Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles.

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV.

Study of a chimeric foot-and-mouth disease virus DNA vaccine containing structural genes of serotype O in a genome backbone of serotype Asia 1 in guinea pigs.

Since foot-and-mouth disease virus (FMDV) serotypes display a great genetic and antigenic diversity, there is a constant requirement to monitor the performance of FMDV vaccines in the field with respect to their antigenic coverage. To avoid possible antigenic changes in field FMDV isolates during their adaptation to BHK-21 cells, a standard step used in production of conventional FMDV vaccines, the custom-made chimeric conventional or DNA vaccines, in which antigenic determinants are replaced with those of appropriate field strains, should be constructed. Using this approach, we made a plasmid-based chimeric FMDV DNA vaccine containing structural genes of serotype O in the genome backbone of serotype Asia 1, all under the control of Human cytomegalovirus (HCMV) immediate early gene promoter. BHK-21 cells transfected with the chimeric DNA vaccine did not show cytopathic effect (CPE), but expressed virus-specific proteins as demonstrated by 35S-methionine labeling and immunoprecipitation. Guinea pigs immunized with the chimeric DNA vaccine produced virus-specific antibodies assayed by ELISA and virus neutralization test (VNT), respectively. The chimeric DNA vaccine showed a partial protection of guinea pigs challenged with the virulent FMDV. Although the chimeric DNA vaccine, in general, was not as effective as a conventional one, this study encourages further work towards the development of genetically engineered custom-made chimeric vaccines against FMDV.

Developmental regulation of heat shock protein 83 in Leishmania. 3' processing and mRNA stability control transcript abundance, and translation id directed by a determinant in the 3'-untranslated region.

Developmental gene regulation in trypanosomatids proceeds exclusively by post-transcriptional mechanisms. Stability and abundance of heat shock protein (HSP)70 and HSP83 transcripts in Leishmania increase at mammalian-like temperatures, and their translation is enhanced. Here we report that the 3'-untranslated region (UTR) of HSP83 (886 nucleotides) confers the temperature-dependent pattern of regulation on a chloramphenicol acetyltransferase (CAT) reporter transcript. We also show that the majority of the 3'-UTR sequences are required for increasing mRNA stability during heat shock. Processing of the HSP70 and HSP83 primary transcripts to poly(A)(+) mRNA was more efficient during heat shock; therefore, even when stability at 33 degrees C was reduced by deletions in the 3'-UTR, transcripts still accumulated to comparable and even higher levels. Translation of heat shock transcripts in Leishmania increases dramatically upon temperature elevation. Unlike in other eukaryotes in which the 5'-UTR confers preferential translation on heat shock transcripts, we show that translational control of HSP83 in Leishmania originates from its 3'-UTR. The 5'-UTR alone cannot induce translation during heat shock, but it has a minor contribution when combined with the HSP83 3'-UTR. We identified an element located between positions 201 and 472 of the 3'-UTR which is essential for increasing translation of the CAT-HSP83 reporter RNA at 33-37 degrees C. This region confers preferential translation during heat shock even in transcripts that were less stable. Thus, investigating the traditionally conserved heat shock response reveals that Leishmania parasites use unique pathways for translational control.

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia.

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.

Post transcriptional control of gene expression in Leishmania.

Leishmania parasites are ancient eukaryotes, characterized by unusual molecular mechanisms. We have used the gene encoding for Hsp83 as a model system for studying regulatory mechanisms that control developmental gene regulation. We previously showed that protein coding genes are regulated exclusively by post-transcriptional mechanisms, while no transcriptional activation could be observed even for the conserved Hsp83 gene. We now show that processing and maturation of the Hsp83 polycistronic primary transcripts is more efficient at elevated temperatures. The mature transcripts are more stable during heat shock, with regulation conferred by 3' UTRs. Poly(A) tails of Hsp83 are approximately 30 nucleotides long, as common for other low eukaryotes. The mechanism that signals differential degradation is still unclear, since it was not possible to detect differences in deadenylation of Hsp83 transcripts at varying temperatures. Heat shock transcripts are preferentially translated at 33-37 degrees C, but unlike Drosophila, translational regulation is controlled by a region within the 3' UTR. Using this traditionally conserved system emphasizes that regulatory mechanisms in Leishmania differ from those prevailing in other eukaryotes.

Effect of acidic pH on heat shock gene expression in Leishmania.

Temperature and pH shifts trigger differential gene expression and stage transformation in Leishmania. The parasites encounter dramatic fluctuations in the extra-cellular pH between the mid-gut of the sand fly (pH>8) and the phagolysosomal vacuole of mammalian macrophages (pH<6). The authors examined the effect of pH shifts on heat shock gene expression in Leishmania amazonensis and Leishmania donovani promastigotes. Acidic pH resulted in preferential stability of the hsp83 transcripts at 26 degrees C, but hsp transcripts were not preferentially translated as observed during heat shock. Pre-conditioning of promastigotes to acidic pH did not alter the temperature threshold for hsp synthesis but lead to an increase in hsp synthesis mainly in L. donovani at 37 degrees C, and to a slight decrease in the arrest of tubulin synthesis in L. amazonensis. The stage specific morphological alterations that take place in vitro correlated with the arrest in tubulin synthesis and occurred at different temperatures in L. donovani and L. amazonensis.