Leishmaniasis and malaria: new tools for epidemiologic analysis.

Parasitic diseases are still prevalent in many parts of the world, causing both human suffering and economic loss. Recent developments in biotechnology, such as the use of monoclonal antibodies and recombinant DNA, have the potential for providing both more extensive and detailed information on the parasite in the infected human and in insect vectors. New methods of detection, both in man and insect vectors, have been developed for two parasitic diseases, leishmaniasis and malaria. These new methodologies will be important in epidemiologic studies on the prevalence and transmission of these parasitic diseases.

Mutating the anchor residues associated with MHC binding inhibits and deviates CD8+ T cell mediated protective immunity against malaria.

We investigated whether immune responses induced by immunization with plasmid DNA are restricted predominantly to immunodominant CD8+ T cell epitopes, or are raised against a breadth of epitopes including subdominant CD8+ and CD4+ T cell epitopes. Site-directed mutagenesis was used to change one or more primary anchor residues of the immunodominant CD8+ T cell epitope on the Plasmodium yoelii circumsporozoite protein, and in vivo protective efficacy and immune responses against defined PyCSP CD8+ and/or CD4+ epitopes were determined. Mutation of the P2 but not P9 or P10 anchor residues decreased protection and completely abrogated the antigen-specific CD8+ CTL activity and CD8+ dependent IFN-gamma responses to the immunodominant CD8+ epitope and overlapping CD8+/CD4+ epitope. Moreover, mutation deviated the immune response towards a CD4+ T cell IFN-gamma dependent profile, with enhanced lymphoproliferative responses to the immunodominant and subdominant CD4+ epitopes and enhanced antibody responses. Responses to the subdominant CD8+ epitope were not induced. Our data demonstrate that protective immunity induced by PyCSP DNA vaccination is directed predominantly against the single immunodominant CD8+ epitope, and that although responses can be induced against other epitopes, these are mediated by CD4+ T cells and are not capable of conferring optimal protection against challenge.

Heterogeneity in the clonal T cell response. Implications for models of T cell activation and cytokine phenotype development.

The T cell can be defined in the context of two properties--the recognition specificity of the T cell receptor (TCR) heterodimer and the functional response of the T cell after TCR stimulation. Once a particular TCR heterodimer is expressed and successfully selected during thymic development, the antigen specificity is fixed for all the clonal progeny of that cell. In contrast, the potential functional responses that may be generated in response to specific antigen in the postthymic environment are quite extensive. These range from programmed cell death to initiation of alternate programs of phenotype development that generate effector populations with distinct cytokine expression patterns and regulatory properties. Recent advances in analytical methods that have permitted multiparametric characterizations of the T cell response at the single cell, rather than population level, have necessitated a modified view of T cell activation and the clonal T cell response, and have generated new insights into the regulation of immunity. In this brief review, we highlight studies that have characterized heterogeneity of the CD4+ T cell clonal response based on single-cell analyses, and discuss implications for models of T cell activation and cytokine phenotype development.

ELISPOT assay for detection of peptide specific interferon-gamma secreting cells in rhesus macaques.

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51Cr-release cytotoxic T cell (CTL) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-gamma (IFN-gamma) ELISPOT assay capable of detecting IFN-gamma secretion in response to stimulation with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/10(6) PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-gamma response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4(+), but not CD8(+), T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10(6) PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-gamma assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

Generation of sequence diversity in the kinetoplast DNA minicircles of Leishmania mexicana amazonensis.

In order to understand the mechanisms which generate minicircle sequence diversity, we sequenced three minicircles belonging to the same or closely related sequence classes from the kinetoplast DNA of Leishmania mexicana amazonensis strains, PH8, Raimundo, and Josefa. Closely related minicircles from PH8 and Raimundo were unexpectedly found to differ at 11% of positions within the evolutionarily conserved region, but at only 3.9% of positions in the variable region. It thus appears that accumulation of point mutations will not account for the wide intra-strain and intra-subspecies divergence of the variable region. Comparison of more distantly related minicircles from PH8 and Josefa revealed only two short stretches of 70% homology within the variable region. These stretches of homology are not located in the same positions relative to the conserved regions in their respective minicircles. They may represent vestiges of recombinational events responsible for the rapid divergence of minicircle variable regions.

Partial protection against malaria by immunization with Leishmania enriettii expressing the Plasmodium yoelii circumsporozoite protein.

Since infection with Leishmania species induces CD4+ and CD8+ anti-leishmania T cells, we assessed protection against malaria by immunization with Leishmania enriettii transfected with the gene encoding the Plasmodium yoelii circumsporozoite protein (PyCSP). The recombinant plasmid appeared to be a circular episome in the host cells. Reverse transcription PCR showed that the PyCSP was trans-spliced by the addition of the 39-bp spliced leader of L. enriettii at its 5' end. The transfectant expressed a protein in a pattern similar to that found in the sporozoite itself. Immunofluorescence and immunoelectron microscopy indicated that PyCSP was abundantly expressed on the surface of the parasite. Mice immunized with the transfectant produced antibodies to sporozoites, had a delay in onset of parasitemia after challenge, and 4 of 22 (18%) were completely protected. The protected mice had cytotoxic T lymphocytes against the PyCSP. Immunization with recombinant vaccinia, Salmonella typhimurium, and pseudorabies virus expressing the PyCSP induces excellent immune responses, but has not been shown to protect against challenge. Thus, the modest protection found in these initial studies represents a step forward. After further work Leishmania may prove to be an important live vector vaccine system for induction of protective immune responses.

Characterization of the gene encoding sporozoite surface protein 2, a protective Plasmodium yoelii sporozoite antigen.

Sporozoite surface protein 2 (SSP2) is a 140-kDa, protective sporozoite surface protein from Plasmodium yoelii distinct from the circumsporozoite protein (CSP). A genomic clone containing the SSP2 gene was isolated and sequenced to determine its size, structural organization and deduced primary amino acid sequence. The coding sequence consists of a single, long open reading frame encoding 826 amino acids. The overall structure of SSP2 is similar to that of the CSP, consisting of a central region of immunogenic amino acid repeats flanked by non-repetitive sequence. SSP2 has one copy of a thrombospondin repeat motif in common with several cell adhesion molecules as well as with the CSP and the thrombospondin related anonymous protein (TRAP) of P. falciparum. Additionally, SSP2 shares substantial sequence similarity to TRAP, suggesting that TRAP is the analogue of SSP2 in P. falciparum.

Detection of Leishmania within sand flies by kinetoplast DNA hybridization.

A kinetoplast DNA hybridization probe method was used to detect Leishmania within sand flies and to distinguish it from the non-pathogenic flagellate, Endotrypanum. Eighty-one sand flies (74 Lutzomyia umbratilis, 1 Lu. anduzei, and 6 Lu. shannoni) collected outside Manaus, Brazil were dissected. Forty-four of these were found to be infected with flagellates, and 2 hybridized with a Leishmania braziliensis probe. Thirty-three of sixty-one flies reprobed with an Endotrypanum probe were positive.

Detection of alprazolam in three cases of methadone/benzodiazepine overdose.

Benzodiazepine abuse is common among clients at methadone maintenance clinics. Diazepam and lorazepam are readily detected by immunological screening methods and confirmed by GC/MS. Alprazolam has been relatively difficult to confirm. We recently reported a modification of an existing serum HPLC procedure which allows us to analyze whole blood. We report here three cases of fatal drug overdose caused by co-ingestion of methadone and alprazolam. In all three cases, alprazolam was detected by HPLC and could not be identified by alkaline extraction GC/MS. Postmortem blood concentrations of methadone were at the lower range or below the concentrations previously identified in methadone overdose fatalities, suggesting an increased risk from co-ingestion of methadone and alprazolam.

Kinetoplast DNA minicircles: regions of extensive sequence divergence.

Previous work has shown that the kinetoplast minicircle DNA of Leishmania species exhibits species-specific sequence divergence and this observation has led to the development of a DNA probe-based diagnostic test for leishmaniasis. In the work reported here, we demonstrate that the minicircle is composed of three types of DNA sequences with differing specificities reflecting different rates of DNA sequence change. A library of cloned fragments of kinetoplast DNA (kDNA) from Leishmania mexicana amazonensis was prepared and the cloned subfragments were found to contain DNA sequences with different taxonomic specificities based on hybridization analysis with various species of Leishmania. Four groups of subfragments were found, those that hybridized with a large number of Leishmania sp. as well as sequences unique to the species, subspecies, or isolate. Analysis of nested deletions of a single, full-length minicircle demonstrates that these different taxonomic specificities are contained within a single minicircle. This implies that different regions of a single minicircle have DNA sequences that diverge at different rates. These sequences represent potentially valuable tools in diagnostic, epidemiologic, and ecological studies of leishmaniasis and provide the basis for a model of kDNA sequence evolution.

Construction and immunogenicity of DNA vaccine plasmids encoding four Plasmodium vivax candidate vaccine antigens.

Plasmodium vivax is the second most common agent of human malaria. Although infection is rarely fatal, it nonetheless imposes a significant burden of illness in endemic areas. A successful vaccine against P. vivax will likely need to induce immune responses against both pre-erythrocytic and erythrocytic stage forms of the parasite. Accordingly, we constructed eight nucleic acid vaccines based on four antigens, the circumsporozoite protein (PvCSP) and sporozoite surface protein 2 (PvSSP2) from the pre-erythrocytic stage, and apical membrane antigen 1 (PvAMA1) and merozoite surface protein 1 (PvMSP1) from the erythrocytic stage. The constructs induced high levels of specific antibody in mice regardless of whether the antigen was expressed in native form or fused to a human tissue plasminogen activator leader peptide. High titer antibodies induced against PvCSP did not react with the protective AGDR epitope within the sequence of this antigen. These results support the immunogenicity of these four vaccine candidate antigens when delivered as nucleic acid vaccines.

Induction of murine cytotoxic T lymphocytes against Plasmodium falciparum sporozoite surface protein 2.

Sporozoite surface protein 2 has been identified as a target of malaria vaccines designed to produce protective CD8+ cytotoxic T lymphocytes (CTL) because mice immunized with mastocytoma cells expressing a fragment of Plasmodium yoelii sporozoite surface protein 2 (PySSP2) are protected against malaria by an immune response that requires CD8+ CTL. To define CTL epitopes in the Plasmodium falciparum sporozoite surface protein 2 (PfSSP2), spleen cells (SC) from mice immunized with irradiated sporozoites (irr spz) were stimulated with synthetic peptides, and these effectors were tested for cytolytic activity against peptide-pulsed, major histocompatibility complex (MHC)-matched targets. Two peptides containing CTL epitopes, A6 (Pf SSP2 3D7 214-233) and BH1 (Pf SSP2 3D7 3-11) were identified in bulk cultures of SC from immune C57BL/6 mice, and by production of CTL lines. Immunization with recombinant vaccinia expressing the full length PfSSP2 induced antigen specific, MHC-restricted, CD8+ T cell-dependent cytolytic activity against these two peptides. Finally, CTL were induced by immunization with a bacteria-derived recombinant fragment of PfSSP2 (rPfSSP2) mixed with a liposomal formulation containing a cationic lipid (Lipofectin Reagent, LPF). Induced CTL lysed target cells pulsed with peptide A6 or with LPF/rPfSSP2, but not targets pulsed with only rPfSSP2. These studies demonstrate that CTL specific to PfSSP2 are present in C57BL/6 mice and that immunization with purified rPfSSP2 delivered with LPF induces a cytotoxic T cell response.

Stage-dependent localization of a novel gene product of the malaria parasite, Plasmodium falciparum.

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

Visualization of antigen-specific T cell activation and cytokine expression in vivo.

A transgenic TCR adoptive transfer system was used to visualize Ag-specific T cell activation and cytokine expression in vivo. After s.c. injection of peptide in adjuvant the entire Ag-specific population up-regulated IL-2 receptor alpha-chain expression, underwent blast transformation, and developed a memory-surface phenotype. A minority of the Ag-specific T cells produced predominantly IL-2 mRNA and localized at the T cell/B cell junction in draining lymph nodes. In the secondary response, a mixed cytokine pattern of both Th1 and Th2 cytokines was demonstrated. When peptide was administered i.v. without adjuvant, 50% of the Ag-specific cells expressed IL-2, but the peak of expression occurred before IL-2 receptor alpha-chain up-regulation, and only a minority of the Ag-specific T cells underwent blast transformation.

Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice.

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (approximately 80%) is much greater than that of Homo sapiens (approximately 59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1(42)) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 microg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

Immunogenicity and protective efficacy of a Plasmodium yoelii Hsp60 DNA vaccine in BALB/c mice.

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 microg of PyHsp60 DNA vaccine alone or in combination with 30 microg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 x 10(3) irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.

Identification and characterization of the protective hepatocyte erythrocyte protein 17 kDa gene of Plasmodium yoelii, homolog of Plasmodium falciparum exported protein 1.

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.