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A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses.

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.

An interesting feature of trypanosome genome organization involves genes transcribed by RNA polymerase III. The U6 small nuclear RNA (snRNA), U-snRNA B (the U3 snRNA homolog), and 7SL RNA genes are closely linked with different, divergently oriented tRNA genes. To test the hypothesis that this association is of functional significance, we generated deletion and block substitution mutants of all three small RNA genes and monitored their effects by transient expression in cultured insect-form cells of Trypanosoma brucei. In each case, two extragenic regulatory elements were mapped to the A and B boxes of the respective companion tRNA gene. In addition, the tRNAThr gene, which is upstream of the U6 snRNA gene, was shown by two diferent tests to be expressed in T. brucei cells, thus confirming its identity as a gene. This association between tRNA and small RNA genes appears to be a general phenomenon in the family Trypanosomatidae, since it is also observed at the U6 snRNA loci in Leishmania pifanoi and Crithidiafasciculata and at the 7SL RNA locus in L. pifanoi. We propose that the A-and B-box elements of small RNA-associated tRNA genes serve a dual role as intragenic promoter elements for the respective tRNA genes and as extragenic regulatory elements for the linked small RNA genes. The possible role of tRNA genes in regulating small RNA gene transcription is discussed.Protozoa of the family Trypanosomatidae include a diverse number of organisms, several of which are parasites of great medical and economical importance. Interest in these cells stems not only from their parasitic lifestyles but also from evolutionary considerations which place the trypanosomatids among the deepest branches of the eukaryotic lineage (23). In recent years, the analysis of trypanosome gene expression has been quite rewarding and has provided evidence for novel mechanisms, including RNA editing, trans splicing, and polycistronic transcription units. Notwithstanding many advances in our understanding of trypanosome biology, our knowledge of promoter structures in these organisms is still limited to a few examples. These include three RNA polymerase (pol) I promoters of the procyclic acidic repetitive protein (PARP) (1, 3), variant surface glycoprotein (27,28), and rRNA (27) genes and one pol III promoter of the trans-spliceosomal U2 small nuclear RNA (snRNA) gene (4). For the protein-coding genes, it has become evident that the eukaryotic rule of one promoter for one gene does not apply. Rather, several protein-coding genes are transcribed as part of complex polycistronic transcription units. This functional economy in terms of usage of promoter signals is accompanied by a high density of genetic information in the nuclear genome, with protein-coding genes being separated only by a hundred to a few hundred nucleotides.Another interesting feature of trypanosome genome organization was noted for some of the genes transcribed by pol III (4, 19). In Trypanosoma brucei, the genes coding for the trans-spliceosomal U6 snRNA, the U3 snRNA homol...