A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1.

The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

Detection of 13 Hypervariable Region 1 (Hv1) SNPs using single-base extension (Sbe) primers in parallel with Sanger sequencing.

The aim of this study was to determine whether single-base extension (SBE) chemistry can be applied to forensic practice of testing the target single nucleotide polymorphisms (SNPs) of the mitochondrial DNA (mtDNA) Hypervariable Region 1 (HV1). Despite itsweak discrimination power, high copy number of mtDNA per cell and its stability against degradation guarantee mtDNA testing a place in modern forensic genetics. In this research, buccal swab samples were obtained from 294 individuals from Bosnia and Herzegovina. Following DNA isolation using QIAamp® DNA Mini Kit, full sequencing of HV1 was completed using chain-termination Sanger sequencing method. SBE reactions were then performed by targeting 13 SNPs that were identified to be the most frequent in the study population. Uniplex SBE reactions for each individual SNP, as well as two multiplex reactions were prepared for both pure and mixed samples. The results showed complete agreement of the Sanger sequencing results with SBE reactions for both uniplex and multiplex reactions. The results obtained with SBE were encouraging in regard to multiplexing and processing of the mixed samples, since the allele of the minor contributor to the sample was observed in SBE electropherogram in all prepared mixtures. SBE method is limited by the fact that only target SNPs of interest will be analyzed, meaning that they must be carefully selected and curated for each population. However, typing with SBE protocol is accurate, as compared to the golden standard of Sanger sequencing, but was more time- and labor-efficient and simpler to analyze, therefore offering a suitable alternative when Sanger sequencing is not available, given that polymorphic SNPs are known for the population of interest.

Poly_NumtS_430 or HSA_NumtS_587 observed in massively parallel sequencing of the mitochondrial HV1 and HV2 regions.

An increasing number of studies on massively parallel sequencing of mitochondrial DNA (mtDNA) have been reporting identification of various types of noise or off-target sequences. Herein, we report that an off-target haplotype (sequence length 192 bp) observed in MiSeq data of mtDNA at nucleotide position 16,209-16,400 was likely caused by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from Volunteers #001-004 and Control DNA 007 were amplified with our multiplex system of the B (15,998-16,172), C (16,209-16,400), and E (30-289) regions using 2000 copies of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original Excel macro to sum up the read counts as the phased variant calls for each region. An off-target haplotype differing at 19 sites against the revised Cambridge reference sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in 9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1], HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome 11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1) which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence of the off-target haplotype was not contained in the latest human reference genome sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically distant from modern human mtDNA and not directly connected to them. In conclusion, observed off-target haplotype amplified by our multiplex system was likely caused by Poly_NumtS_430 or HSA_NumtS_587.

A bivalent HCV peptide vaccine elicits pan-genotypic neutralizing antibodies in mice.

Vaccine development for antigenically variable pathogens has faltered because extreme genetic diversity precludes induction of broadly neutralizing antibodies (nAB) with classical vaccines. Here, using the most variable epitope of any known human pathogen (HVR1 of HCV), we describe a novel approach capable of eliciting broadly neutralizing antibodies targeting highly variable epitopes. Our proof-of-concept vaccine elicited pan-genotypic nAB against HCV variants differing from the immunogen sequences by more than 70% at the amino acid level. These findings suggest broadly nAB to highly variable pathogens can be elicited by vaccines designed to target physicochemically conserved residues within hypervariable epitopes.

Hypervariable Region 1 in Envelope Protein 2 of Hepatitis C Virus: A Linchpin in Neutralizing Antibody Evasion and Viral Entry.

Chronic hepatitis C virus (HCV) infection is the cause of about 400,000 annual liver disease-related deaths. The global spread of this important human pathogen can potentially be prevented through the development of a vaccine, but this challenge has proven difficult, and much remains unknown about the multitude of mechanisms by which this heterogeneous RNA virus evades inactivation by neutralizing antibodies (NAbs). The N-terminal motif of envelope protein 2 (E2), termed hypervariable region 1 (HVR1), changes rapidly in immunoglobulin-competent patients due to antibody-driven antigenic drift. HVR1 contains NAb epitopes and is directly involved in protecting diverse antibody-specific epitopes on E1, E2, and E1/E2 through incompletely understood mechanisms. The ability of HVR1 to protect HCV from NAbs appears linked with modulation of HCV entry co-receptor interactions. Thus, removal of HVR1 increases interaction with CD81, while altering interaction with scavenger receptor class B, type I (SR-BI) in a complex fashion, and decreasing interaction with low-density lipoprotein receptor. Despite intensive efforts this modulation of receptor interactions by HVR1 remains incompletely understood. SR-BI has received the most attention and it appears that HVR1 is involved in a multimodal HCV/SR-BI interaction involving high-density-lipoprotein associated ApoCI, which may prime the virus for later entry events by exposing conserved NAb epitopes, like those in the CD81 binding site. To fully elucidate the multifunctional role of HVR1 in HCV entry and NAb evasion, improved E1/E2 models and comparative studies with other NAb evasion strategies are needed. Derived knowledge may be instrumental in the development of a prophylactic HCV vaccine.

Association study of mitochondrial DNA polymorphisms with type 2 diabetes in Tunisian population.

Mitochondrial DNA (mtDNA) variation may play an important role in the pathogenesis of type 2 diabetes (T2Ds). In this study, we aimed to explore whether mtDNA variants contribute to the susceptibility to T2Ds in a Tunisian population. The hypervariable region 1 (HVS1) of the mtDNA of 64 T2Ds patients and 77 healthy controls was amplified and sequenced. Statistical analysis was performed using the STATA program. Analysis of the total screened variants (N = 88) from the HVS1 region showed no significant difference in the distribution of all polymorphisms between T2Ds and controls, except for the variant G16390A which was more frequent in T2Ds (15.9%) than in controls (5.4%) (p = 0.04). The association of G16390A was not detected after multivariate regression analysis. Similarly, analysis of the distribution of mitochondrial haplogroups within our dataset showed 18 distinct major haplogroups with no significant difference between T2Ds and controls. Except, the weakly association found for the G16390A variant, our results showed that none of the tested polymorphisms from the HVS1 region have a major role in T2Ds pathogenesis in the studied Tunisian population even when taking into account the population stratification.

Analysis of mitochondrial DNA HVR1 haplotype of pure-bred domestic dogs in Japan.

To develop DNA markers for forensic analysis, we examined the hypervariable region 1 (HVR1) sequences of 447 pure-bred domestic dogs (Canis lupus familiaris) that had been bred and raised in Japan. HVR1 is a 660-bp stretch of mitochondrial (mt) DNA. Among the 447 HVR1 sequences examined, we identified 58 haplotypes from 47 single nucleotide polymorphisms (SNPs) and two insertion-deletion (InDel) polymorphisms. The haplotype diversity inferred from inter-breed analysis (N=154, 88 breeds) was 0.929±0.011. Intra-breed analysis showed that the haplotype diversity of Golden Retrievers (N=53), Labrador Retrievers (N=67), Miniature Dachshunds (N=61), Toy Poodles (N=62), and Welsh Corgis (N=50) was 0.624±0.052, 0.722±0.029, 0.922±0.010, 0.877±0.020, and 0.443±0.084, respectively. The results of this genotype analysis were used to construct a dataset consisting of dog mtDNA HVR1 sequences for use in forensic applications in Japan.