Phenotypic and genetic characterization of a next generation live-attenuated yellow fever vaccine candidate.

We assessed the genetic and phenotypic characteristics of a yellow fever vaccine candidate, which was cloned from a YF-VAX substrain selected for growth in Vero cells (vYF-247), during the manufacturing process from the master seed lot (MSL) and working seed lot (WSL) through to the drug substance (DS) stage. There were nine minor nucleotide variants observed from the MSL to the DS stage, of which five led to amino acid changes. The variant positions were, however, not known risks for any virulence modification. vYF-247 exhibits a homogenous plaque size profile (as expected for a cloned vaccine candidate) composed of small plaques (<1 mm) that remained consistent throughout the manufacturing process. In addition, there was no change in the viral replication rate. Of note, the DS sequences across the two manufacturing campaigns (2018 and 2019) were very similar suggesting a high batch-to-batch consistency. All MSL, WSL and DS batches exhibited similar neurovirulence profiles in mice and had a more attenuated neurovirulence phenotype than the YF-VAX (egg-based vaccine) comparator. Overall, the neurovirulence phenotype of vYF-247 does not change from MSL, WSL to DS. These data collectively support the safety and genetic stability of vYF-247 during the production process.

Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study.

During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells.

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve NK cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein; it also binds to WNV virus-like particles. These WNV-virus-like particles and WNV-domain III of WNV envelope protein directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK degranulation. Finally, WNV infection of cells results in increased binding of rNKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFN-gamma secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 Abs. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein.

Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein.

Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA.