Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Family-specific degenerate primer design: a tool to design consensus degenerated oligonucleotides.

Designing degenerate PCR primers for templates of unknown nucleotide sequence may be a very difficult task. In this paper, we present a new method to design degenerate primers, implemented in family-specific degenerate primer design (FAS-DPD) computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. To assess their efficiency, four different genome collections were used, covering a wide range of genomic lengths: Arenavirus (10 × 10(4) nucleotides), Baculovirus (0.9 × 10(5) to 1.8 × 10(5) bp), Lactobacillus sp. (1 × 10(6) to 2 × 10(6) bp), and Pseudomonas sp. (4 × 10(6) to 7 × 10(6) bp). In each case, FAS-DPD designed primers were tested computationally to measure specificity. Designed primers for Arenavirus and Baculovirus were tested experimentally. The method presented here is useful for designing degenerate primers on collections of related protein sequences, allowing detection of new family members.

Molecular analysis of the virulence attenuation process in Junín virus vaccine genealogy.

The Junín virus strain Candid#1 was developed as a live attenuated vaccine for Argentine hemorrhagic fever. In this article, we report sequence information of the L and S RNAs of Junín virus Candid#1 and XJ#44 strains, and show the comparisons with the XJ13 wild-type strain and with other Junín virus strains, like Romero, IV4454 and MC2 strains, and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of all genes of three strains from the same vaccine genealogy, revealed different point mutations that could be associated with the attenuated phenotype. A 91% of the mutations found are consistent with a hypothesis of progressive attenuation of virulence from XJ13 to XJ#44 and to Candid#1; 39% of mutations were observed in XJ#44 and conserved in Candid#1, while another 52% of the mutations appeared only in Candid#1 strain. The remaining 9% corresponded to reverse mutations in the L gene. In summary, the present work shows a set of mutations that could be related to the virulence attenuation phenomenon. This information will serve as a starting point to study this biological phenomenon, provided that a reverse genetics system for Junín virus is developed to allow the generation of infectious virions with specific mutations.