Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis.

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.

Molecular cloning of a developmentally regulated protein isolated from excretory-secretory products of larval Dirofilaria immitis.

Three proteins isolated from the excretory-secretory products (ES) of larval Dirofilaria immitis have been previously characterized and termed the 20, 22L and 22U kDa proteins. Two of the proteins (20 and 22L) were produced and released around the time of the third molt and were specifically recognized by immune dog sera. An amino acid sequence common to both proteins was used to synthesize a DNA probe to molecularly clone these molecules from a 48-h third stage larval cDNA library. The DNA sequence of the isolated clones encoded a 17.5 kDa protein with a 21 amino acid hydrophobic leader sequence that when removed yielded a 15.3 kDa protein starting with the N-terminal sequence obtained from the 20 kDa protein and containing all sequences obtained from tryptic peptides of the 20 and 22L kDa proteins. It was hypothesized that the 20 and 22L kDa proteins were the same, differing only by a 21 amino acid hydrophobic leader sequence which was later cleaved. The calculated molecular masses were consistent with those determined by reducing Tris-tricine SDS-PAGE. Expression of the protein without the leader sequence was accomplished in Escherichia coli. Antiserum raised against the expressed protein demonstrated the presence of the protein in L3 and L4, but not in adults or microfilariae. Expression of the protein with the leader sequence using a baculovirus system demonstrated processing of the signal sequence at the same time as found in larval D. immitis ES. Sera from dogs immune to infection were reactive with the D. immitis proteins expressed in either E. coli or insect cells.

Vaccine research and development for the prevention of filarial nematode infections.

The development of vaccines for the prevention of filarial nematode infections is in a state of relative infancy in comparison to vaccines for other parasitic diseases, such as schistosomiasis and malaria. There are many reasons for this slow start. Some of the principal problems are: (1) the lengthy and complex life cycle of these organisms with attendant complex immune responses, (2) the unique characteristics associated with a relatively large number of different pathogens, (3) the lack of suitable model systems for study of medically important infections, (4) the paucity of parasite material for antigen discovery and recombinant library construction, (5) the lack of substantial evidence suggesting the natural occurrence of protective immune responses, and (6) the limited data on mechanisms responsible for protective immunity. As technical hurdles are considered, it is also critical to focus on the characteristics of a vaccine necessary for its eventual utility. In the case of a vaccine for D. immitis a completely successful product will need to approach a 99+% efficacy. This is because of the 99+% efficacy of competitive chemotherapeutic products and the fact that microfilaremia observed on blood examination, resulting from as few as two worms, would present as a vaccine failure. Although very low worm burdens in large dogs could be perceived as success in the context of protection from clinical disease, because of the option of virtually complete chemoprophylactic protection, the typical veterinary practitioner would probably fail to appreciate less than complete vaccine protection. In contrast, a vaccine that produced a reduction in adult worm burdens without complete protection in either lymphatic filariasis or onchocerciasis would be very important. Highly effective chemoprophylactic agents are not widely available for prevention of the human filariases, and dramatically reduced clinical disease provided by less than a completely effective vaccine could occur as the result of fewer adult worms. The importance of developing these vaccines has outweighed the obstacles to this research. There has been a great deal of epidemiological and experimental evidence to suggest a vaccine is feasible and antigen discovery has progressed relatively rapidly within just the past few years. Efforts to generate appropriate larval cDNA libraries are beginning to yield dividends and a variety of fascinating vaccine candidates have been cloned. Additional antigen discovery, research on appropriate modalities for overexpression of genes from these parasites, and the complex tasks associated with vaccinology remain as significant research and development obstacles.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts.

Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs as an inverted repeat in a 55-kb multicopy, circular DNA in Leishmania infantum ITMAP263. The sequence is also found with a different genomic organization, possibly a tandem array, within a 1.5-Mb chromosome in all Leishmania isolates. About 26 stable transcripts of LD1 sequence, ranging from 0.6 to 15 kb, are found in ITMAP263. Transcripts were detected from both strands of the entire LD1 sequence, but the inverted repeat nature of the circular molecule prevented determination of whether transcription proceeded in one or both directions. Nine abundant transcripts (0.6-8.4 kb) from adjacent regions on the same strand of the repeat unit may represent mature mRNAs. One of these transcripts was shown to contain the 39-nucleotide spliced leader sequence characteristic of the 5' termini of trypanosomatid mRNAs. Several transcripts from the other strand of the repeat unit are also abundant and contain sequence complementary to some of the putative mRNAs. Less abundant, larger transcripts that span sequences encoding abundant mRNAs are also present, suggesting that transcription of LD1 is polycistronic.

A DNA sequence (LD1) which occurs in several genomic organizations in Leishmania.

Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs in all 91 stocks of twelve New and Old World Leishmania species examined; related sequences are present in some other kinetoplastid species. LD1 has no homology to several DNA sequences that are amplified in drug-resistant Leishmania. LD1 occurs in 3 different genomic organizations in Leishmania, depending on the stock. It is present within large (1.5-2 megabase) chromosomes in all stocks, and 74 stocks contain only this form. In 12 other stocks, LD1 also occurs in smaller (less than 550 kb) chromosomes, some of which are multicopy. Five stocks contain LD1 in multicopy circular DNA molecules in addition to the sequences found in the larger chromosome(s). Restriction fragment length polymorphisms of LD1 sequences correlate with taxonomic grouping, suggsting that LD1 is an endogenous sequence.

Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library.

Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.