Genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses.

A tetravalent dengue vaccine based on four live, attenuated, chimeric viruses (CYD1-4), constructed by replacing the genes coding for premembrane (prM) and envelope (E) proteins of the yellow fever (YF)-17D vaccine strain with those of the four serotypes of dengue virus, is in clinical phase III evaluation. We assessed the vaccine's genetic stability by fully sequencing each vaccine virus throughout the development and manufacturing process. The four viruses displayed complete genetic stability, with no change from premaster seed lots to bulk lots. When pursuing the virus growth beyond bulk lots, a few genetic variations were observed. Usually both the initial nucleotide and the new one persisted, and mutations appeared after a relatively high number of virus duplication cycles (65-200, depending on position). Variations were concentrated in the prM-E and non-structural (NS)4B regions. PrM-E variations had no impact on lysis-plaque size or neurovirulence in mice. None of the variations located in the YF-17D-derived genes corresponded with reversion to the wild-type Yellow Fever sequence. Variations in NS4B likely reflect virus adaptation to Vero cells growth. A low to undetectable viremia has been reported previously [1-3] in vaccinated non-human and human primates. Combined with the data reported here about the genetic stability of the vaccine strains, the probability of in vivo emergence of mutant viruses appears very low.

High stability of yellow fever 17D-204 vaccine: a 12-year restrospective analysis of large-scale production.

We have retrospectively analyzed 12 bulk lots of yellow fever vaccine Stamaril, produced between 1990 and 2002 and prepared from the same seed lot that has been in continuous use since 1990. All vaccine batches displayed identical genome sequence. Only four nucleotide substitutions were observed, compared to previously published sequence, with no incidence at amino-acid level. Fine analysis of viral plaque size distribution was used as an additional marker for genetic stability and demonstrated a remarkable homogeneity of the viral population. The total virus load, measured by qRT-PCR, was also homogeneous pointing out reproducibility of the vaccine production process. Mice inoculated intracerebrally with the different bulks exhibited a similar average survival time, and ratio between in vitro potency and mouse LD(50) titers remained constant from batch-to-batch. Taken together, these data demonstrate the genetic stability of the strain at mass production level over a period of 12 years and reinforce the generally admitted idea of the safety of YF17D-based vaccines.

Human T cell leukaemia virus type I env gene-transfected HeLa cells display a decrease in cell fusion ability.

The envelope (env) gene of human T cell leukaemia virus type I (HTLV-I) was inserted into an expression vector, referred to as phMTenv, under the transcriptional control of the human metallothionein IIa gene promoter (hMT-IIa). When this vector was transiently transfected in HeLa cells treated with hMT-IIa inducers, formation of multinucleated cells was observed, indicating the expression of functional surface and transmembrane glycoproteins. Of several HeLa cell clones transfected with phMTenv together with a plasmid carrying the neomycin resistance gene and isolated after selection in G418-containing medium, env mRNA was detected in only two, in the presence of hMT-IIa inducers. Viral glycoproteins were found to be weakly expressed as detected in immunoprecipitation assays of 125I-surface-labelled cells. These env-transfected HeLa cell clones, although unable to form syncytia when cocultivated with untransfected control HeLa cells, retained the capacity to fuse with HTLV-I-producing C91PL T cells. However, a significant decrease in their fusogenic ability was observed, after treatment with hMT-IIa inducers. Under identical experimental conditions, control HeLa cell clones stably transformed with the same plasmid, but lacking the env gene, were still able to fuse with C91PL cells. These observations suggest that a post-transcriptional step in HTLV-I env expression is impaired, probably leading to the establishment of superinfection interference.

Critical involvement of human T cell leukaemia virus type I virions in mediating the viral mitogenic effect.

Human T cell leukaemia virus type I (HTLV-I) is a direct activator of human resting T lymphocytes. The present study was undertaken to delineate further the role of viral particles and to define the involvement of envelope glycoproteins in the induction of T cell mitogenic stimulation. Virus-producing cells treated with paraformaldehyde (PFA) were found to be unable to induce the formation of syncytia, but still able to trigger the proliferation of resting T cells. Likewise, PFA-treated virus particles were still mitogenic. These results suggest that the mitogenic event is triggered before the fusion of the envelope with the cell membrane. Furthermore, HTLV-I envelope-expressing cells obtained after infection of C8166/45 cells (HTLV-I-transformed, but defective in virion production) with an HTLV-I envelope recombinant vaccinia virus were unable to activate normal T cells. Human immuno-deficiency virus type 1 particles produced by C8166/45 cells were also devoid of mitogenic ability. However, when HTLV-I viral preparations were purified by chromatography, only the virion-containing fractions were found to be mitogenic for human resting T lymphocytes. This mitogenic activity was partially abolished by preincubating the purified virus with a monoclonal antibody directed to the surface envelope glycoprotein. Finally, treatment of HTLV-I-transformed cells by tunicamycin, an inhibitor of N-linked glycosylation, led to the production of virus particles with a decreased mitogenic activity. Collectively, these observations suggest that the HTLV-I mitogenic activity is triggered by the contact of HTLV-I virions with T cells.