Étude de la réponse immunitaire et de l'évolution de la quasiespèce du virus de l'hépatite C (VHC) durant la grossesse

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Structural Basis for Broad Neutralization of Hepatitis C Virus Quasispecies

Monoclonal antibodies directed against hepatitis C virus (HCV) E2 protein can neutralize cell-cultured HCV and pseudoparticles expressing envelopes derived from multiple HCV subtypes. For example, based on antibody blocking experiments and alanine scanning mutagenesis, it was proposed that the AR3B monoclonal antibody recognized a discontinuous conformational epitope comprised of amino acid residues 396–424, 436–447, and 523–540 of HCV E2 envelope protein. Intriguingly, one of these segments (436–447) overlapped with hypervariable region 3 (HVR3), a domain that exhibited significant intrahost and interhost genetic diversity. To reconcile these observations, amino-acid sequence variability was examined and homology-based structural modelling of E2 based on tick-borne encephalitis virus (TBEV) E protein was performed based on 413 HCV sequences derived from 18 subjects with chronic hepatitis C. Here we report that despite a high degree of amino-acid sequence variability, the three-dimensional structure of E2 is remarkably conserved, suggesting broad recognition of structural determinants rather than specific residues. Regions 396–424 and 523–540 were largely exposed and in close spatial proximity at the surface of E2. In contrast, region 436–447, which overlaps with HVR3, was >35 Å away, and estimates of buried surface were inconsistent with HVR3 being part of the AR3B binding interface. High-throughput structural analysis of HCV quasispecies could facilitate the development of novel vaccines that target conserved structural features of HCV envelope and elicit neutralizing antibody responses that are less vulnerable to viral escape

Phylogenetic analysis of HCV quasispecies derived from HCV-infected subjects.

<p>391 independently-obtained HCV E2 nucleic acid sequences derived from HCV-infected subjects were analyzed using the neighbor-joining method, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026981#s4" target="_blank">Materials and Methods</a>. 500 bootstrap re-samplings were performed to ascertain tree topology. Scale bar represents 0.1 nucleotide substitution per site.</p

Putative binding site of the AR3B monoclonal antibody on E2.

<p>A. Structural analysis of the proposed AR3B binding site on E2 (orange, red, and green) revealed that it would bury between 1092 and 1794 Ų at the surface of E2. Regions 396–424 (red) and 523–540 (orange) are closely associated compared with region 436–447 (green). B. Analysis of the 396–424 and 523–540 regions (magnified) showed that critical residues involved in AR3B binding (Ser 424, Gly 530, Asp 535 and Val 538) were largely surface-exposed and lied in close proximity to one another.</p

HCV E2 amino-acid sequence variability in HCV quasispecies derived from HCV-infected subjects.

<p>A. Consensus E2 amino-acid sequences were determined in 17 HCV-infected subjects and in the HCV-1a infected serum donor from ref. 14 based on the identity of the most frequent amino-acid residue at each position. 1: R or H; 2: V or I; 3: A or T; 4: R or Q. B. Variability at each amino acid position was computed using the Entropy-ONE Web tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026981#pone.0026981-Korber1" target="_blank">[58]</a>. C. Amino-acid segments that were shown to be important for binding of the AR3B antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026981#pone.0026981-Law1" target="_blank">[14]</a>.</p

Dendrogram of E2 structure clustering in HCV quasispecies derived from HCV-infected subjects.

<p>A. Analysis of the structural distance (RMSD) matrix of modelled E2 structures from HCV-infected subjects (n = 391) using the neighbour-joining algorithm showed a clustering of genotype 1 variants (boxed), with the exception of singular outlier structures (circled) and genotype 3a variants. B. Subtype 1a and 1b clustered separately but were structurally similar. E2 structures derived from patients infected with the same HCV subtype formed distinct clusters.</p

Differing Patterns of Liver Disease Progression and Hepatitis C Virus (HCV) Quasispecies Evolution in Children Vertically Coinfected with HCV and Human Immunodeficiency Virus Type 1

Hepatitis C virus (HCV) quasispeciation was studied in two children vertically coinfected with HCV and human immunodeficiency virus type 1 (HIV-1). HCV quasispecies diversification and liver injury were more significant in patient C1, who was immunocompetent with anti-HIV therapy, than in patient C2, who was immunosuppressed, in consistency with modulation of HCV quasispeciation and liver injury by immunocompetence in coinfected children