Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

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.

Functional genomic technologies applied to the control of the human malaria parasite, Plasmodium falciparum.

Infection with any of the four species of Plasmodium single cell parasites that infects humans causes the clinical disease, malaria. Of these, it is Plasmodium falciparum that is responsible for the majority of the 1.5-2.3 million deaths due to this disease each year. Worldwide there are between 300-500 million cases of malaria annually. To date there is no licensed vaccine and resistance to most of the available drugs used to prevent and/or treat malaria is spreading. There is therefore an urgent need to develop new and effective drugs and vaccines against this devastating parasite. We have outlined a strategy using a combination of DNA-based vaccines and the data derived from the soon-to-be completed P. falciparum genome and the genomes of other species of Plasmodium to develop new vaccines against malaria. Much of the technology that we are developing for vaccine target identification is directly applicable to the identification of potential targets for drug discovery. The publicly available genome sequence data also provides a means for researchers whose focus may not be primarily malaria to leverage their research on cancer, yeast biology and other research areas to the biological problems of malaria.

Plasmodium yoelii: cloning and characterization of the gene encoding for the mitochondrial heat shock protein 60.

Heat shock proteins are a highly conserved group of proteins required for the correct folding, transport, and degradation of other proteins in vivo. The Hsp70, Hsp90, and Hsp60 families are among the most widely studied families. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where, in cooperation with Hsp10, it participates in protein folding and translocation of proteins to the organelles. We have cloned and characterized the Hsp60 gene of Plasmodium yoelii (PyHsp60). PyHsp60 is a single-copy gene, located on chromosome 9, 10, or 11. The PyHsp60 cDNA sequence showed an open reading frame of 1737 nucleotides that codes for a polypeptide of 579 amino acids, with 93% amino acid identity to Plasmodium-falciparum Hsp60 (PfHsp60). Cloning and sequencing of a genomic PCR clone showed the presence of a 201-bp intron, located 141 bp downstream of the ATG codon. A single, heat-inducible, 2.3-kb transcript was detected in Northern blots of RNA isolated from blood stage parasites. Mouse antisera raised against a DNA vaccine vector that expresses PyHsp60 recognized sporozoites and liver- and blood-stage parasites by indirect fluorescent antibody test (IFAT). By Western blot, these antisera reacted with the mycobacterial Hsp65 and recognized a protein of approximately 65 kDa in P. yoelii sporozoites and P. falciparum blood stages. These results show that PyHsp60 and PfHsp60 genes are homologous and that of the PyHsp60 gene encodes a heat-inducible, intracellular protein that is expressed in several of the developmental stages of P. yoelii.