NO327859B1 - Babesia vaccine as well as its preparation and substance for use in a vaccine and antibody. - Google Patents

Description

The present invention relates to a Babesia vaccine as well as a method for its production and a substance for use in a vaccine and antibody.

The parasite Babesia divergens causes serious economic losses among cattle. The genus Babesia to which Babesia divergens belongs also includes the species B. bovis, B. canis and B. bigemina. In Europe, Babesia divergens is the most pathogenic Babes/a species affecting cattle (Kuttler, K.L., in M. Ristic (ed.), Babesiosis of domestic animals and man. CRC Press, Inc., Boca Raton, Fla. 1988) . The parasite is transmitted by the three-host tick, Ixodes ricinus. Babesia divergens causes anemia in cattle which in severe cases leads to death. The disease is also called "blood piss" since in an advanced state it causes bloody urine. The problem of bovine babesiosis has been a long-standing problem: vaccination against bovine babesiosis in Sweden, where blood from cattle that had had acute babesiosis the previous summer, was already recommended by local veterinary authorities in 1920. However, despite all efforts, there is still no safe and effective commercially available vaccine, although babesiosis in Sweden alone costs farmers more than 875,000 dollars each year. It has been shown that babesiosis in cattle can be controlled by vaccination with live attenuated vaccines. However, the live vaccines that are currently available have the disadvantage that their use requires supervision by a veterinarian. This is due to their variable infectivity and morbidity. In animals in poor health, an attenuated live vaccine causes a virulent infection leading to disease. However, it is known that vaccination with live vaccines is not necessary. It has been shown that the so-called SPA preparations (Soluble Parasite Antigen) are capable of inducing an immune response which, although it does not necessarily affect the parasite, sufficiently reduces the degree of clinical manifestations after infection. (Rev.: Sonetters et al., in Parasitology today 11:456-462 (1995)).

Valentine et al.; Infection and Immunity, vol. 63, No. 3, 1995, pp. 811-817 describes vaccination experiments with Babesia divergens exoantigens. Sera from vaccinated animals are used in immunoprecipitation analyzes of parasitic proteins.

EP A1 0417524 describes the antigen 12D3 from Babesia bovis and B. bigemina which was isolated from a sonicate of infected erythrocytes.

A further analysis of the different components found in SPA has revealed several proteins that possibly play a role in inducing protection against infection, or at least against the clinical manifestations of the infection. When the SPA proteins were separated into four separate groups F1-F4, on the basis of their molecular weight, they surprisingly found that all four groups contained a compound that provided at least some degree of protection against infection. (Précigout et al., Experimental Parasitology 77:425-434 (1993)).

Of the proteins found in the F4 fraction, a specific Babesia divergens protein, the 17 kD Merozoite membrane protein, has already been studied for several years with a view to application in vaccines. For this protein, it has been strongly argued that it plays a very important role, if not the main role, in inducing immunity against Babesia. This has been based, among other things, on on the finding that monoclonal antibodies against the 17 kD protein were induced after live infection and in vitro could drastically inhibit the growth of the parasite (Précigout et al., (1993), Exp. Parasitol., 77(4):425-34).

The other two proteins, which occur in the same fraction as the one comprising the 17 kD protein, i.e. the F4 fraction, and which are also recognized by antiserum against Babesia divergens, are a 50 kD protein and a 37 kD protein (Carcy et al. , Infect. and Immun. 63:811-817 (1995)). The detection of both the 50 kD and 37 kD protein was based on the development of antibodies against these proteins in vivo. However, this is the case with many proteins of the SPA. Virtually all proteins are capable of generating antibodies, but only one or a smaller number of proteins play a role in the induction of neutralizing antibodies.

Only antibodies against the 17 kD protein were known to have a protective effect. One should therefore expect that this 17 kD protein was the protein of the F4 group that was responsible for the induction of immunity. The possible role of other F4 proteins, e.g. The 37 kD and 50 kD protein has therefore always been unclear.

It is an object of the present invention to provide a vaccine that induces protection against infection of cattle with B. divergens or clinical manifestations of the infection, characterized in that it comprises two or more different purified proteins of the 37 kD protein family, and a pharmaceutically acceptable carrier.

The surprising discovery has been made here that the 37 kD protein can be found in at least two different variant forms: a 37 kD form and a slightly shorter form, a 35 kD form. Other slightly smaller or larger forms have also been seen. The shape depends on the Babesia divergens strain from which the gene was isolated. It may therefore be correct to speak of a 37 kD protein family rather than just a 37 kD protein. The reason for the difference in size of the various members lies in the fact that certain areas of the protein can obviously be deleted without drastically changing the protein's function (see below). Many different Babesia divergens strains that really show such variations in the molecular weight of the 37 kD protein have so far been isolated. It will be clear that the molecular weight of the proteins is determined on a PAGE gel in relation to marker proteins. It is therefore correct to understand 37 kD as 37 kD (+/-3 kD) and 35 kD as 35 kD (+/- 3 kD).

For the determination of the level of homology between two members of the 37 kD protein family, it is clear that a DNA fragment present in one protein and deleted in the other contributes to decrease the level of homology even in the case where all still occurring nucleic acids would be 100% homologous. The following may serve as an example: if the genes encoding a 37 kD protein and a 35 kD protein are compared, there is a non-homology of 5% which is only due to the deletion in the shorter of the two genes. To compensate for the differences in homology due to differences in the size of the proteins of the 37 kD protein family, the minimum level of total homology between the genes encoding the various proteins within the 37 kD protein family is therefore estimated to be 60%. Therefore, the 37 kD protein family includes those proteins where the genes that code for them have at least 60% homology with the genes that code for the 37 kD protein family whose nucleic acid sequences are listed in SEQ ID NO. ID. NFt:1, 2 and 3. These sequences are provided solely as examples of proteins of the 37 kD protein family. The algorithm used for the determination of the degree of nucleic acid homology is known as "Clustal W" and is described by Thompson et al., in Nucleic Acid Research 22:4673-4680 (1994). The program can be found in several places on the Internet. All the genes that code for a protein of the 37 kD protein family that have at least 60% homology with the nucleic acid sequences indicated in the SEQ ID NO. ID. NR:1, 2 or 3, are considered to be members of the 37 kD protein family. For applications e.g. for vaccine (see below) the homology between a selected gene encoding a protein of the 37 kD protein family and the SEQ. ID. NR: 1, 2 or 3, preferably be somewhat higher than 60%. Although a homology of 60% with SEQ. ID. NR:1, 2 or 3, would be acceptable, a homology of >70% would be preferred. A homology of >80% is more preferred, while a homology of >90% is most preferred.

In exceptional cases, a member of the 37 kD protein family may be found to have a nucleic acid homology less than the level indicated above. This can e.g. caused by an exceptionally large deletion. Nevertheless, such genes belong to the gene family that codes for a protein of the 37 kD protein family, if under stringent conditions they hybridize with any of those genes as nucleic acid sequences! is indicated for in SEQ. ID. NR: 1, 2 or 3. The conditions for hybridization are stated in Example 1.

Another way of determining whether a protein belongs to the proteins of the 37 kD protein family is based on binding of the protein to a specific antibody. This method of characterization of proteins of the 37 kD protein family is set forth below. The characterization is based on the specific reaction between proteins of the 37 kD protein family and the monoclonal antibody F4.2F8. This monoclonal antibody binds to most of the proteins of the 37 kD protein family that have been investigated so far. The monoclonal antibody is very specific, a common characteristic of all monoclonal antibodies. A hybridoma cell line producing the monoclonal antibody F4.2F8 has been deposited at the European Collection of Cell Cultures (ECACC), Center for Applied Microbiology & Research (CAMR), Salisbury, Wiltshire SP4 OJG UK, under accession number 99031816. All proteins that bind to F4. 2F8 is therefore considered to be members of the 37 kD protein family.

It cannot be ruled out that a strain is occasionally isolated that has a protein of the 37 kD protein family that does not bind to monoclonal antibody F4.2F8.1 in such exceptional cases, however, the gene that codes for the protein will still hybridize to a gene whose nucleic acid sequence is indicated in SEQ . ID. NR: 1, 2 or 3, or the gene will show a homology of at least 60% with the nucleic acid sequence indicated in SEQ. ID. NO: 1,2 or 3.

In one embodiment, the invention provides a vaccine comprising 50-75% of a 37 kD member of the 37 kD protein family and 50-25% of a 35 kD protein member of the 37 kD protein family.

By sequence analysis of the genes that code for the 37 kD protein family in the various strains, it has been shown that the difference in size is often due to the presence, absence or polymorphism of small nucleic acid deletions in the gene. This is shown in Fig. 1. This figure shows a sequence comparison between B. divergens strain Rouen 1987 (R), Weybridge 8843 (W) and Y5. It appears that strains W and Y5 encode a 35 kD variant form, while strain R encodes a 37 kD variant.

Next to the presence/absence of the deletion in W and Y5 compared to R, small variations occur in the total nucleic acid sequence of the gene encoding a protein of the 37 kD protein family in the respective Babesia strains. These variations need not have any effect on the amino acid sequences of the polypeptide if the modification is such that the triplet variant codes for the same amino acid. This reason for variation is based on the phenomenon of the degeneracy of the genetic code. For example, it happens that G in the triplet CTG, which codes for the amino acid Leucine, as a result of natural mutation, is replaced by a C, which also codes for Leucine, or that G in GAG, which codes for glutamic acid, is replaced with an A, which is a triplet that also codes for glutamic acid. Such a mutation is a silent mutation, i.e. it does not manifest itself at the amino acid level. Such silent modifications occur very often in nature, by comparison e.g. of two different field isolates of Babesia divergens. This phenomenon applies to all amino acids, except Met and Trp. It is thus clear that the protein family described above cannot only be encoded by the nucleotide sequences indicated in SEQ ID NO. ID. NR: 1-3, but also of a very large selection of other sequences that all code for the same protein.

It will be clear that for the particular proteins in question here, natural variations may exist between individual Babes/a parasites or strains. These variations can be detected by amino acid difference(s) in the total sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid^) in said sequence. Amino acid substitutions that do not significantly change biological and immunological effects have been described, e.g. by Neurath et al., in "The Proteins" Academic Press New York (1979). Amino acid substitutions between related amino acids or substitutions that have often occurred during evolution include, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, Vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/lle, LeuA/al and Ala/Glu . On the basis of this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science 227,1435-1441,1985) and determination of the functional similarity between homologous proteins. Amino acid substitutions may occur in the exemplified embodiments of this invention, as well as variations having deletions and/or insertions. Variations that do not significantly affect the immunogenicity of the protein compared to the wild-type protein as shown in SEQ ID NO. ID. NR: 4-6 may occur. The variations in the amino acid sequence of a particular 37 kD protein according to the invention which still provide a protein which can induce protection against infection with B. divergens, or at least against the clinical manifestations of the infection, are considered to "not significantly affect the immunogenicity".

A definition of immunogenic fragments of a protein of the 37 kD protein family will be given below, where another vaccination is discussed.

In general, the term "protein" refers to a molecular chain of amino acids with biological activity. The protein is not of a specific length and can, if necessary, be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation, and among other things peptides, oligopeptides and polypeptides are thus included in this definition.

By using expression systems to express the gene that codes for the protein of the 37 kD protein family, it is possible to obtain the proteins in sufficient quantities.

An essential requirement for the expression of the nucleic acid sequence is an adequate promoter operatively linked to the nucleic acid sequence. It will be clear to the person skilled in the art that the choice of a promoter includes any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells used as host cells for protein expression. Regulatory sequences have been added to the recombinant DNA molecules which enable expression of the relevant nucleic acid sequence. This can be achieved e.g. using standard molecular biology techniques.

(Maniatis/Sambrook (Sambrook, J. Molecular donation; a laboratory manual, 1989, ISBN 0-87969-309-6).

When the host cells are bacteria, expression control sequences that can be used include the Trp promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057, 1980); the lac promoter and operator (Chang et al., Nature, 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. & Inouge, M., EMBO J. 1, 771-775, 1982); the bacteriophage lambda promoters and operators (Remaut, E., et al., Nucl. Acids Res., 11, 4677-4688, 1983); the α-amylase (S. subtilis) promoter and operator, termination sequences and other expression enhancers and control sequences compatible with the chosen host cell.

When the host cell is yeast, suitable expression control sequences include e.g. a-feeding factor. For insect cells, polyhedrin or p10 promoters of baculovirus can be used (Smith, G.E., et al., Mol. Cell. Biol. 3, 2156-65, 1983). When the host cell is of mammalian origin, suitable expression control sequences include the SV-40 promoter (Berman, P.W., et al., Science 222, 524-527,1983) or the metallothionein promoter (Brinster, R-L, Nature, 296, 39-42,1982) or a heat shock promoter

(Voellmy et al., Proe. Nati. Acad. Sci., USA, 82, 4949-53, 1985). Alternatively, expression control sequences occurring in Babesia can also be used. Regarding maximization of gene expression, see also Roberts & Lauer (Methods in Enzymology, 68, 473, 1979).

Bacterial, yeast, fungal, insect and mammalian cell expression systems are very frequently used systems. Such systems are well known in the field and readily available, e.g. commercially through Clontech Laboratories, Inc., 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Next to these expression systems, parasite-based expression systems are very attractive expression systems. Such systems are e.g. described in French patent application publication number 2,714,074 and in US NTIS publication number US 08/043109 (Hoffman, S. & Rogers, W.: Public. Date 1 December 1993).

LRC (Live Recombinant Carrier) microorganisms comprise a gene that codes for a protein of the 37 kD protein family. Such microorganisms are e.g. bacteria and viruses. These LRC microorganisms are microorganisms into which additional genetic information has been cloned. Animals infected with such LRCs will produce an immunogenic response, not only against the immunogens of the vector, but also against the immunogenic parts of the polypeptides for which the genetic code is further cloned into this LRC, e.g. the 37 kD protein.

An example of bacterial LRC that can be used with advantage are known weakened Salmonella strains.

Live recombinant carrier parasites have been described i.a. by Vermeulen, A.N., (Int. Journ. Parasitol. 28:1121-1130 (1998)).

LRC virus can also be used as a way to transfer the nucleic acid sequence to a target cell. Live recombinant carrier viruses are also called vector viruses. The site of integration of the gene encoding a 37 kD protein can be a site in a viral genome that is not essential for the virus, or a site in an intergenic region. Viruses often used as vectors are Vaccinia virus (Panicali et al., Proe. Nati. Acad. Sei. USA. 79:4927 (1982), Herpesvirus (E.P.A. 0473210A2) and retroviruses (Valerio, D. et al., in Baum, S.J., Dicke, K.A., Lotzova, E & Pluznik, D.H. (eds), Experimental Haematology today - 1988, Springer Verlag, New York, pp. 92-99 (1989)).

The well-known homologous in vivo recombination technique can be used to introduce a recombinant nucleic acid sequence into the genome of a preferred bacterium, parasite or virus, which is capable of inducing expression of the inserted nucleic acid sequence in the host animal.

The present invention also relates to substances for use in a vaccine which induces protection against infection of cattle with B. divergens or the clinical manifestations of the infection where

the substance is:

a. a nucleic acid sequence of B. divergens which codes for a protein of the 37 kD protein family, where said nucleic acid sequence has at least 60% homology with the nucleic acid sequences given in SEQ. ID NO.: 1, 2 and 3; b. a recombinant DNA molecule comprising said nucleic acid sequence and regulatory sequences that enable expression of said nucleic acid sequence; c. a live recombinant carrier microorganism comprising said nucleic acid sequence, or d. a host cell comprising said nucleic acid sequence, said recombinant DNA molecule or said live recombinant carrier microorganism.

Antibody against a protein of the 37 kD family has also been described for use in a vaccine that protects against infection of cattle with B. divergens or clinical manifestations of the infection.

An object of the invention as stated above is to provide an effective vaccine against Babesia divergens infection, or at least against the clinical manifestations of the infection. It has now surprisingly been shown that an immunological response that provides immunity to infection with the Babes/a parasite, or at least an immunological response that sufficiently reduces the level of clinical manifestations after infection (as shown for example by a decrease in the hematocrit value ), can be achieved by vaccination with vaccines comprising a protein of the 37 kD protein family or an immunogenic fragment thereof.

When a polypeptide is used e.g. for vaccination purposes or for the development of

antibodies, however, it is not necessary to use the entire polypeptide. It is also possible to use a fragment of the polypeptide in question as itself, or linked to a carrier such as e.g. KLH, is capable of inducing an immune response against the relevant polypeptide, a so-called immunogenic fragment. An "immunogenic fragment" is to be understood as a fragment of the full-length protein of the 37 kD protein family which has retained its ability to induce an immune response in the host, i.e. comprises a B- or T-cell epitope. Currently, a number of techniques are too easy to identify DNA fragments that

codes for antigenic fragments (determinants) available. The procedure described by Geysen et al. (Patent Application WO 84/03564, Patent Application WO 86/06487, US Patent 4,833,092, Proe. Natl. Acad. Sei. 81:3998-4002 (1984), J. Imm. Meth. 102, 259-274 (1987 ), the so-called PEPSCAN method is a fast and well-established method that is easy to perform, for the detection of epitopes; the immunologically important protein regions. The method is used all over the world and is thus well known to the person skilled in the art. This (empirical) method is particularly suitable for detection of B-cell epitopes Moreover, when the sequence of the gene encoding any protein is known, computer algorithms are able to construct specific polypeptide fragments as the immunologically important epitopes, on the basis of their sequence and/or structural agreement with epitopes now known. The determination of these regions is based on a combination of the hydrophilicity criteria of Hopp & Woods (Proe. Nati. Acad. Sei. 78:38248-3828 (1981)) and the secondary structure aspects of Chou & Fasman ( Advances in Enzymology 47:45-148 (1987) and

US Patent 4,554,101). T-cell epitopes can also be deduced from the sequence by computer based on Berzofsky's amphiphilicity criterion (Science 235,1059-1062 (1987) and US Patent Application NTIS US 07/005,885). A condensed review can be found in: Shan Lu on general principles: Tibtech 9: 238-242 (1991), Good et al., on malaria epitopes; Science 235:1059-1062 (1987), Lu, for a review; Vaccine 10:3-7 (1992), Berzowsky regarding HIV epitopes; the FASEB Journal 5:2412-2418

(1991).

The preferred protein of the 37 kD protein family, or the gene that codes for the protein in question, can be a 35 kD protein or a 37 kD protein or a mixture of the two. The protein or the gene that codes for the protein can e.g. is selected from Babesia divergens strain Rouen 1987 or Weybridge 8843 or any other variant from other isolates.

More surprisingly, it has also been found that proteins of the 37 kD protein family are able to induce antibodies that not only protect against infection with a homologous Babesia divergens strain, but also with heterologous strains. This means i.a. that a vaccine based on a 35 kD protein of e.g. B. divergens strain Weybridge, induces protection against a 37 kD protein of e.g. B. divergens strain Rouen and vice versa.

Unexpectedly, it was also found that antibodies against a protein of the 37 kD protein family are able to confer in vivo immunity against both homologous and heterologous exposures.

This is even more surprising for the following reasons: antibodies against the above-mentioned 17 kD protein are found abundantly after experimental infection and after immunization with fraction 4. Moreover, α-Bd17, a monoclonal antibody against the 17 kD protein, has been described as the relevant vaccine component by passive vaccination against Babesia (Précigout et al., Experimental Parasitology 77:425-434

(1993), see above) on the basis that it dramatically inhibits growth of the parasite in vitro. On this basis, the 17 kD protein was expected to be the relevant vaccine component. When this antibody was used as a reference in in vivo experiments, however, it did not provide any protection at all either in a homologous or in a heterologous exposure experiment (as will be seen from Example 4).

Even more surprisingly, it was found that a vaccine comprising a mixture of two or more different proteins of the 37 kD family exhibits a synergistic effect in relation to vaccines comprising the same amount of protein, but only from one member of the 37 kD protein family. This has the advantage that vaccines comprising a mixture of two or more different proteins of the 37 kD protein family can comprise a lower total amount of antigenic material compared to vaccines comprising a protein of only one member of the 37 kD protein family. This embodiment of the invention relates in a preferred form, thus to vaccines comprising two or more different proteins of the 37 kD protein family.

The following may serve as an example: a vaccine comprising a 35 kD protein and a 37 kD protein exhibits a synergistic effect compared to vaccines comprising the same amount of protein, but only from the 37 or 35 kD protein. A vaccine comprising both 50-75% of a 37 kD protein and 25-50% of a 35 kD protein provides a degree of protection against disease that is between 33% and 60% better than what is achieved when the same amount of pure 37 kD protein or 35 kD protein was used as vaccine. This applies to exposure to strains comprising a 37 kD protein as well as to strains comprising a 35 kD protein. This has the advantage that relatively small amounts of immunogenic material can be used to still achieve a sufficiently high immune response.

The present invention further relates to a method for producing a vaccine, characterized in that said method comprises mixing a substance according to claim 4 or antibodies according to claim 5, and a pharmaceutically acceptable carrier.

There is also described a method for producing a vaccine according to claim 1, characterized in that said method comprises a mixture of two different proteins of the 37 kD protein family or immunogenic fragments thereof and a pharmaceutically acceptable carrier.

Such vaccines can therefore be prepared, e.g. by mixing the desired amounts of purified proteins, but also by mixing the desired amounts of cells comprising the various proteins. In a more preferred form, this embodiment of the invention therefore relates to vaccines comprising both 50-75% of a 37 kD protein of the 37 kD protein family and 25-50% of a 35 kD protein of the 37 kD protein family.

Such vaccines can alternatively be prepared by mixing live recombinant carriers that code for the 37 kD protein and code for the 35 kD protein according to the invention. Alternatively, in the case of DNA vaccines, mixtures of recombinant DNA molecules can be prepared, each capable of expressing one of the proteins.

It is clear that a vaccine for the protection of cattle against Babesia divergens infection or against the clinical manifestations thereof can also be based on the administration of antibodies against a protein of the 37 kD protein family. As an antibody source, a polyclonal antiserum developed e.g. in rabbits against a member of the 37 kD protein family, is used. As described above, antisera against one protein of the 37 kD protein family show cross-protection against other proteins of the 37 kD protein family. Any protein of the 37 kD protein family can therefore be used for the induction of such a polyclonal antiserum. Moreover, neutralization of monoclonal antibodies against a protein of the 37 kD protein family, such as e.g. the monoclonal antibody F4.2F8 is used. The invention thus also relates to vaccines that protect cattle against infection with B. divergens, or the clinical manifestations of the infection, which include antibodies against a protein of the 37 kD protein family.

As an alternative to the administration of vaccines comprising proteins of

37 kD protein family, vaccines comprising an LRC carrying a gene encoding a protein of the 37 kD protein family, or an immunogenic fragment thereof, can be administered. The advantage of such a method is that such an LRC invades certain target cells in the host organism. Once inside the target cells, all the LRC genes are expressed, including the gene encoding the protein of the 37 kD protein family carried by the LRC. The result is that the protein of the 37 kD protein family is expressed and

presented to the immune system in a manner that closely mimics the natural mode of expression and immune presentation. Vaccination with an LRC that carries a gene of the 37 kD protein family, or an immunogenic fragment thereof, can therefore be used with advantage.

When a vaccine is prepared based on a protein of the 37 kD protein family, or an immunogenic fragment thereof, obtained from an expression system, the protein can in principle be purified from the proteins in the host cells of the expression system. However, this is not always necessary. It is quite possible to incorporate in a vaccine the cells that have been used for expression of the preferred protein from the 37 kD protein family.

The vaccine may also include one or more immunogenic proteins of other pathogens. This is advantageous since vaccination with such a vaccine makes it possible to immunize an animal against two or more diseases in one vaccination step.

There are several ways to obtain such a vaccine. One is to mix a protein with one or more immunogenic proteins of other pathogens. Another possibility is the cloning of a heterologous DNA sequence encoding an immunogenic protein of another pathogen into a live recombinant carrier.

Preferably, the immunogenic protein is selected from the group of bovine pathogens consisting of bovine herpesvirus, bovine viral diarrhea virus, parainfluenza type 3 virus, bovine paramyxovirus, foot-and-mouth disease virus, Pasteurella haemolytica, bovine respiratory syncytial virus, Theileria parva, Theileria annulata, Babesia bovis, Babesia bigemina, Babesia major, Trypanosoma aria, Anaplasma marginale, Anaplasma centrale or Neospora caninum.

The heterologous DNA sequence can also code for a cytokine, such as interleukins, TNF and interferons. Several cytokines, e.g. interferons, are known to play an important role as immunomodulators. It may therefore be advantageous to include genetic information for this type of molecule in the aforementioned section.

It is clear that a heterologous DNA sequence can be introduced at a specific site in said section, e.g. in a restriction site, without deleting nucleotides from the section. On the other hand, it is possible to replace one or more nucleotides with heterologous DNA sequences of the same or different length.

Vaccines according to the present invention can be prepared, for example, by a pure mixture of a protein of the 37 kD protein family, or an immunogenic fragment thereof, either as such or linked to a suitable carrier molecule, and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is to be understood as a compound which does not adversely affect the health of the animal to be vaccinated, at least not to the extent that the adverse effect is worse than the effects seen when the animal is not vaccinated. A pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological saline solution. In a more complex form, the carrier can e.g. be a buffer.

The vaccine can also be prepared by mixing a nucleic acid sequence that codes for a protein of the 37 kD family, a recombinant DNA molecule that codes for a protein of the 37 kD family, a live recombinant carrier or a host cell according to the invention, and a pharmaceutically acceptable carrier.

The vaccine according to the present invention can in a preferred preparation also contain an adjuvant. Adjuvants generally include substances that enhance the host's immune response. A number of different adjuvants are known in this area. Examples of adjuvants are Freund's complete and incomplete adjuvant, vitamin E, non-ionic block polymers and polyamines, such as e.g. dextran sulfate, carbopol and pyran. In addition, surface-active substances such as Quil A® are very suitable.

Saponins are preferred adjuvants. Saponins are preferably added to the vaccine in an amount of between 20 and 150 (ig/mL. Within the group of saponins, the saponin Quil A is a more preferred adjuvant.

In addition, peptides such as myramyl dipeptides, dimethylglycine, tuftsin are often used. Alongside these adjuvants, immunostimulating complexes (ISCOMS), mineral oil, e.g. Bayol® or Markol®, vegetable oils or emulsions thereof, as well as Diluvac® Forte. The vaccine can also include a so-called "vehicle". A vehicle is a compound to which the polypeptide adheres without being covalently bound to it. Frequently used vehicle connections are e.g. aluminum hydroxide, phosphate, sulphate or oxide, silica, kaolin and bentonite. A special form of such a vehicle, where the antigen is partially embedded in the vehicle, are the so-called ISCOMs (EP 109,942, EO 180,564, EP 242,380).

Often the vaccine is mixed with stabilizers, e.g. to protect degradation-prone polypeptides from degradation, to improve the storage time of the vaccine or to improve the effect of freeze-drying. Suitable stabilizers are i.a. SPGA (Bovarnik et al.; J. Bacteriology 59:509 (1950)), skimmed milk, gelatin, bovine serum albumin, carbohydrates, e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or their breakdown products, as well as buffers such as alkali metal phosphates. Freeze-drying is an effective method of preservation. Freeze-dried material can be stored and kept alive for many years. Storage temperatures for freeze-dried materials can be well above 0°C without this damaging the material. Freeze-drying can be carried out according to well-known standard freeze-drying procedures.

In a preferred embodiment, the vaccine is therefore in a freeze-dried form. Furthermore, the vaccine can be suspended in a physiologically acceptable diluent. Such a diluent can e.g. be as simple as sterile water or a physiological saline solution. It goes without saying that other ways of achieving an adjuvant effect, adding vehicle compounds or diluents, emulsifying or stabilizing a polypeptide, are also covered by the present invention.

The vaccine according to the invention can be administered according to a conventional active immunization schedule: a single or repeated administration in a manner compatible with the dosage formulation and in an amount that will be prophylactically effective, i.e. the amount of immunizing antigen or recombinant microorganism, capable of expressing said antigen, which will induce immunity in cattle against virulent Babesia divergens parasites. Immunity is defined as the induction of a significant degree of protection in a cattle population after vaccination compared to an unvaccinated group.

For live viral vector vaccines, the dosage per animal can vary from 10<3> to 10<8> pfu (but even <1000 pfu may be sufficient, e.g. for carrier virus of bovine herpesvirus). A typical subunit vaccine according to the invention comprises 0.1 to 100 µg of the polypeptide (or variant or fragment thereof) according to the invention. Preferably at least 5 u.g will be included. Such vaccines can be administered e.g. intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, oral or intranasal.

An alternative and effective method of vaccination is direct vaccination with DNA that codes for the relevant antigen. Direct vaccination with protein-coding DNA has been successful for many different proteins. (As discussed in review by, e.g., Donnelly et al., The Immunologist 2:20-26 (1993)). In the area of anti-parasite vaccines, protection against e.g. Plasmodium yoelii has been obtained by DNA vaccination with the Plasmodium yoelii circumsporozoite gene (Vaccine 12:1529-1533

(1994)). Protection against Leishmania major has been achieved with DNA vaccination with the Leishmania major surface glycoprotein gp63 gene (Vaccine 12:1534-1536

(1994)).

This method of vaccination is also very attractive for vaccinating cattle against babesiosis. The invention therefore also relates to vaccines comprising DNA which codes for one or more proteins of the 37 kD protein family or an immunogenic fragment thereof.

Since the infection with Babesia divergens can develop relatively slowly, it may take some time before the diagnosis of Babesia divergens infection is made. In many cases, Babasia divergens infection is found in sick animals as the cause of the disease after the occurrence of hemoglobinuria (visible as it turns the urine red), which means that the disease is already far advanced.

Babesiosis infection also exists in various forms. One form is characterized by Babes/a antigens being present in the plasma without antibodies having yet been formed. This situation is found in acutely infected animals.

Another form is where the Babes/a parasites are eradicated and the plasma contains antibodies against Babes/a antigens, but no Babes/a antigens. This is the situation that occurs in animals that have developed a sterile immunity to Babesia.

A third form is the one where both antigens and antibodies are found in the plasma. Animals with this form of infection are immune to Babesia, but are also carriers of it

the parasite. These animals are a potential source of infection of other animals.

To determine the Babes/a infection state in an animal, it is therefore important to determine both the Babes/a antigen levels and the anti-Babes/a antibody level in serum. Therefore, diagnostic aids, which are preferably able to differentiate between the different forms, are highly desired.

A diagnostic test for the detection of Babesia divergens antibodies in sera, can e.g. be a simple ELISA test, where purified protein of the 37 kD protein family, or an antigenic fragment thereof, is coated on the wall of the wells in an ELISA plate. Incubation with serum from mammals to be tested, followed by e.g. of incubation with a labeled antibody against the relevant mammalian antibody, can then reveal the presence or absence of antibodies against the 37 kD protein.

Another example of a diagnostic test system is e.g. the incubation of a western blot comprising 37 kD protein, with the serum of the mammal to be tested, followed by analysis of the blot.

A diagnostic test for the Babes/a antigens and which is therefore suitable for the detection of Babesia oVVergens parasites, can e.g. also be a standard ELISA test, where the walls of the wells in an ELISA plate are covered with antibodies directed against a protein of the 37 kD protein family.

A quantitative, or competitive ELISA that not only indicates the presence or absence of Babes/a antigens, but also indicates the amount of Babes/a antigens, is also part of the invention. Such an ELISA is well known in the field.

This embodiment of the invention thus relates to competitive ELISA.

Antibodies or derivatives thereof (e.g. fragments such as Fab, F(ab')2 or Fv fragments), which are directed against proteins of the 37 kD protein family and which have potential applications in passive immunotherapy, diagnostic immunoassays and in the development of anti-idiotypic antibodies can be prepared according to standard techniques as indicated below.

The proteins or immunogenic fragments thereof can be used to produce antibodies, which can be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for the production and processing of polyclonal sera are known in the field (e.g. Mayer & Walter, ed. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).

Monoclonal antibodies reactive towards the protein (or variants or fragments thereof) can be produced by immunization of inbred mice using known techniques (Kohler & Milstein, Nature, 256, 495-497, 1975).

Antibodies against any of the polypeptides produced e.g. in one of the ways described above, can be used i.a. for vaccination purposes, especially in immunocompromised animals.

All kinds of antibodies against a protein of the 37 kDa protein family can be used in the diagnostic tests described above. Polyclonal antibodies obtained by injecting the Bd37 protein into an animal have a high affinity towards all proteins of the 37 kDa protein family. Nevertheless, the preferred antibodies are specific antibodies produced with the hybridoma cell line deposited at ECACC under accession number 99031816. They have high affinity towards all proteins of the 37 kDa protein family tested so far.

The diagnostic tests for the detection of antibodies against Babesia divergens preferably take the form of a kit, which comprises the 37 kD protein in a purified form. The antigens could e.g. be purified using standard protein separation techniques over a suitable column. Another possibility is separation on a PAGE gel, followed by western blotting. On the western blot, the 37 kD protein will form a specific band, separated from other Babesia divergens protein bands, and is thus considered to be purified. A pure form of the protein can also be obtained by expressing one of the nucleic acid sequences.

In principle, the easiest way to prepare such a diagnostic test system is to use purified whole 37 kD protein as explained above. However, it is very well possible to use only a part of the 37 kD protein. This applies with the proviso that the fragment used still comprises an antigenic determinant of the protein. All antigenic determinants of the 37 kD protein will by definition induce antibodies. Therefore, the use of a 37 kD protein fragment comprising even a single antigenic determinant of the 37 kD protein will be able to bind to anti-37 kD protein antibodies.

As mentioned above, the protein can be purified directly from cells infected with the parasite. However, it is also possible to use standard expression systems, such as bacterial, parasitic, yeast, baculovirus or mammalian expression systems, of which a large selection has been described, for the expression of the 37 kD protein. The thus expressed protein can be easily purified.

EXAMPLES

Example 1

Cultivation of Babesia divergens and genomic DNA extraction

An in vitro culture of Babesia divergens grown according to Grande et al., (Parasitology, 115, 81-84 (1997)).

After the parasitemia from in vitro culture reached 30%, B. divergens parasitized erythrocytes were pelleted by centrifugation for 5 min. at 2500 g.

The supernatant was centrifuged for 20 min. at 15,000 g; and the pellet, which contained merozoites, was washed twice in RPMI.

Infected red blood cells were lysed in 5 volumes of lysis buffer (5 mM EDTA; 100 mM NaCl; 20 mM Tris, pH 7.5; 0.5% Triton X-100; 0.5% SDS) supplemented with proteinase K to a final concentration of 1 mg/mL, 1 hour at 37°C.

DNA extraction was carried out in three steps with phenol, phenol-chloroform and finally with chloroform:isoamyl alcohol (24:1): addition of 1 volume of phenol or phenol-chloroform or chloroform:isoamyl alcohol (24:1), vortex-mixed for 1 min ., centrifuged for 5 min. at 10,000 g, whereupon the upper aqueous phase was transferred to a new tube.

DNA was then precipitated with 2 volumes of cold 100% ethanol and 1/10 volume of 3M sodium acetate, pH 5.6, 1 hour at -80°C. After centrifugation 15 min. at 10,000 g at 4°C, the DNA pellet was washed with ethanol 70%, dried and resuspended in sterile water.

Plasmid miniprep

A single plasmid colony was transferred to a tube containing 1.5 ml_ LB medium (10 g bactotryptone, 5 g yeast extract and 5 g NaCl in 1 liter distilled water, mixed and autoclaved), containing the appropriate antibiotic and incubated overnight at 37° C. Cells, pelleted by centrifugation for 30 seconds at 10,000 g, were first dissolved in 200 µl of a solution containing 100 mM Tris-HCl, pH 7.5; 10 mM EDTA, then in 200 |iL of a solution containing 200 mM NaOH, 1% SDS and in the third step after 5 min. at room temperature, in 200 uL of a solution containing 3M potassium acetate and 5M acetic acid. After incubation for 5 min. at 4°C and centrifugation for 5 min. at 10,000 g, the plasmid DNA contained in the supernatant was precipitated with 0.7 volumes of isopropanol. It was then dissolved in 200 µL TE buffer (10 mM Tris-HCl,

pH 7.5; 1 mM EDTA). For sequencing, a phenol/chloroform extraction and an ethanol precipitation were carried out as previously described.

cDNA library construction

A cDNA library was obtained from mRNA derived from B. divergens Rouen 1987 cultures as follows: 10<9> infected erythrocytes were pelleted by centrifugation of the culture and treated with a lytic agent containing guanidine thiocyanate using the RNA agents Total RNA Isolation System® Kit (Promega). Total RNA was then purified by phenol:chloroform extraction and precipitated with isopropanol. With total RNA as starting material, the poly(A) mRNA fraction was isolated by hybridization to a biotinylated oligo(dT) primer using the polyA Tract mRNA Isolation System Kit (Promega). The hybrids were collected and washed under high stringency using streptavidin coupled to paramagnetic particles and a magnetic separation setup. mRNA was eluted with water, and cDNA was synthesized using a ZAP Express™ cDNA Synthesis Kit (Stratagene).

cDNA synthesis was performed by reverse transcription using a primer containing a poly(dT) sequence and an XhoI restriction site. £cof?/ adapters were added to the 5' end of 0.4-4 kbp cDNA fragments (size fractionated on Sephacryl S-500 spin Columns) after Xho/ digestion. cDNA was ligated into the X-ZAPII Express vector in a sense orientation {EcoRI-Xho\) relative to the lacZ promoter using a ratio of 150 ng cDNA to 1 µg vector. Recombinant lambda phage were then packaged in Gigapack II packaging extracts from Stratagene, and plated on NZY agar plates using XL1-Blue MRF' cells, to be amplified and titrated as recommended by the manufacturer (Stratagene).

cDNA library screening

The polyclonal antibody directed against the 37 kDa protein family (oc-Bd37) was used for screening the cDNA library of B. divergens Rouen 1987.

For screening, 4000 recombinants of the cDNA library were plated using XL1-Blue MRF' cells. After growth for 8 hours at 37°C, recombinant β-galactosidase from each clone was blotted onto nitrocellulose membranes saturated with 10 mM IPTG over 3 hours. Slices were then saturated overnight in TBS (150 mM NaCl, 20 mM Tris-HCl, pH 7.2) + 5% skim milk at 4°C, washed in TBS and incubated for 1 h with α-Bd37 at a dilution of 1 :250. Positive plaques were visualized using goat anti-rabbit IgG conjugated to peroxidase, at a dilution of 1:500. Positive plates were transferred into 500 µL of SM buffer, incubated for 1 hour at 37°C and stored at 4°C. After purification of these positive plaques, the cDNA encoding the 37 kDa protein was excised from the A,-ZAPII phage in the form of the kanamycin-resistant pBK-CMV phagemid vector using a helper phage, as recommended by Stratagene.

cDNA sequencing

4 u.g plasmids that were carriers of cDNA that codes for the 37 kDa protein of B. divergence Rouen, was sequenced using the ^Sequencing™ Kit (Pharmacia Biotech). After denaturation with NaOH, neutralization and ethanol precipitation, the cDNA was pelleted by centrifugation and resuspended in 10 µl water. Renaturation was carried out using 10 pmol of primer, after which the DNA was sequenced using the dideoxy method of Sanger et al., ((1977), Proe. Nati. Acad. Sei. USA 74 (12):5463-7), by use reagents and methods according to Pharmacia Biotech. Primers suitable for reading the entire sequence were synthesized by Eurogentec.

Analysis of the cDNA sequence that codes for the 37 kDa protein

The sequence of the cDNA encoding the 37 kDa protein (1208 bp between the EcoRI and XhoI restriction sites) showed the presence of a poly(A)i8 tail and a coding region of 1023 deoxyribonucleotides, providing a single open reading frame of 341 amino acids without a repetitive motif .

Analysis of the hydrophilicity profile revealed two hydrophobic sequences at each end of the protein; from amino acids 1 to 18 at the N-terminus and from amino acids 321 to 341 at the C-terminus of the protein. The hydrophobic segment at the N-end could agree with the signal sequence of the 37 kDa protein, and the presence of such a cleavable N-terminal signal sequence indicated that the 37 kDa protein is a secreted protein. However, the presence of another hydrophobic segment at the C-end of the molecule indicates that the 37 kDa protein could interact or be anchored in the membrane of parasitic vesicles during the secretory pathway, to be on the surface of the parasite at the end of this process. These observations are consistent with the immunofluorescence pattern obtained on B. d/Vergens-infected erythrocytes, since the labeling was observed both inside parasitic vesicles and on the surface of the parasite. The finding of the 37 kDa protein in the supernatant of an in vitro culture could result in a cleavage of the molecule either from the surface of the parasite or from secretory vesicles.

Cloning of the 37 kDa protein gDNA from different isolates of B. divergens in pGEM®-T vector

Genomic DNA (gDNA) of B. divergens Rouen 1987, Weybridge 8843 and Y5 isolates was amplified by PCR (Polymerase Chain Reaction) and cloned into pGEM®-T Vector System II (Promega) to be sequenced as described above. gDNA from B. divergens Rouen 1987 and Weybridge 8843 was amplified using primer Bd37-5 (sense oligonucleotide),

5'-GAATTCACGACCATACGAATA-3' and Bd37-Low (antisense oligonucleotide), 5'-ACAGGATCCAAAAGCTACATAGCTGTCCACT-3', while gDNA from B. divergens Y5 was amplified using primer Bd37-Upp (sense oligonucleotide), 5'- CAAGGATCCTCTAAGTACGATGAAAACCAGTAA-3' and Bd37-Low. To perform PCR, 100 ng of each gDNA was incubated with 0.5 µL of each primer at 10 µM, 4 µL of dNTPs (1.25 mM of each), 2.5 µL of 10X enzyme buffer, and 1 unit of Taq DNA polymerase (Ozyme ) in a final volume of 25 jiL. The following conditions were used: 3 min. at 94°C, 3 rounds of 1 min. at 94°C, 1 min. at 50°C and 2 min. at 72°C and then 5 min. at 72°C.

PCR products were purified by electroelution by subjecting them to a 1% agarose gel electrophoresis. The expected bands were visualized under UV, excised and subjected to electroelution in 400 µL TAE buffer at 100 mA during 1 hour. The electroeluted materials were phenol-extracted and ethanol-precipitated as described above.

The ligation reaction was carried out by adding 50 ng of each gDNA to 50 ng of pGEM vector and 3 units of T4 DNA ligase in 1 µL of the approx. 10X appropriate buffer, over 5 hours at 16°C. 2 µL of each ligation mixture was used for the transformation reaction in JM109 High Efficiency Competent Cells, as recommended by Promega. Recombinant vectors were visualized using blue-white screening, with individual white colonies being picked and transferred into 50 µL of sterile water. After vortexing, the tubes were boiled for 5 min. and centrifuged for 1 min. at 10,000 g, during which 10 µL of the supernatant was used for a PCR reaction as described above with T3 and T7 primers to verify the presence of the insert. For sequencing the insert, a culture of 1.5 ml in LB of the recombinant vector for a plasmid miniprep was used, as previously described.

Characterization of genes encoding a protein of the 37 kD protein family by hybridization

For hybridization experiments, 4 µg genomic DNA is digested with 20 units of PstI restriction enzyme for 3 hours at 37°C, and digested products are subjected to 0.8% agarose electrophoresis. After denaturation and neutralization, the DNA is transferred to Hybond-N membrane (Amersham) and fixed by UV The DNA to be tested is labeled using the Nick Translation Kit (Boehringer Mannheim), with Redivue™

[a-<32>P]dCTP (3000 Ci/mmol) at a final concentration of 20 uCi (Amersham) and then purified using the Probe Purification after labeling kit (Jetnick) as recommended by the manufacturer. Membranes are first incubated in a prehybridization solution (5xSSC, 0.5% SDS and 1 g/L Ficoll type 400, polyvinylpyrrolidone and bovine serum albumin), 1 hour at 65°C and then hybridized overnight at 65°C in 10 mL of the same solution , supplemented with 1 mg of herring sperm DNA and the denatured labeled probe. Membranes are washed three times for 15 min. at 65°C in 6XSSC and exposed to Biomax MR-1 film (Polylabo) at -80°C for 48 hours.

DNA that hybridizes to DNA on the Hybond-N membrane comprises a gene that codes for a protein of the 37 kD protein family.

Example 2

Cloning of Bd37 cDNA into the pGEX-A vector

To express a recombinant Bd37 protein, the cDNA was subcloned into the E. coli vector pGEX-A. In a first step, the entire coding region of this cDNA was amplified by PCR using the primers Bd37-Up (sense oligonucleotide) and Bd37-Low (antisense oligonucleotide) containing SamH/ restriction sites. The PCR products were then subcloned into the pGEM®-T vector, as described above. In a second step, 500 ng of the pGEX-A vector and 500 ng of the pGEM®-T vector containing the Bd37 cDNA were cut with 30 units of the BamH/ restriction enzyme,

3 hours at 37°C, in the appropriate buffer (Life technologies) to be ligated.

After phenol/chloroform extraction and ethanol precipitation, the pGEX-A vector was dephosphorylated using 0.0015 units of Calf Intestinal Alkaline Phosphatase (Promega), 1 hour at 37°C in the appropriate buffer and reprecipitated. The cleaved cDNA fragment was purified by electroelution as described above.

The ligation of 100 ng of Bam/-//-cut phosphatase-treated pGEX-A vector with 100 ng of Bd37 cDNA fragment was performed using 15 units of T4 DNA ligase, in the appropriate buffer, for 1 hour at 25° C. Of the ligation mixture, 1 µL was used to transform 75 µL of Epicurian Coli® SURE®2 supercompetent cells, as recommended by the manufacturer (Stratagene).

Recombinants were determined by hybridization: Hybond-N (Amersham) filters were placed on plates for 30 seconds, denatured for 1 min. with 1.5M NaCl, 0.5N NaOH and neutralized with 1.5M NaCl, 0.5M Tris-HCl, pH 7.5. Filters were cleaned in 2xSSC, air dried and exposed, DNA side up, to UV (X>25 nm) for 3 min.

A PCR was performed on B. divergens Rouen 1987 cDNA using the internal primers Bd37-E1 (sense oligonucleotide),

5'-CAAGGTGGTGCGAATTCAAAG-3' and Bd37-E4 (antisense oligonucleotide), 5'-ATACAATGATACCGAATTCAATGG-3': of the electroeluted PCR fragment, 0.2 µg was used to synthesize a nucleotide probe using the Nick Translation Kit (Boehringer Mannheim). The probe was labeled with Redivue™ [oc-<32>P]dCTP

(3000 Ci/mmol) in a final concentration of 20 uCi (Amersham). The excess of triphosphate-deoxyribonucleosides was removed on a column of glass beads (Glasperlen 0.25-0.3 u. from Braun) and Sephadex®G50 superfine (Pharmacia). Membranes were prehybridized in 6xSSC + 2.5% skim milk, 1 hour at 65°C, and hybridized overnight at 65°C in 6xSSC + 2.5% skim milk containing herring sperm DNA as 100 µg/ml_, and the denatured labeled probe. The membranes were washed three times for 15 minutes at 65°C in 6xSSC and exposed to Biomax MR-1 film (Polylabo) at -80°C for 2 hours.

The correct orientation of the Bd37 fragment in the positive recombinant pGEX-A vector was checked by performing PCR on the purified recombinant vector using the 5' pGEX primer (Pharmacia), combined with the internal Bd37-E4 primer.

The same procedure has been followed using 100 ng of genomic DNA from B. divergens, Weybridge 8843 as starting material, to clone a gene encoding a 35 kDa protein in the pGEX-A vector and express a recombinant Bd35 protein.

Finally, as a control, 100 ng of native pGEX-A vector was used to transform 75 uL of Epicurian Coli®SURE®2, supercompetent cells to produce native glutathione-S-transferase (GST).

Production of recombinant GST, GST-Bd37 and GST-Bd35 proteins for a vaccine test

The various constructs expressing GST or a fusion protein of Bd37 or Bd35 protein with GST were induced by IPTG. The bacterial cultures were grown overnight and diluted 1:10 with LB medium supplemented with ampicillin to 50 µg/ml_. The cultures were incubated for 1 hour at 37°C and then induced with 10"<4>M IPTG for 3 hours. The bacteria were lysed by sonication in MTPBS (150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.3) - 1% Triton X-100, and the recombinant proteins affinity purified on glutathione-agarose beads and eluted by competition with reduced glutathione (Smith, D.B. & Johnson, K.S. (1988), Gene 67(1):31-40).

Production of a recombinant 37 kDa protein in insect cells, for a vaccine test

For the production of the recombinant baculovirus DNA, the cDNA encoding the 37 kDa protein was cloned according to standard procedures into the Bac-to-Bac™ vector from Life Technologies, 8717 Grovemont Circle, P.O. box 6009, Gaithersburg, MD 20884-9980, USA.

Six T75 flasks were used for culturing infected and uninfected SF9 cells, each containing 7.5 x 10<6> SF9 cells in a final volume of 15 mL HYQ CCM3 medium (Hyclone Europe NV, Belgium). Three of these flasks were infected with 52.5 µL of Bd37-1 virus (6.9 x 10 7 pfu/mL), which corresponds to an infection of SF9 cells at an MOI (multiplicity of infection) of 0.5.

Infected and uninfected cells (NIC) were collected 60 h post-infection in a final volume of 7.5 mL CCM3 medium and treated with 0.3% Triton X-100.1 h at room temperature. An aliquot of 100 µL of infected and uninfected cells was dissociated in 10 µL of 10X sample buffer and boiled for 5 min. Two aliquots of 25 µL from each sample (one used for a Coomassie blue stain and one used for an immunoblotting using the monoclonal antibody F4.2F8 at a dilution of 1:50) were subjected to a 12% SDS-PAGE. These infected cells expressing the 37 kDa protein, and uninfected cells, have been used to immunize gerbils to test the immunoprotective effect of the 37 kDa protein.

Example 3

Vaccination test with a recombinant 35 and 37 kD protein of the 37 kDa protein family

The vaccination test has been carried out on 15 groups of 10 gerbils to test the immunoprotective effect of the recombinant Bd37 protein, either expressed in the prokaryotic system (using the fusion proteins GST-Bd37 or GST-Bd35) or in the eukaryotic Bac-to-Bac system (using the recombinant Bac-Bd37). Three different doses of recombinant proteins, comprising 5 µg, 25 µg and 125 µg, have been tested (see Table 1). Negative control groups received only the equivalent of native GST or uninfected insect cells. Each vaccine preparation has been supplemented with the Quil-A adjuvant. A positive control group has received 300 uL of supernatant from the B. divergens Rouen 1987 in vitro culture. An exposure-control group received only 10<6> parasitized erythrocytes.

Animals

Desert rats (7-8 weeks old) were used. The animals received feed and water ad libitum.

Parasites

The exposure strain used was B. divergens Rouen 1987, and each animal received 10<6> parasitized erythrocytes in a final volume of 100 uL.

Vaccine production

Recombinant proteins (GST, GST-Bd35 or GST-Bd37 protein, infected or uninfected insect cell lysate) were prepared as described above (conf. Fig. 3). Supernatant from B. divergens Rouen 1987 in vitro culture was harvested when the parasitemia reached 30-40% in RPMI-Albumax, centrifuged at 10,000 g for 15 min., passed through a filter membrane (0.22 µm pore size; Gelman Sciences) and stored at - 80°C until use.

Each preparation (GST, GST-Bd35 or GST-Bd37 protein, infected or uninfected insect cell lysate and supernatant from in vitro culture) was diluted in RPMI to obtain 12 doses each containing the expected amount of recombinant protein (or equivalent) in a final volume of 3.4 mL. 100 uL of a Quil A stock solution of 2.8 mg/mL was added to each vaccine preparation. A dose of 300 µl was injected subcutaneously into each gerbil.

Vaccination procedure

Priming and booster infections have been made subcutaneously at an interval of 3 weeks. Animals were challenged with B. divergens Rouen 1987 intraperitoneally, 3 weeks after the booster injection, with 10<6> parasitized erythrocytes (100 uL/gerbil).

On days D-43, D-22 and D-1, blood samples were taken from the animals in each group. Serum samples from each group were tested by immunoprecipitation and immunofluorescence to analyze the humoral response of the gerbils to the recombinant 37 kDa protein.

Results

Immunoprecipitation

Serum from each gerbil from D-43, D-22 and D-1 was used for immunoprecipitation of <35>S-radiolabeled B. divergens Rouen 1987 total antigens.

Figure 4 shows that the uninfected insect cells and native GST are not immunogenic, in the sense that the humoral response does not cross-react with B. divergens antigens (groups 5 to 7 and 11 to 13). The observed pattern corresponds to the non-specific response.

For all gerbil sera vaccinated with the 37 kDa recombinant protein, a strong humoral response against the protein is specifically observed (groups 1 to 4 and 8 to 10), while controls vaccinated with NIC (non-infected cells) or native GST are negative.

Protection

Table 2: Protection with recombinant 37 kDa protein expressed in a prokaryotic (A) or eukaryotic (B) system

Conclusion

As can be seen in Table 2A, gerbils vaccinated with GST-Bd37 and GST-Bd35 are given a total immune protection against a homologous or heterologous exposure, respectively, to B. divergens Rouen 1987 (which encodes a 37 kDa protein). Desert rats showed no drop in hematocrit and some positive parasitemia. These results were obtained with very low doses (5 µg) as well as with high doses (125 µg) of recombinant protein. In contrast, all control desert rats vaccinated with native GST died.

From Table 2B it also appears that very good results were obtained for gerbils vaccinated with recombinant Bac-Bd37, even when it is taken into account that the Bd37 protein was not purified. Even if the gerbils received a high dose of insect cell lysate, the level of protection reached 80 to 100%, depending on the dose examined, while control gerbils, vaccinated with uninfected insect cells, all but one died. It can be concluded that protein of the 37 kD protein family provides a very high degree of protection regardless of which expression system is used.

Example 4

Passive immunization with the monoclonal antibodies F4.2F8 and homologous or heterologous exposure

Hybridomas producing mAb F4.2F8 were cultured and the monoclonal antibodies were purified according to standard procedures for the production of monoclonal antibodies. The monoclonal antibodies were injected intraperitoneally into gerbils and the concentrations of F4.2F8 were maintained by several injections, starting one day before the exposure and continuing several days after.

Since an antibody concentration of 100 µg/ml_ (1% of the total gerbil immunoglobulins) is needed, each gerbil was injected daily with 0.5 mg of antibody in culture medium. The experiment was continued 7 days after the exposure.

Two different exposure strains were used. B. divergens Rouen 1987 (comprising the 37 kDa protein) and Weybridge 8843 (comprising the 35 kDa protein). The desert rats were exposed to a high dose (10<6> parasitized gerbil erythrocytes) or a low dose (10<3> parasitized gerbil erythrocytes). The volume of the exposure dose (10<6> PE or 10<3> PE) was 100 uL.

For each exposure strain, a first control group (isotype control) received an unrelated mouse monoclonal antibody lgG2a, which is an antibody against a non-37 kDa protein that, however, has the same isotype as F4.2F8, to test the nonspecific activity . A second control group (antibody control) received the monoclonal cc-Bd17 directed against another B. divergens protein, the 17 kDa protein described above (Précigout era/., (1993), Exp. Parasitol. 77(4):425-34). A final control, to check the virulence of the strains, did not receive any injection of antibody (control without antibody).

Results

Table 3: results after homologous (A) and heterologous (B) exposure after passive immunization.

Conclusion

Results of homologous exposure

As is clear from Table 3A, vaccination with antibodies developed specifically against the 37 kDa protein provides full protection against infection with a homologous Babesia divergens strain. This confirms that the 37 kDa protein according to the invention is able to trigger antibodies that provide full protection against a homologous exposure.

Results of heterologous exposure

As can be seen from Table 3B, the 37 kDa antibodies provide not only full protection against homologous exposure (a strain expressing the 37 kDa protein), but also against heterologous exposure (a strain expressing the 35 kDa protein). It can therefore be concluded that the proteins of the 37 kDa protein family according to the invention induce an antibody response which provides protection against exposure to both homologous Babesia divergens strains and against heterologous strains.

Example 5

Synergistic effect of vaccines comprising both a 35 kD protein and a 37 kD protein of the 37 kD protein family

Vaccine production

B. divergens Rouen and Weybridge parasites were propagated in human erythrocytes in RPM11640 medium supplemented with 5 g/L Albumax®. Culture supernatants were harvested at 30-40% parasitemia and passed through a 0.22 µm filter. The total volume was kept at 250 uL. One vaccine dose contained 23.3 u.g of Quil A.

Immunization procedure

Vaccinations of gerbils were carried out with volumes of 250 µL, comprising Rouen/Weybridge supernatant in ratios of 0/100, 25/75, 50/50, 75/25 and 100/0. Vaccination was carried out twice with an interval of 3 weeks.

Exposure

Desert rats were intraperitoneally exposed to 10<3> or 10<6> parasitized erythrocytes/250 uL/ desert rat 3 weeks after booster injection.

Exposure strain 7107b was passed through gerbils to prepare an exposure inoculum. After the development of babesiosis, the animals were euthanized and the degree of parasitemia determined.

Analysis of ranking scores

Survival, minimal PCV (Packed Cell Volume = hematocrit) and parasite load were subjected to rank score analysis. Groups were ranked for each parameter. The best group was ranked as 1, the second best as 2 and so on. After ranking the different parameters, the total score for each group was determined. The total net rank score was expressed as the difference between the control and the vaccinated groups. This total score can be described as a new parameter: protection based on the various parameters mentioned above.

Results

As is clear from Figure 5, a vaccine based on a mixture of both 35 kD and 37 kD proteins according to the invention provides better protection upon exposure with both strains comprising 35 kD and 37 kD protein, compared to a vaccine based on the same amount of protein from only a 35 kD or 37 kD protein.

Conclusion

Vaccination with a mixed vaccine comprising both 35 kD and 37 kD protein has a synergistic effect. Such a vaccine protects when given at low (ie lower than optimally protective) doses, even better when exposed to both homologous and heterologous strains than comparable low dose vaccines based on pure 35 kD or 37 kD protein.

Figure headings

Figure 1: Comparative arrangement of nucleic acid sequences that code for protein of the 37 kDa protein family in B. divergens strain Rouen 1987, Weybridge and Y5, respectively SEKV. ID. NR: 1, 2 and 3 according to the Clustal method. Figure 2: Comparative arrangement of amino acid sequences of the 37 kDa protein family in B. divergens strain Rouen 1987, Weybridge and Y5, respectively SEQ.

ID. NR: 4, 5 and 6 according to the Clustal method.

Figure 3: Recombinant proteins of the 37 kD protein family were produced either in a prokaryotic system (A) or in a eukaryotic baculovirus (Bac-to-Bac) system (B). The following proteins are visible on the gel: purified GST-Bd37 (lane 1) and GST-Bd35 (lane 2) fusion proteins, infected cell lysate containing Bac-Bd37 protein (lane 3) and uninfected insect cell lysate (lane 4). Figure 4: Immunoprecipitation of antigens from B. divergens Rouen 1987 with sera from gerbils vaccinated with recombinant Bd37 proteins. Figure 5: Overall net rank score of minimal PCV, parasite load and survival in gerbils vaccinated with a mixture of Weybridge and Rouen SPA (Soluble Parasite Antigen) challenged with a high or low exposure dose.

Claims (7)

1. Vaccine that induces protection against infection of cattle with B. divergens or clinical manifestations of the infection, characterized in that it comprises two or more different purified proteins of the 37 kD protein family, and a pharmaceutically acceptable carrier. 2. Vaccine according to claim 1, characterized in that it comprises 50-75% of a 37 kD member of the 37 kD protein family and 50-25% of a 35 kD protein member of the 37 kD protein family. 3. Vaccine according to claims 1 or 2, characterized in that at least one of the two or more different purified proteins of the 37 kD protein family is a fusion protein with glutathione S-transferase. 4. Substance for use in a vaccine that induces protection against infection of cattle with B. divergens or the clinical manifestations of the infection where the substance is: a. a nucleic acid sequence of B. divergens which codes for a protein of the 37 kD protein family, where said nucleic acid sequence have at least 60% homology with the nucleic acid sequences given in SEQ. ID NO.: 1, 2 and 3; b. a recombinant DNA molecule comprising said nucleic acid sequence and regulatory sequences that enable expression of said nucleic acid sequence; c. a live recombinant carrier microorganism comprising said nucleic acid sequence, or d. a host cell comprising said nucleic acid sequence, said recombinant DNA molecule or said live recombinant carrier microorganism. 5. Antibody against a protein of the 37 kD family for use in a vaccine that protects against infection of cattle with B. divergens or clinical manifestations of the infection. 6. Procedure for the production of a vaccine, characterized in that said method comprises mixing a substance according to claim 4 or antibodies according to claim 5, and a pharmaceutically acceptable carrier. 7. Method for producing a vaccine according to claim 1, characterized in that said method comprises mixing two different proteins of the 37 kD protein family or immunogenic fragments thereof and a pharmaceutically acceptable carrier.