Antigenic variation of a cysteine-rich protein in Giardia lamblia.

The WB isolate of Giardia lamblia expresses a cysteine-rich 170-kD surface antigen (CRP170) that undergoes antigenic variation. An (6E7), cytotoxic for isolates expressing CRP170, was used in another study to select antigenic variants from clones of the WB isolate of Giardia. CRP170 was replaced by surface-labeled bands ranging in size from approximately 50 to 170 kD. In this study, mAb 6E7 was used to isolate a 1-kb portion of the CRP170 gene (M2-1) from a lambda gt 11 expression library. The M2-1 clone hybridized to a 5.4-kb transcript from isolates expressing CRP170 but did not hybridize to RNA from antigenic variants. Evidence was found for frequent rearrangements at the CRP170 gene locus. DNA sequencing of the M2-1 clone revealed the presence of long tandem repeats. The putative amino acid sequence of M2-1 reveals a 12% cysteine content, and CRP170 is readily labeled in vivo with cysteine.

Structurally distinct, stage-specific ribosomes occur in Plasmodium.

Two structurally distinct nuclear genes code for cytoplasmic small subunit ribosomal RNA's in the parasite Plasmodium berghei. Stable transcripts from one of the ribosomal RNA genes are found almost exclusively in those stages of the life cycle that develop in the mosquito. When the parasite infects the mammalian host, transcripts from the second gene become the predominant small subunit ribosomal RNA species.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Variation among circumsporozoite protein genes from rodent malarias.

We investigated the effect of long term passage of parasites in naive animals on the circumsporozoite protein (CSP) gene of Plasmodium yoelii. The CSP gene sequence was determined from a non-lethal cloned line of P. yoelii and compared to the CSP gene sequence from a lethal strain of P. yoelii. The two parasite lines were originally derived from the same isolate, but were separated 17 years ago followed by continued passage. The sequence of the CSP gene and its surroundings from the non-lethal line remains identical to that from the lethal isolate except for a deletion within the repeated central domain. This result contrasts with the results obtained by sequencing a number of clones from different geographical field isolates of Plasmodium falciparum where there appears to be rapid accumulation of sense mutations within putative functional domains. These observations are consistent with the suggestion that strong biological pressure in a field environment results in selection of parasite types on the basis of different CSP gene sequences.

Structure of the circumsporozoite gene of Plasmodium malariae.

The sequence of the gene encoding the circumsporozoite protein of Plasmodium malariae was determined. The central immunodominant region of the protein consists of 45 copies of the sequence Asn-Ala-Ala-Gly and 6 copies of the sequence Asn-Asp-Ala-Gly. The CSP of the monkey parasite Plasmodium brasilianum contains the same repetitive sequences. Further comparison of the two genes in regions outside the immunodominant domains reveals only three nucleotide differences and each results in an amino acid change. One is centered in a putative T-cell determinant bearing region, the second is in the putative liver binding site, and the third is part of a degenerate repeat at the start of the immunodominant region.

Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum.

We have determined the complete sequence of two structurally distinct 18S ribosomal RNA genes from the malarial parasite Plasmodium falciparum. S1 nuclease analyses demonstrate that only one of the genes is represented in stable rRNA populations isolated from blood-stage parasites. Comparisons of homologous rRNA genes from Plasmodium berghei and P. falciparum reveal that they are identical at 86% of their positions. From comparisons of the Plasmodium genes to that of humans, it was possible to design genus-specific as well as species-specific oligonucleotide probes that can be used to distinguish the parasite 18S ribosomal RNA from that of its host. The utilization of these probes as diagnostic reagents is discussed.

Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae.

The DNA sequence of approximately 80% of the transcribed region of the kinetoplast maxicircle DNA of Leishmania tarentolae was obtained, and structural genes were localized by comparison of the translated amino acid sequences with those of known mitochondrial genes from other organisms. By this method, the genes for cytochrome oxidase subunits I, II, and III, cytochrome b, and human mitochondrial unidentified reading frames 4 and 5 were identified. By comparing the amino acid sequences of the putative L. tarentolae genes with those of known genes, we conclude that TGA codes for tryptophan, as in most other mitochondrial systems. This is the only apparent change from the universal genetic code. The six identified structural genes show various degrees of divergence from the homologous genes in other species, with cytochrome oxidase subunit I being the most conserved and cytochrome oxidase subunit III being the least conserved. A comparison of the cytochrome b genes from L. tarentolae and Trypanosoma brucei showed that the ratio of transversions to transitions is 1:1, suggesting that these species diverged from each other more than 80 X 10(6) years ago. Several as yet unidentified open reading frames were also present in the maxicircle sequence. These data confirm that maxicircle DNA has a coding potential which typifies other mitochondrial systems.

Identifying antigenic T-cell sites.

Computer algorithms that have been used successfully on protein sequences for the prediction of antigenic T-cell sites have been collected into a single computer software package called TSites.

Sequence variation in putative functional domains of the circumsporozoite protein of Plasmodium falciparum. Implications for vaccine development.

Sequences of the circumsporozoite protein gene from five isolates of the human malaria parasite Plasmodium falciparum are compared, and the extent of sequence variability within putative functional domains is assessed in terms relating to vaccine efficacy. Nucleotide substitutions were observed outside of the immunodominant domain. Of the substitutions observed outside of the repeat domain, none were silent. The substitutions correlated with biologically functional regions, such as a helper T cell epitope (Th2R) and a region (N1) which may be important in liver invasion. Contrary to previous impressions, the small numbers of amino acid changes in these areas of the protein seem potentially very significant. The immunodominant repeat region displays several characteristics that implicate a rapid evolutionary mechanism, most probably involving recombination. The data supporting this are 1) variable numbers of repeats, 2) a shifting pattern of substitutions among the isolates, and 3) codon bias. The region thus has the potential for very rapid change should an effective anti-repeat vaccine come into use. We conclude that strain variability is significant, that the potential for large scale variation in the repeats is great, and that regions that may be critical for an effective vaccine are polymorphic. Their potential impact on malaria vaccine development must be addressed.

Immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage.

Repeat regions of the circumsporozoite protein gene of Plasmodium falciparum were cloned into the pIII gene of a filamentous phage. These genetically engineered filamentous phage display the recombinant proteins on their surface. We demonstrate that they are both antigenic and immunogenic in rabbits. The recombinant phage were shown to be useful as a source of antigen for this scarce malaria protein, for producing carrier-hapten conjugates for obtaining immunological reagents in rabbits, and for B epitope mapping. In addition, in mice the antibody response to the cloned antigens seems to be controlled by immune response genes. Therefore this system also has the potential for use in helper T cell epitope mapping using inbred mouse strains. This advantage will be of use in vaccine development.

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines.

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.

Primary sequence and partial secondary structure of the 12S kinetoplast (mitochondrial) ribosomal RNA from Leishmania tarentolae: conservation of peptidyl-transferase structural elements.

The sequence of the 1173 nt 12S kinetoplast ribosomal RNA from Leishmania tarentolae was determined from the maxicircle DNA sequence, and the 5' and 3' ends localized by primer runoff and S1 nuclease protection experiments. The gene was shown to be free of introns by S1 nuclease analysis. A partial secondary structure model of the 12S RNA molecule is presented which is equivalent in certain respects to the corresponding portions of the Escherichia coli 23S ribosomal RNA model. Domain II of the E. coli model is completely missing in the kinetoplast model with the exception of several phylogenetically conserved stems and one loop. There is a striking conservation of the functionally important peptidyl-transferase region except for the deletion of a few stems and loops. The 12S RNA is the smallest large subunit ribosomal RNA described to date.

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.

A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.

The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66.

The immunologic significance of variation within malaria circumsporozoite protein sequences.

We have previously suggested that variation within the circumsporozoite protein of the malaria parasite Plasmodium falciparum was the result of selection by immune T cells. Our hypothesis has been supported by experiments documenting a lack of cross-reactivity between variant peptides from the C-terminal region for murine T cells primed by 7G8-specific sequences. Now, by using a murine model we have found that peptides representing variant regions (amino acid residues 326-343 and 361-380) of two other parasite clones (Wel and LE5) are also immunodominant for murine T cells. However, there were distinct changes in response profiles. For example, whereas lymph node cells from H-2d and H mice immunized with peptides from the 326-343 region of all three variants proliferated in vitro after homologous challenge, only lymph node cells from H-2b mice immunized with LE5 peptide proliferate after homologous challenge. In contrast, only LE5 did not induce lymphoproliferation against homologous challenge in the H-2s background. These data suggest that the naturally occurring substitutions affect agretopic (i.e., Ia). Peptides from all variants representing the 361-380 domain were recognized only by T cells from H-2k mice. Also, in nearly all cases, T cells primed by one sequence did not recognize variant sequences. The immunodominance of these domains from three different clones and the lack of significant cross-reactivity further supports the hypothesis that variation is the result of T cell immune pressure.

Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.

Mapping and 5' end determination of kinetoplast maxicircle gene transcripts from Leishmania tarentolae.

Transcripts for six Leishmania tarentolae maxicircle structural genes (cytochrome oxidase subunits I, II and III, cytochrome b, human mitochondrial unidentified reading frames 4 and 5) and several unidentified open reading frames were mapped, and the locations of the 5' ends determined by primer runoff analysis. All genes studied here are transcribed from the same strand as the 12S and 9S ribosomal RNAs except for the cytochrome oxidase subunit I gene. In two cases (ORF3 and ORF4, ORF5 and ORF6), a single transcript covers two contiguous overlapping reading frames. The 5' ends of the RNAs are located 20-64 nt from the putative translation initiation codons. Primary transcripts from a mitochondrial RNA preparation were 5' end-labeled with guanylyltransferase and alpha -32P-GTP; the major labeled species comigrated with the 12S and 9S mitochondrial rRNAs, and in addition there were at least four higher molecular weight labeled species.

Immunization against parasites: bridging the gap between attenuated and non-living vaccines.

While great progress has been made in the last decade in defining parasite antigens which are targets of host protective responses, only limited success has been achieved in the use of these molecules as effective vaccines. A consistent problem is the failure of the candidate immunogens to induce levels of protection comparable to those obtained with attenuated vaccines against the same organisms. One explanation is that the purified or recombinant molecules employed have not been presented in a form or route which induces the correct T cell or cytokine response necessary for protection. As summarized in this overview, optimal immunization with attenuated vaccines is associated with characteristic patterns of T cell subset and cytokine induction and in at least several examples has been shown to be altered by exogenous cytokine. We hypothesize that cytokine manipulation may offer a useful strategy for improving the action of existing nonliving vaccines. The gap in efficacy between attenuated and dead vaccines could also be bridged by the use of live recombinant vaccine vectors. We have previously reported that paramyosin admixed with BCG can induce partial protection against Schistosoma mansoni in mice. Our preliminary results in the construction and testing of a recombinant BCG vector incorporating schistosome paramyosin are described.