Characterization of recombinant yellow fever-dengue vaccine viruses with human monoclonal antibodies targeting key conformational epitopes.

The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these laboratory markers relate to vaccine efficacy and safety.

Characterization of the structural modifications accompanying the loss of HBsAg particle immunogenicity.

The aim of this work was to further understand the relationship between the immunogenicity and the structure of Hepatitis B surface antigen (HBsAg) particles used in Hepatitis B vaccines. To reach this aim, we compared by using a large range of techniques, the structure and properties of untreated particles with those of particles stored for 3 weeks at +60°C, a treatment which resulted in a loss of HBsAg antigenicity (toward RF-1 mAb) and immunogenicity (in mice). While untreated particles imaged by electron microscopy and atomic force microscopy appeared as isolated nanoparticles of ∼ 20nm, heated particles appeared as long chains of particle aggregates with a partial loss of their protein protrusions. Moreover, infrared spectroscopy and circular dichroism revealed that the secondary structure of the S proteins was significantly affected, with a loss of 10% of their α-helix content. Steady-state and time-resolved fluorescence data further revealed strong modifications of the most emitting Trp residues at the particle surface, confirming significant changes in the conformation of the S proteins. Moreover, modifications in the organization of both the lipid core and lipid membrane surface of the heated particles were evidenced by environment-sensitive 3-hydroxyflavone probes. Taken together, our data evidenced a clear relationship between the bona fide S protein structure and lipid organization notably at the particle surface and the particle immunogenicity.

Method for the simultaneous assay of the different poliovirus types using surface plasmon resonance technology.

The inactivated polio vaccine (IPV) contains viral samples that belong to serotypes 1, 2 and 3. We report here a surface plasmon resonance (SPR)-based technique that permits the simultaneous assay of the individual viral types in the IPV as well as in different bulk intermediates from the industrial vaccine production process. Monoclonal antibodies specific to each of the 3 viral types along with a negative control antibody are captured via an anti-IgG antibody on the surface of the 4 flow cells of the SPR instrument. The viral samples are then injected over these flow cells and the increase in resonance units as a result of virus binding is measured. The method was calibrated by an analysis of the European Working Standard (EWS) for poliovirus vaccines. We show that the antibodies used recognize viruses with functional affinities in the picomolar range permitting an effective capture of the antigen. In addition we demonstrate that the antibodies are highly specific to a given virus type and that the heat induced destruction of the D-antigen abolishes antibody recognition entirely. The technique was found to be reproducible and robust and its response was linear to the antigen concentration. Due to the rapidity of analysis this technique permits an almost real-time follow-up of the industrial production process and may present an alternative to the established ELISA assay for the analysis of the intermediates and the final product.