Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain. ß

The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falkiparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels ofinfection with the highly virulent FVO isolate of P. falckarum. However, the B-and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.

Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology.

Plasmodium falciparum merozoites have variable surface proteins that are processed from a 190‐kd precursor protein (p190). The gene encoding p190 exists in two allelic forms and cross‐over events occurring mainly near the 5′ end, combined with isolate‐specific tripeptide repeats, contribute to its antigen diversity. We have sequenced a large portion of the p190 gene from the parasite isolate RO‐33 (Ghana). Remarkably, the typical N‐terminal tripeptide repeat structure is lacking. Apart from mutations in the variable parts, the gene appears identical to the MAD‐20 allele (Papua, New Guinea). Southern blot analysis detects p190 genes similar to RO‐33 in other parasite isolates independent of their geographical origin. The lack of p190 repeats in RO‐33 eliminates the possibility that they are involved in host cell recognition or integration and restricts their function to immune escape.

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