Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate.

Gozar, M. M. G., Muratova, O., Keister, D. B., Kensil, C. R., Price, V. L., and Kaslow, D. C. 2001. Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate. Experimental Parasitology 97, 61-69. The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice. Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits. Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21. As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations. Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28. The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28.

Saccharomyces cerevisiae-secreted fusion proteins Pfs25 and Pfs28 elicit potent Plasmodium falciparum transmission-blocking antibodies in mice.

Transmission-blocking vaccines based on sexual-stage surface antigens of Plasmodium falciparum may assist in the control of this lethal form of human malaria. Two vaccine candidates, Pfs25 and Pfs28, were produced as single recombinant fusion proteins. The 39-kDa chimeric proteins, having a C-terminal His6 tag, were secreted by Saccharomyces cerevisiae, using the prepro-alpha-factor leader sequence. Pfs25-28 fusion proteins were significantly more potent than either Pfs25 or Pfs28 alone in eliciting antibodies in mice that blocked oocyst development in Anopheles freeborni mosquitoes: complete inhibition of oocyst development in the mosquito midgut was achieved with fewer vaccinations, at a lower dose, and for a longer duration than with either Pfs25 or Pfs28 alone. Increased antigen-specific immunoglobulin G titers and highly significant lymphoproliferative stimulation by Pfs28-containing antigens suggest the presence of an immunodominant helper T-cell epitope in the Pfs28 portion of the fusion proteins. This epitope may be responsible for the enhanced humoral response to both Pfs25 and Pfs28 antigens. Protein production of the fusion protein was improved 12-fold by converting Pfs28 codons to yeast-preferred codons (TBV28), using a modified ADH2 promoter and incorporating a (Glu-Ala)2 repeat after the Kex2 cleavage site.

Sequence polymorphism in two novel Plasmodium vivax ookinete surface proteins, Pvs25 and Pvs28, that are malaria transmission-blocking vaccine candidates.

BACKGROUND: For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and P. vivax. Work on P. vivax transmission-blocking vaccines has been hampered by the inability to clone the vaccine candidate genes from this parasite. MATERIALS AND METHODS: To search for genes encoding the ookinete surface proteins from P. vivax, the DNA sequences of the eight known proteins in the P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in the P21/28 subfamily (Pfs28, Pgs28, Pys21, Pbs21) of zygote/ookinete surface proteins were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. Genomic DNA from the Sal I strain of P. vivax and genomic and splinkerette DNA libraries were used as PCR templates. To characterize the polymorphisms of Pvs25 and Pvs28, these two genes were PCR amplified and the DNA sequences were determined from genomic DNA extracted from patients infected with P. vivax. RESULTS: Analysis of the deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four epidermal growth factor (EGF)-like domains, six copies of the heptad amino acid repeat (GSGGE/D), and a short hydrophobic region. Because the fourth EGF-like domain has four rather than six cysteines, the gene designated Pvs28 is the presumed homologue of P21/28 subfamily members. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to that of Pvs28. The presence of six rather than four cysteines in the fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily members. Several regions of genetic polymorphisms in Pvs25 and Pvs28 were identified in field isolates of P. vivax. CONCLUSIONS: The genes encoding two ookinete surface proteins, Pvs28 and Pvs25, from P. vivax have been isolated and sequenced. Comparison of the primary structures of Pvs25, Pvs28, Pfs25, and Pfs28 suggest that there are regions of genetic polymorphism in the P25 and P21/28 subfamilies.

An organelle-like small subunit ribosomal RNA gene from Babesia bovis: nucleotide sequence, secondary structure of the transcript and preliminary phylogenetic analysis.

Investigations aimed at identifying the mitochondrial genome of Babesia bovis using the polymerase chain reaction (PCR) have established the existence of an organelle-like small subunit ribosomal RNA (SSU rRNA) gene in the parasite. The sequence, compiled from three main PCR products, was 1448 bp in length (including the primer regions), had a 73% A+T content and showed significant similarity (68% sequence identity) to the "organellar" SSU rRNA gene from Plasmodium falciparum. The proposed secondary structure of the transcript showed several features which were consistent with a eubacterial origin for the organelle-like gene. The presence of putative binding sites for streptomycin and tetracycline also supported an "organellar" location for the gene and suggested that the SSU rRNA transcript is functional in protein synthesis because tetracycline has anti-babesial activity. Phylogenetic analyses based on the conserved regions of the SSU-like rRNA genes from a wide variety of organisms showed only a weak association of the babesial sequence with its mitochondrial homologues and an even weaker association with the corresponding genes of plastid origin. The origin of this organelle-like gene in B. bovis therefore remains unresolved, as is the case for its homologue from P. falciparum.

Identification of a Babesia bovis gene with homology to the small subunit ribosomal RNA gene from the 35-kilobase circular DNA of Plasmodium falciparum.

Isolation of a 552-base pair (bp) fragment of a putative extrachromosomal small subunit ribosomal RNA (SSrRNA) gene from Babesia bovis was achieved using the polymerase chain reaction (PCR) followed by cloning and sequencing of the PCR product. The sequences of the oligonucleotide primers used for the PCR were derived from selected known sequences in the organellar SSrRNA gene which is encoded within the 35-kilobase (kb) circular DNA from Plasmodium falciparum. Comparison of the sequence of the 552-bp fragment from B. bovis with gene sequences from other organisms showed 71% identity with the organellar SSrRNA gene from P. falciparum and up to 65% identity with the plastid SSrRNA gene sequences from various other organisms. We conclude that the 552-bp fragment amplified by PCR from B. bovis is possibly derived from an organellar genome of this parasite.

Mitochondrial function in Babesia bovis.

A variety of anti-mitochondrial drugs that had previously been found to inhibit the growth of the malarial parasite Plasmodium falciparum were tested on Babesia bovis in vitro. Several of these drugs were found to be non-toxic towards B. bovis. However, those drugs that were found to inhibit babesial growth included compounds (shown in parentheses) that have the following putative mitochondrial targets in the parasite: ATP synthetase complex (rhodamine 123, oligomycin, Janus Green); ATP-ADP translocase (bongkrekic acid); electron transport (rotenone, n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), antimycin A); ubiquinone (CoQ) function (BW58C, menoctone); protein synthesis (tetracycline); and the proton pump (CCCP). We have also investigated the effects of some of these drugs on pyrimidine biosynthesis de novo by following the incorporation of [14C]bicarbonate into pyrimidine nucleotides and into the pyrimidine moieties of nucleic acids. The ubiquinone analogues BW58C and menoctone inhibited this pathway in the nM-microM range of concentrations. Inhibitors of electron transport (antimycin A and oligomycin) and an uncoupler (CCCP) were also effective inhibitors of pyrimidine biosynthesis de novo. We conclude that B. bovis has a functional mitochondrion that contributes significantly to pyrimidine biosynthesis de novo and to the overall energy metabolism of the parasite.