Immune-driven adaptation of hepatitis B virus genotype D involves preferential alteration in B-cell epitopes and replicative attenuation—an insight from human immunodeficiency virus/hepatitis B virus coinfection

Abstract

An important driving force behind the sequence diversity of hepatitis B virus (HBV) is viral adaptation to host immune responses. To gain an insight into the impact of host immunity on genetic diversification and properties of HBV, we characterized HBV of genotype D from treatment-naive hepatitis B e antigen-positive (EP) and hepatitis B e antigen-negative (EN) patients with chronic hepatitis B (CHB), where HBV is under stronger immune pressure, with that of HBV derived from human immunodeficiency virus (HIV)/HBV-coinfected individuals, where HIV infection has significantly weakened the immune system. Full-length sequence analysis showed that HBV heterogeneity was most extensive in EN-CHB followed by EP-CHB and HIV/HBV coinfection. The relative magnitude of non-synonymous changes within B-cell epitopes was greater than that in T-cell epitopes of HBV open reading frames (ORFs) in both EP-CHB and EN-CHB. Nine amino acid substitutions were identified in B-cell epitopes and one in a T-cell epitope of HBV in EN-CHB, most of which resulted in altered hydrophobicities, as determined using the Kyte and Doolittle method, relative to wild-type residues found in HBV from the HIV-positive group. Additionally, 19 substitutions occurred at significantly higher frequencies in non-epitope regions of HBV ORF-P in EN-CHB than HIV/HBV-coinfected patients. In vitro replication assay demonstrated that the substitutions, particularly in reverse transcriptase and RNaseH domains of ORF-P, resulted in a decline in replication capacity of HBV. Hence, our results indicate that HBV adapts to increasing immune pressure through preferential mutations in B-cell epitopes and by replicative attenuation. The viral epitopes linked to immune response identified in this study bear important implications for future HBV vaccine studies.

The Correlation Between Hepatitis B Virus Precore/Core Mutations and the Progression of Severe Liver Disease

“…The location of mutations that are associated with HBV-related disease progression are variable; Kim et al ( 2012 ) found that such core protein mutations were primarily located within CD4 + T-cell epitopes that were designated MHC class II restricted regions (Kim et al, 2012 ), while Carman et al ( 1997 ) found that HBcAg mutations associated with progressive disease were concentrated in B-cell epitopes (Carman et al, 1997 ). A separate study of Indian patients infected with HBV genotype D identified advantageous mutations in B-cell epitopes during the progression of chronic hepatitis B infection (Mondal et al, 2015 ). Similarly, Assar et al ( 2012 ) reported several mutations responsible for immune evasion in T helper and B-cell epitopes in Iranian patients infected with HBV genotype D (Assar et al, 2012 ).…”

Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

“…Our in vitro replication assay depicted a significant reduction in the replication efficiency of HBV in presence of these rt and rh mutations, which could partly explain the low copy numbers of HBV in OBI-positive patients. Further, the slow replication rates are often advantageous for viruses as by generating fewer virus-derived peptides, they could avoid being eliminated by host immune system and thereby have a greater likelihood of persistence in the host 34 .…”

“…To determine the genetic affiliation of OHBV strains, the full-length sequences obtained in the study were compared with representative sequences of ten HBV genotypes (A–J) retrieved from GenBank. Alignments were carried out using CLUSTALX software and a phylogenetic tree was constructed by the neighbour joining (NJ) method using the Jukes–Cantor model in MEGA software version 6 34 . To identify the potential mutations in HBV that are associated with occult status, the complete sequences of OHBV belonging to genotype D (OHBV/D) were compared with 167 HBV/D sequences derived from HBsAg-positive carriers that comprised of 62 sequences from Eastern India (EI) and 105 sequences from other geographical locations (WRLD), all of which are available in GenBank.…”

“…All OHBV-specific mutations, identified in this study, except those in ORF-P, were introduced separately in wt-HBV clone (pJET-HBV-wt) by Site-Directed Mutagenesis (Agilent Technologies) with specific mutagenic oligonucleotides (Supplementary Table S3 ) and confirmed by sequencing. The substitutions detected in reverse transcriptase (rt) and RNaseH (rh) domains of ORF-P of OHBV were introduced in replication competent HBV/D cloned in pTriEX-Mod vector (pTriEX-Mod-HBV-D) (a kind gift from Prof. Fabien Zoulim, INSERM, France) by cassette insertion method 34 . Briefly, an 1802 bp region of ORF-P (nt.3164–1783) was amplified using primers F4 and R10 (Supplementary Table S2 ) from pJET-HBV-wt and from previously sequenced OHBV/D DNA included in the study.…”

“…Forty-eight hours post-transfection, Huh7 cells were harvested and HBV-DNA was isolated from both intracellular core particles 34 as well as from culture supernatant. To isolate the intracellular HBV DNA from the viral core particles, HBV transfected Huh7 cells were lysed with lysis buffer (50 mM Tris-HCL, pH = 8; 1 mM EDTA; 1% NP40), centrifuged at a speed of 10,000 rpm for 1 minute at room temperature followed by DNase I (Roche) and RNase A (Fermentas) digestion at 37 °C for 3 hours.…”