The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoiteexpressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.genetically attenuated parasites ͉ malaria vaccine ͉ P36 ͉ P52 ͉ sporozoite M alaria is a formidable global health problem, affecting 300 million to 500 million people worldwide annually (1). The resulting Ϸ1 million deaths per year are mainly caused by Plasmodium falciparum infections. Eradication of malaria will in large part depend on an effective vaccine that prevents infection by Plasmodium, but such a vaccine has remained elusive. The parasites' preerythrocytic stages, encompassing the mosquito-inoculated sporozoites and liver stages that develop from sporozoites after their invasion of hepatocytes, are attractive targets for antiinfection vaccines, because at this stage the number of infected host cells is low, and further transmission of the parasite is not yet possible. Occurrence of blood-stage infection after sporozoite challenge is completely preventable by immunization with radiation-attenuated sporozoites in mouse models of malaria (2). This was a landmark finding that set the standards for malaria preerythrocytic vaccine development. Radiation-attenuated sporozoites arrest in development during hepatocyte infection, but their safety and efficacy are dependent on a precise irradiation dose. Humans immunized with P. falciparum radiation-attenuated sporozoites have been effectively protected from subsequent challenge with homologous an...

The idenficaton of apparently fastidious microorganisms is often problematic. DNA from a rickettsialike agent (called the ELB agent) present in cat fleas could be amplified by PCR with conserved primers derived from rickettsial 17-kDa common protein antigen and citrate synthase genes but not spotted fever group 190-kDa antigen gene. Alm I sites in both the 17-kDa and citrate synthase PCR products obtained with the rickettsia-like agent and Rickkesia lyphi were different even though both agents reacted with monoclonal antibodies previously thought specific for R. typhi. The DNA sequence of a portion of the 17-kDa PCR product of the rickettsia-like agent differed significantly from all known rickettsial sequences and resembled the 17-kDa sequences oftyphus more than spotted fever group rickettsiae. The rare stable transovarial maintenance of this rickettsia in cat fleas has important implications for the disease potential of cat fleas.

The new ligand 6,6′-dihydroxy-2,2′-bipyridyl (dhbp) was synthesized via its tautomer, and this provides an efficient route to novel metal complexes of dhbp. In ruthenium complexes of dhbp, these OH groups enhance water solubility and may play a role in aqueous transfer hydrogenation with formate/formic acid as the hydrogen source. A series of cationic catalysts, [(η 6 -arene)Ru(N,N)Cl]-Cl (arene = cymene, C 6 Me 6 ; N,N = bipyridyl with OH, OMe, or H at the 6-and 6′-positions), were synthesized, fully characterized, and tested for transfer hydrogenation activity in various polar protic media. In aqueous media (90/10 water/ methanol), Ru complexes of dhbp outperform the other catalysts tested (all at 1 mol %), and high percentage conversion of aromatic ketones to the corresponding alcohols is observed in 6 h. The OH groups appear to be essential for use of water as a green solvent and can potentially allow for metal−ligand bifunctional catalysis.

Identification of ELB agent-infected fleas and rodents within several foci of murine typhus in the United States has prompted a retrospective investigation for this agent among human murine typhus patients. This agent is a recently described rickettsia which is indistinguishable from Rickettsia typhi with currently available serologic reagents. Molecular analysis of the 17-kDa antigen gene and the citrate synthase gene has discriminated this bacterium from other typhus group and spotted fever group rickettsiae. Current sequencing of its 16S ribosomal DNA gene indicates a homology of 98.5% with R. typhi and 99.5% with R. rickettsii. Through a combination of restriction fragment length polymorphism and Southern hybridization analysis of rickettsiaspecific PCR products, one of five tested patient blood samples was shown to be infected with ELB while R. typhi infections were confirmed in the remaining samples. This is the first reported observation of a human infection by the ELB agent and underscores the utility of PCR-facilitated diagnosis and discrimination of these closely related rickettsial infections.

In total, data from 532 volunteers demonstrate that experimental challenge is safe and results in predictable incubation and prepatent periods. Our findings support the continued use of this method for testing efficacy of vaccines and drugs against P. falciparum.