Live, attenuated vaccines remain the safest, most cost-effective intervention against viral infections. Because live vaccine strains are generated empirically and the basis for attenuation is usually ill defined, many important viruses lack an efficient live vaccine. Here, we present a general strategy for the rational design of safe and effective live vaccines that harnesses the microRNA-based gene silencing machinery to control viral replication. Using poliovirus as a model, we demonstrate that insertion of small miRNA homology sequences into a viral genome can restrict its tissue tropism, thereby preventing pathogenicity and yielding an attenuated viral strain. Poliovirus strains engineered to become targets of neuronal-specific miRNAs lost their ability to replicate in the central nervous system, leading to significant attenuation of neurovirulence in infected animals. Importantly, these viruses retained the ability to replicate in non-neuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

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Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endotheium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P.fakiparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. fakiparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdrl gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdrl and to no longer over-produce the protein product termed Pglycoprotein homologue 1 (Pghl). The pfmdrl gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the Pglycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdrl. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfimdrl gene and the level of chloroquine and mefloquine resistance.

Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5'- or 3'-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5'- and 3'-UTRs. In general, the 5'-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5'-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3'-UTR had little impact on mRNA and protein accumulation, as long as a 3'-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5'-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

Peripheral blood gene expression profiling (GEP) in SLE allows patients to be categorised into two groups based on a high or low IFN gene signature. Disease activity measured using the SLEDAI-2K is correlated with the high IFN gene signature, indicating that GEP may be a useful biomarker of disease activity in SLE.

A classic hypothesis for enzyme catalysis is the induction of strain in the substrate. This notion was first expressed by Haldane with the lock and key analogys"the key does not fit the lock perfectly but exercises a certain strain on it" (1). This mechanism has often been invoked to explain the catalytic efficiency of enzymes but has been difficult to establish conclusively (2-7). Here we describe X-ray crystallographic and mutational studies of an antibody metal chelatase which strongly support the notion that this antibody catalyzes metal ion insertion into the porphyrin ring by inducing strain. Analysis of the germline precursor suggests that this strain mechanism arose during the process of affinity maturation in response to a conformationally distorted N-alkylmesoporphyrin.N-Alkylporphyrins are metalated at rates which are orders of magnitude faster than their nonalkylated analogues (8). This is thought to result from distortion of the planar porphyrin ring toward a transition state-like geometry by the N-alkyl substituent. N-Alkylporphyrins are also strong inhibitors of the enzyme ferrochelatase (9), which catalyzes the insertion of metal ions into porphyrin, a key step in heme biosynthesis. These observations have led to the proposal that ferrochelatase functions by straining the porphyrin ring toward a distorted conformation in which the two pyrrole N-σ electrons are more accessible to metal ions (10). Consistent with this notion, antibody 7G12, raised against a distorted N-alkylmesoporphyrin 1, was found to catalyze Cu(II) insertion into mesoporphyrin substrate 2 with a catalytic efficiency approaching that of ferrochelatase (Figure 1) (11). To establish the structural basis for catalysis by antibody 7G12, X-ray crystallographic and mutagenesis studies were carried out along with characterization of mutations along the path of affinity maturation from the germline precursor antibody to 7G12. EXPERIMENTAL PROCEDURESAntibody Cloning and Mutagenesis. Total RNA was isolated from the hybridoma cell line by the method of Chomczynski and Sacchi (12). Total RNA was enriched for messenger RNA coding for 7G12 or 5A5 variable regions by affinity chromatography with oligo(dT) cellulose (Pharmacia). Constant region 3′ primers were used to reverse transcribe cDNA. PCR 1 amplification with the 3′ constant region primer and the 5′ primers described by Huse et al. (13) yielded sufficient DNA for cloning and sequencing. After determination of the J κ and J H regions used by 7G12 and 5A5, 3′ primers corresponding to the respective J regions and containing restriction sites were synthesized and used in conjunction with the 5′ primers to PCR amplify DNA