, or 20998-AP/1 5.07.92 A. 53 ATC- CTG ATC CTT TCG CTC GTA AAC CTG GCC ATT TTS GTC GGC ACC GTS CCT TCC GAG TCG AST CAB TTG CAA SAC ACT TTC CAA cCs TCC ACT BAC CA GTG CST AAA CTS GTC GAT CCG TAT CCG AAA GAG GAG GTT SAC CCA AST TCC CAC ASS TTT CTG AiCA SOT TAT CCT CCTi CTC ACA GTT AAA AGA AAC AGA SOC CTT GTT CCG AAT CCT CAC GAA G06 AAC TTT SAC CTA CTG AAT CCA AAr AAT G ATC ATT SAC TCA TTC AAG TCA GAG STC CCG TCC -TTT 3AC GCC SGG CG ATG TOT TGC SAC ACT CBS CAC AAC GAT AAT AAC TG GAG SAC OCT *CCT G AT i-GT TCO ACT CCG TAT AAiS AGC COOz CCG CCC TTC AAA TCA SAC TTT ACC TCA OAA ACC CCC AAT ACT i-AC CTA TSC TAT GTC TOT GSC CCT ACC CCT ACT AST. SAG CGA TCA GTS CAS GTC TTT TOC SOT SAT AAA AAC ASGA AAT CCT CTC CCT CCC ATT ATC CCT GTT TTC TCT COC GTO TCG CAT GTC CCG AAA CCC AAiS CTT TAC ATC OTT AAA CCA AGO SAC ACC ACT AAA AAA 550 TTC CTA ABC AAiS 20998-AP/1 5.07.92 *J I -54 A process for selecting a DNA or RNA sequence coding for an antigen according to any one of claims 1 to 22 which process comprises providing one or more DNA or RNA sequences, determining which of the sequence hybridizes with a DNA or RNA sequence known to code for an antigen according to any one of claims 1 to 22 or providing an anti-serum to the antigen and indentifying host-vector combinations that express the antigen. 31. A recombinant DNA molecule comprising a DNA sequence according to any one of claims 25 to 29 and vector DNA. 32. A recombinant DNA molecule according to claim 31 additionally comprising an expression control sequence operatively linked to the DNA sequence. 33. A recombinant DNA molecule according to claim 32 wherein the expression control sequence is selected from: the S-galactosidase gene of E. coli, the tryptophan operon, the Tra-T gene of E. coli, the leftward promoter of bacteriophage lambda, the tac promoter, the Cup 1 promoter and the SV40 early promoter. 20 34. A recombinant DNA molecule according to any one of claims 31 to 33 wherein the vector DNA is plasmid DNA. 35. A recombinant DNA molecule according to claim 34 wherein the plasmid DNA is selected from: pUR290, pUC18, pYEUC114 and derivatives thereof. 25 36. A recombinant DNA molecule according to any one of claims 31 to 33 wherein the vector DNA is bacteriophage DNA. 37. A recombinant DNA molecule according to claim 36 wherein the bacteriophage DNA is selected from: bacteriophage lambda and derivatives thereof, including lambda gtlO and lambda gtll. S20998-AP/15.07.92 L 38. pYEUC30B4E as hereinbefore defined. 39. A fused gene comprising a promoter, a translation start signal and a DNA sequence according to any one of claims 25 to 29. 40. A process for the preparation of a recombinant DNA molecule according to any one of claims 31 to 37 which process comprises providing a DNA insert comprising a DNA sequence according to any one of claims 25 to 29 and introducing the DNA insert into vector DNA. 41. A process according to claim 40 wherein the DNA insert is introduced into the cloning vector in correct spacing and correct reading frame with respect to an expression control sequence. 42. A transformed host, transformed with at least one recombinant DNA molecule according to any one of claims 31 to 38. 43. A transformed host according to claim 42 wherein the host is capable of expressing an antigen according to any one of claims 1 to 22. 44. A transformed host according to claim 42 or 43 Saccharomyces cerevisiae strain CL13-ABSY86, other fungus, vertebrate cell, insect cell, plant cell, human cell, human tissue cell, live virus or a whole eukaryotic 25 organism. e• A transformed host according to claim 44 wherein the "'live virus is vaccinia or baculovirus. ee *46. A transformed host according to claim 42 or 43 S wherein the host is E. coli, an enteric organism other /1 30: than E. coli, a Pseudomonas or Bacillus species. 20998-AP/1 5.07.92 1, i 56 47. A transformed host according to claim 46 wherein the host is an E. coli K12 derivative selected from JM109 and Y1090. 48. BTA 1689 as hereinbefore defined. 49. BTA 1691 as hereinbefore defined. BTA 1690 as hereinbefore defined. 51. A process for transforming a host to provide a transformed host according to any one of claims 42 to which process comprises providing a host, making the host competent for transformation, and introducing into the host a recombinant DNA molecule according to any one of claims 31 to 38. 52. An expression product of a transformed host according to any one of claims 42 to 50 which product comprises an antigen according to any one of claims 1 to 22. 53. An expression product according to claim 52 in substantially purified form. SJ." 54. An expression product according to claim 52 or 20 claim 53 comprising a first polypeptide sequence homologous to the host and a second polypeptide sequence which is an amino acid sequence coding for an antigen according to any one of claims 1 to 22. 55. An expression product according to claim 54 wherein the first polypeptide sequence is all or part of E-galactosidase or Tra-T and the host is E. coli. 56. A process for the biosynthesis of a proteinaceous IQ^ product comprising an antigen according to any one of claims 1 to 22 which process comprises: transforming a 20998-AP/15.07.92 -57 host with a recombinant DNA molecule according to any one of claims 31 to 38 so that the host is capable of expressing a proteinaceous product which includes an antigen according to any one of claims 1 to 22; culturing the host to obtain expression; and collecting the proteinaceous product. 57. An epitope of an antigen according to any one of claims 1 to 22 which epitope is responsible for the protective immune response. 58. An antibody raised against an epitope according to claim 57. 59. An anti-idiotype antibody raised against the variable region of an antibody according to claim 58. A vaccine comprising an effective amount of one or more: antigens according to any one of claims 1 to 22; expression products according to any one of claims 52 to 55; epitopes according to claim 57; and/or anti-idiotype antibodies according to claim 59, together with a pharmaceutically acceptable carrier, diluent, 20 excipient and/or adjuvant. 61. A vaccine according to claim 60 suitable for oral administration or in injectable form. 62. A vaccine comprising an effective amount of an antigen selected from: Tc Ad ESA1, Tc Ad ESA2, Tc Ad 25 ESA3, Tc Ad ESA4, or Tc Ad ESA5 as hereinbefore defined, i or combination of all or some of these antigens together with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. 63. An antibody prepared as a result of vaccination of a host by administration of one or more antigens according to any one of claims 1 to 22, expression products 20998-AP/15.07.92 1; 58 according to any one of claims 52 to 55, epitopes according to claim 57, anti-idiotype antibodies according to claim 59 and/or vaccines according to any one of claims 60 to 62 to the host. 64. An antibody composition comprising an effective amount of at least one antibody according to claims 58 or 63 together with a pharmaceutically acceptable carrier, diluent and/or excipient. A process for the preparation of an antigen according to any one of claims 1 to 22 which process comprises: collecting excretory/secretory fluids from a parasitic stage of a parasitic nematode species; fractionating the fluids by lentil lectin chromatography with methyl mannoside as eluent; collecting the bound and unbound fractions; further fractionating by SDS-gel electrophoresis; and electroeluting the antigen. 66. A process for the preparation of a fused gene according to claim 39 which process comprises providing a promoter, a translation start signal and a DNA sequence 20 according to any one of claims 25 to 29 and operatively linking the promoter, translation start signal and DNA sequence. 67. A process for the preparation of a vaccine according to any one of claims 60 to 62 which process comprises admixing an effective amount of at least one antigen according to any one of claims 1 to 22 and/or expression products according to any one of claims 52 to 54, and/or epitopes according to claim 57, and/or anti-idiotype antibodies according to claim 59, with a pharmaceutically 30 acceptable carrier, diluent, excipient and/or adjuvant. 68. A process for the preparation of an antibody according to claim 58 or 63 which process comprises: immunising an immunoresponsive host with an effective 20998-AP/15.07.92 I ~1 _ii-ILili--~. .i i 59 amount of antigen according to any one of claims 1 to 22, an expression product according to any one of claims 52 to 55, an epitope according to claim 57, an anti-idiotype antibody according to claim 59 or a vaccine according to any one of claims 60 to 62. 69. A process for the preparation of an anti-idiotype antibody according to claim 59 which process comprises immunizing an immunoresponsive host with an anti-epitope antibody according to claim 58 or antibody composition according to claim 64. A process for the preparation of an antibody composition according to claim 64 which process comprises admixing an effective amount of at least one antibody according to claim 58 or 63 with a pharmaceutically acceptable carrier, diluent and/or excipient. 71. A method of protecting a host in need of such treatment from infection by a parasitic nematode which method comprises vaccinating the host with at least one antigen according to any one of claims 1 to 22, 20 expression product according to any one of claims 52 to 55, epitope according to claim 57, anti-idiotype antibodies according to claim 59, and/or vaccines according to any one of claims 60 to 62. 72. A method of passively protecting a host in need of such treatment against infection by a parasitic nematode which method comprises passively vaccinating the host with at least one antibody according to claim 58 or 63 and/or antibody composition according to claim 64. DATED this 28th day of July 1992 BIOTECHNOLOGY AUSTRALIA PTY LTD and COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By their Patent Attorneys S"GRIFFITH HACK CO 0998-AP/1 5.07.92 I i; WO 90/03433 PCT/AU89/0041 6 1/-l MW 93 m 67.~ 14 L L+ L L -U FIGURE 1 A WO90033 WO 9003433PC'I/AU89/00416 1~ 2/7 1c Ad E SA FIGURE 2 7 Mi'O 90/03433 PCT/AU89/00416 31/7 FIGURE 3 Fi 4 c1 -oa N-emiu 0oLne eune TGT GG.GC.ATGAGCACAGCA.TC.AG.AG.CG.AT.TG.AG.TC.GT.CAATTCGGG Cys ly la Mt Sr Tr Al Il Ly LysGinsn er Sr Sr Vl Gi Ph Gl F ~trctue fTrTTcdSAAuso IGTGG.C.ATG.AGAC.GCA.AC.AAAG.AA.AGATTGAGTGGACA1CGG CGly Ala MerT Ser Thr Ala lie Lys Lys Gin e e e a i h l I I CTC AAT TGT TTC ACT TGT TTT, 20998-API1 5.07.92 IvI.4WO 90/03433 PCT/AU89/00416 5/-7 (S) copt -11 1, I-' N IC) I 0C Probe TcAdESA1 Probe TcAdESA4 U. ~4 t Z: 1~. WL w 1' -s Bill? HBP Bill? 11c Tc Dji BIIP JIBI? BlIP 1ic Tc Di B Bani III 11 Hlind III P Pst I l1c laemonchus contortus Tc Trichostrongylus colubriforns Di Dirofilaria inuitis FIGURE 6 00J E S4 20998-AP/1 5.07.92 A p U 4400 2350- 2020 1400- 1353- 650 OEM9 a a a a a *aa. B a a a a. a a a a a a a. M HcTc Oo OcDi M: Positions of DNA size markers (sizes are indicated to the left in numbec of bases) Ho: Haemonchus contortus Tc: Trichostrongylus colubriformis Qo: Ostertagia ostdrtagi. OC:, Ostertagia circurncincta Di: Dirofilari~a irwuitis .L ZaA 20998-AP/1 5.07.92 f I I Y b IN'IgRNATIONAL SEARCH REPOWP International Application No. [Cr/AU 89/00416 I. CLASSMFCATIctN OF SUB=~ MAT (if several classification symboLs apply, indicate all) 6 According to InternationaL Patent Classification or to both National Classification and IPC Int. Cl. CJ.2N 15/00, 1/16, 1/20; C07K 13/00, 7/10, 3/28, 15/08, 15/12; C12P 21/02; C07H 21/04; C12Q 1/68; A61K 39/00, 37/02 CM2 1/20: CM2 1/19; C12N 1/16: C12R 1/865I nI. FIMD &FAP I Minimum Documentation Searched 7 Classification system IClassification Symbols IPC 4 I Keywords: mematode or Trichostrngylus or CAS 82 I Haaxxxichu and antigen or allergen Documentation Searched other than Minimum Documentation to the Extent that such Documents are IncLuded in the Fields Searched 8 AUl: C07K 13/00, 7/10, 15/08, 15/12, 15/14; C07G 7/00; C07C 103/52; CM2 15/00; I Genbank Data base; NWR nucleic acid1 data base; RM Data base; III. DOCEROM =nMW~ M1 BE RE"ANT 9 Category* Citation of Document, with indication, where appropriate, IReLevant to of the reLevant passages 12 CLaim No 13 X'Y I3=1.139(July 1987) MaizeIs R.M. et al., 'Shared carbohydrate I (49,50,55,6011 disoesona tinct surface and secreted antigens of the parasitic 1 (1,3-5,51-53,56, I I I rwnatode Taxocaris canis see pages 207-214 I61-64)I I X,Y Molecular Paradigns for Eradicating Hebsinthic Parasites. LIUA I(49,50,55,60) I Isymposia vol.60 (McImis A.J. A.R. Liss publicationi, New (1,3-6,51-53,56 I IYork (pubi.)) (1987) Maizels R.M. et al., "Glycoconjugate I57,61-64) I I ~antigens f rom paraitic nematodes" I I I IM I pca caeore ociedouet:1 T Lardcmntpb iseafrth Secrialctgresrie documents 10 ubihd no lt document piuLshred ae the afe h international filing date divnonanor pricorsitd te n A document defininga thgr al states of irte ocannot in conitrewit th e aiain bu cIis art which i osidrdt eo cited to unestabnds the principle ortheoyp puE' e arlier docue buat bhed cton or 'Y document of particuLar reLevance; theI I other special reason (as specified) cLaimed invention cannot be considered to S 0' document referring to an oraL discLosure, invoLve an inventive step when the document use, exhibition or other means is combined with one or more other such P document published prior to the documents, such combination being obvious to I International filing date but Later than a person skilled in the art. I the priority date claimed *V document member of the same patent family IV. CIEICATIN I Date of the Actual CompLetion of the IInternational Search 13 Januar 1990 (13.01,90) Date of MaiLing of this InternationaL Search Report i International searching Authority hnstralim Patent Office Form PCT/ISA/210 (second sheet) (Jay I Signature of Autt IJ. W.ASHMANA C.Laims L ro 22 wnicni process comprises: transforming a 20998-AP/1 5.07.92 JI 4 InternationaL AppLic...ion No. PM/hUf 89/00j416 Ml. D3MMS CLWSM TO BE R&ANT (QtMTIU M 7M* SIM MET) Category* Citation of Document, with indication, where appropriate, ReLevant to of the reLevant passages ICL*1* No AJ,A, 76729/87 (ALLU.JX IWC) 18 February 1988 (18.02.88) Proc. Nat. Acad. Sci. (USA) 85(10) (1988), Nilsen et al., 'Cladng and characterization of a potentially protective antigen in lyuiiatic flariasis", pages 3604-3607 IMUMzO1 Cell Biol. 65 (1987), Monroy F.G. and Dobson "Mice vaccinated against Neatospiroides dubius wi.th antigens isolated by affinity chrcsItogra~iy froim adult vom", pages 223-230 Int. J. Parasitol. 15 (1985), O'Don~nell I.J. et al., "Attempts to probe the antigens and protective imziogens of Trichostromgylus; colubrifois An inmzznoblots; with sera from infected an hyperixz sheep and h~-and 1cow-responder guinea pigs".* pages 129-136 AU,A, 1999i/88 (Biotecbrm1ogy Auistralia Pty Ltd and Commonealth Scientific and Ixbitrial Research Organization).. 12 Jarnwary 1989 (12.01.89) AIJ-B-36095/84 (Patent 582129), (T1he Washingtonl University) 6 Junie 1985 (06.06.85) Curr. Topics in nmaznl. 120 (1985), Almon~dN.M. and Paddose "Nemtode Antigensm pages 173-203 (1;3-6,49-64) (1,3-6,17-19,23-30 32-39,40-56,58-64) 51-53, 56,61-64) (1-8,51-53,56 61-64) (1,3-6,17-19, 23-30,31-39, 43-53,55-64) 1,3-6,49-53,55 56,60,61-64 1,3-6,51-53,56 61-64 Form PCT/ISA/910 (extra sheet) (January 1985) 20998-AP/1 5.07.92 3 I ~4 I ANNEX TO MhE INTEHNATONAL SEARCH REPOR~T ON D1UERNATIONAL APPLICATION NO. r AU 89/00416 This Annex Ijsts the known publication level patent family nmnbers relating to the patent documents cited in the above-uentioned international search report. The Australian Patent Office is in no way liable for these particulars which are mrerely given for the purpose of information. Patent Document Cited in Search Patent Family Members Report AU 76729/87 JP 63057599 US 4842999 AU 36095/84 AU 582129 CA 1232849 US 4839275 AU 19998/88 WO 8900163 EP 330681 END OF ANNEX OIL-