Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.

Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.

Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91.

Cattle were vaccinated either with a single recombinant tick antigen, Bm86 or with a combination of two recombinant antigens, Bm86 and Bm91 from the tick Boophilus microplus. In three experiments, the responses of cattle to subsequent challenge with the tick were assessed. The addition of the Bm91 antigen enhanced the efficacy of the vaccination over that with Bm86 alone to a statistically significant degree. Moreover, co-vaccination with two antigens did not impair the response of cattle to the Bm86 antigen. Finally, responses of individual cattle to the two antigens were independent. All of these results may be relevant to the increase in efficacy expected from a dual antigen vaccine.

Commercialisation of a recombinant vaccine against Boophilus microplus.

Increasingly, there is need for methods to control cattle tick (Boophilus microplus) infestations by the use of non-chemical technology. This need is brought about by a mixture of market forces and the failure or inadequacy of existing technology. A recombinant vaccine has now been developed against the tick. This vaccine relies on the uptake with the blood meal of antibody directed against a critical protein in the tick gut. The isolation of the vaccine antigen, Bm86, and its production as a recombinant protein is briefly described. The vaccine has been tested in the field, has been taken through the full registration process and is now in commercial use in Australia. A related development has occurred in Cuba. The potential for improvement of the current vaccine and for the development of similar vaccines against other haematophagous parasites is discussed.

A protective "concealed" antigen from Boophilus microplus. Purification, localization, and possible function.

A membrane protein that can be used successfully to vaccinate cattle against the tick Boophilus microplus has been purified and characterized. The mature protein, which is referred to as Bm91, has an apparent m.w. of 86,000, an isoelectric point of 4.8 to 5.2, and is glycosylated, with an affinity for lentil lectin. Bm91 is of relatively low abundance, with approximately 300 to 400 micrograms being recovered from 1 kg of semiengorged adult female ticks. The protein is located largely in the salivary gland and gut of these ticks. Partial amino acid sequence data for the protein show striking similarities to that of mammalian angiotensin-converting enzyme, suggesting that the Ag may have an enzymatic function. The protein seems not to be recognized by sera from cattle with extensive exposure to ticks under natural conditions. The immunity induced by vaccination, therefore, represents another example of vaccination against a hematophagous parasite with "concealed" Ags.

Successful vaccination against Boophilus microplus and Babesia bovis using recombinant antigens.

Current methods for the control of the cattle tick Boophilus microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.

Successful vaccination against Boophilus microplus and Babesia bovis using recombinat antigens

Current methods for the control of the cattle tick Boophils microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials

Veterinary parasite vaccines.

This paper overviews the new veterinary parasite vaccines being developed by Biotech Australia Pty Ltd (one of Australia's largest biotechnology companies) in conjunction with other Australian research organisations. The research strategy adopted by the company is given.

Purified glutathione S-transferases from parasites as candidate protective antigens.

Glutathione S-transferase (GST) activity was detected in larvae of the Australian sheep blowfly Lucilia cuprina, and in the nematode Haemonchus contortus. A specific inhibitor of the enzyme was shown to affect survival of both species of parasite in vitro. GST from both parasites has been purified and partially characterized. Antisera raised to the purified enzymes were shown to inhibit the enzyme activity in vitro. However, the antisera had no effect on the survival of either parasite.

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.

Chromatography and generation of specific antisera to synthetic peptides from a protective Boophilus microplus antigen.

Four oligopeptides corresponding to predicted antigenic regions of the protective Bm86 glycoprotein of the cattle tick Boophilus microplus were synthesized and purified. Three were conjugated to carrier proteins and antisera raised in rabbits and cows. All elicited antipeptide antibodies that recognized Bm86 and recombinant derived products in Western blots; however, only one produced antiserum capable of recognizing native Bm86 in an indirect immunofluorescence assay. Ticks fed in vitro on this antiserum showed no obvious gut damage.

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.

Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen.

A cDNA clone coding for a mite allergen of mol wt approximately 14,000 has been isolated and its DNA sequence determined. The native component from mite extracts encoded by this DNA was identified by immunoprobing blots of mite body extract with human IgE eluted from the electroblotted cloned fusion polypeptides derived from the expressed cDNA clone. The clone encodes a polypeptide with a deduced mol wt of 17,460. The deduced amino acid sequence was not homologous to any known protein sequences and it contains no cysteine or tryptophan. On blots, 21 of 38 sera from mite-allergic subjects recognized the cloned material, and this recognition strongly correlated with IgE-binding to the native component on protein blots of mite extract.

Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus.

Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum.

Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid.

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA. The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and has a pre-pro region of 60 amino acids. The mature B subunit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa form of inhibin also isolated from bovine follicular fluid, with subunits of 20 kDa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.

A novel species of double stranded RNA in mitochondria of Saccharomyces cerevisiae.

A double stranded RNA species has been detected in guanidine hydrochloride extracts of mitochondria from respiratory competent cells of Saccharomyces cerevisiae. This novel mitochondrial RNA, termed mtdsRNA, has been purified in a Cs2SO4 density gradient where it bands at a density of 1.58 g/ml. The mtdsRNA runs as a single slow moving band on agarose gels. Its double stranded RNA character was evidenced by its sensitivity to digestion by RNase III, but not by RNase H, or DNase I. Moreover the mtdsRNA hybridized to each separated strand of a petite mtDNA. It is concluded that mtdsRNA contains long transcripts derived from most regions of yeast mtDNA, because 1) its weight-average length as determined by electron microscopy was 4.5 micrometer (about 14 kb, or 20% of the wild type mtDNA genome), and 2) it hybridized to each of a series of eight petite mtDNA probes carrying sequences derived from widely different segments of mtDNA. It is proposed that prolonged transcription of both strands of yeast mtDNA can occur and that mtdsRNA arises from hybridization of these long complementary transcripts.

Biogenesis of mitochondria: Mapping of transcripts from the oli2 region of mitochondrial DNA in two grande strains of Saccharomyces cerevisiae.

A series of 14 contiguous restriction fragments of yeast mitochondrial DNA that cover a 5.2 kb segment including the oli2 gene provided a set of hybridization probes that were used to analyze gel fractionated mitochondrial RNA immobilized on diazotized paper. Of the nine oli2 region transcripts in grande strain J69-1B (class I), the five largest (5,100, 4,500 (most prominent), 3,900, 3,600 and 3,000 nucleotides) contain the oli2 coding sequence; all appear to have the same 3' end. The four smallest J69-1B transcripts (1,900, 1,700, 800 and 600 nucleotides) contain only sequences down-stream of the oli2 gene. A 1.8 kb DNA region specifying these four transcripts is deleted from grande strain JM6 (class 11); these transcripts are thus dispensible for respiratory function. The five oli2 transcripts of JM6 (3,000, 2,400 (most prominent), 2,000, 1,700 and 1,150 nucleotides) all contain the oli2 coding sequence and are each related to a corresponding J69-1B transcript; the 5' and 3' ends match in each case, but the JM6 transcripts are about 2,000 nucleotides shorter. In particular, the putative oli2 mRNA has a 5' leader of about 1,400 nucleotides, a coding region of 780 nucleotides, and a variant 3' tail that is about 2,300 nucleotides in J69-1 B, and about 200 nucleotides in JM6.

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast.

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moieyt has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants in Saccharomyces cervisiae. The drugs also cause a loss of mtDNA from the cells in vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replication per se, as shown by direct in vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA in S. cervisiae.