Evaluating the effect of formulation on the uptake of a ZIKA subunit vaccine candidate by antigen-presenting cells

A major issue with vaccination for Zika, Dengue and other flaviviruses is the potential for antibody-dependent enhancement (ADE) of disease, caused by the generation or boosting of infection-enhancing antibodies. To address this concern, a subunit vaccine is being developed against the Zika virus using a modified version of the envelope protein as the antigen which has been modified with glycan residues to mask the fusion loop region of the protein (Figure 1), which is a cross-reactive and immunodominant site strongly implicated in the generation of antibodies capable of ADE. With this subunit vaccine approach, there is a need to formulate with an appropriate adjuvant to enhance the immunogenicity of the modified envelope protein. In this study we have evaluated a range of adjuvants using flow cytometry and fluorescence microscopy and have determined the relative uptake by human Antigen-presenting cells (APCs). Various combinations of clinically acceptable adjuvant materials: Alhydrogel®, 3D-(6-acyl) PHADTM (a synthetic analogue of MPL) and QS21, were tested using liposomal formulations. In addition, the modified Zika envelope protein was compared to that of wild type Zika antigen, similarly formulated. Please click Additional Files below to see the full abstract

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

<div><p>Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.</p> </div

Density fraction neutralization of WT viruses by patient IgG, anti-SR-BI, anti-CD81, anti-LDL and anti-HDL.

<p>WT viruses were resolved using an iodixanol step gradient. (A) Infections of individual fractions in the presence of patient IgG, anti-SR-BI (C167) or anti-CD81 (JS-81). (B) Similar infections as described in (A) were performed with pre-incubated viruses with anti-ApoE, anti-HDL or anti-LDL. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of two independent experiments.</p

Dose-dependent inhibition of alanine mutants by conformational anti-E2 AR3A antibodies and patient-derived IgG.

<p>Huh-7.5 cells were infected with Luc-Jc1 WT or with the indicated E2 mutant viruses as follows: viruses were with pre-incubated with anti-E2 AR3A antibodies (A & B) or patient-derived IgG (C & D), at the given concentrations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of three independent experiments. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD).</p

Buoyant density infectivity and RNA of WT and mutant viruses.

<p>The indicated viruses were resolved using an iodixanol step gradient. For each fraction HCV infectivity in Huh-7.5 cells and RNA (RT-qPCR) were determined. Values are plotted against the density of the respective fraction. The infectivity is expressed as a percentage of the total infectivity obtained from all fractions. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of two independent experiments.</p

HCV E2 glycoprotein contains conserved basic residues in different regions.

<p>(A) Scheme of E2 putative GAG-binding sites and other regions important for entry and for proper protein folding. Amino acid numbers refer to positions in the polyprotein sequence of the H77 prototype isolate. N: glycosylation sites, HVR1, 2, 3: hyper-variable region 1, 2, 3, TM: transmembrane domain. (B) Frequency of basic residues at the positions analyzed in this study. The height of the box in each bar indicates the frequency of histidine (H, white box), lysine (K, light grey box) and arginine (R, dark grey box). The frequency of the basic residues at each position was calculated by dividing the number of basic residues by the total number of sequences (2073 sequences) and is expressed as a percentage.</p

Effects of alanine substitutions for E2 basic amino acids on HCVcc infectivity, heparin neutralization, core release and anti-ApoE inhibition.

<p>(A) HCVcc infectivity of Luc-Jc1 WT or mutants. Infectivity of each mutant is expressed as a percentage of the infectivity level observed for the WT. The cut-off was set according to the infectivity observed for the E1AA mutant. Values shown represent the mean for three assays (± SD). Columns next to infectivity rates represent percentage (%) of E2 protein immunoprecipitated (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#pone-0052651-g002" target="_blank">Fig. 2</a>) and core supernatant release relative to WT virus. (B) Huh-7.5 cells were infected with Luc-Jc1 WT or with the indicated E2 mutant viruses in the presence of GAG antagonists (chondroitin sulfate or heparin, 200 µg/ml). The heparin neutralization scale was set relative to chondroitin sulfate neutralization. (C & D) Dose-dependent inhibition of mutantś entry by anti-ApoE. Infections were performed with WT or mutant viruses as follows: viruses were pre-incubated with anti-ApoE antibodies at the given concentrations and then added to the cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of three independent experiments. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD).^a NA, not applicable.</p