Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF), and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC). Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc) derived capsid-like particles (CLPs) to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.     

丙型肝炎病毒高变区1模拟表位的交叉反应性分析

研究丙型肝炎病毒(Hepatitis C virus,HCV)高变区1(Hypervariable region 1,HVR1)抗原表位的交叉反应性,获取高反应性的抗原表位.设计并合成5种HVR1模拟表位基因,构建编码HVR1模拟表位的表达载体,表达并纯化表位蛋白.ELISA法检测表位蛋白与35份HCV抗体阳性血清的交叉反应性.包装HCV假病毒(HCV pseudotype particles,HCVpp),评价表位蛋白免疫BALB/c鼠血清在假病毒感染Huh7.5细胞中的作用.结果表明,表达纯化的5种表位蛋白(P1、P2、P5、P6、P8)均可与HCV抗体阳性血清反应,阳性反应率分别为54.3%(P1)、62.9%(P2)、80%(P5)、68.6%(P6)、54.3%(P8).表位蛋白P6、P8免疫BALB/c鼠血清对HCV假病毒感染Huh7.5细胞具明显的抑制作用.结果提示,选取的HVR1模拟表位在HCV感染免疫与疫苗研制中可能具有潜在的价值.     

丙型肝炎病毒高变区基因变异特点初探

对两例ＨＣＶＲＮＡ持续阳性者分三个时间点随访五年,测定了ＨＣＶ的高变区基因序列,每个时间点平均测定２８个克隆,总共测定了１６８个克隆。研究发现ＨＣＶ高变区基因变异有五个明显特点：（１）变异程度大,高变区至少有８９％的核苷酸位点都可能发生变异;（２）变异形式多样,除常见的替代突变外,缺失突变（缺失１、２或３个核苷酸）的克隆数占总克隆数的２２．５％;（３）优势克隆明显,即有若干个克隆高变区的核苷酸和氨基酸序列相同;（４）类似株现象严重;（５）变异幅度大。该研究说明ＨＣＶ高变区基因变异具有高度复杂多样性。     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(kappa)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The V(H) of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the V(L) kappa sequences were highly homologous. The affinity (K(d)) of 2P24 and 15H4 (10(-9) or 10(-8) M with two immunizing peptides and 10(-8) M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.     

Evolution of Hypervariable Region 1 of Hepatitis C Virus in Primary Infection

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [K_a], synonymous mutations per synonymous site [K_s], K_a/K_s ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2.11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rK_a, rK_s, rK_a/rK_s ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCV HVR1模拟表位融合蛋白.用Western blot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况.皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应.结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35).融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCV HVR1合成肽发生交叉反应.本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值.     

Long-Term Evolution of the Hypervariable Region of Hepatitis C Virus in a Common-Source-Infected Cohort

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.     

丙型肝炎病毒高变区及其变异机制

丙型肝炎病毒(HCV)包膜糖蛋白E2所含的3个高变区(HVR1/2/3)是HCV基因组中变异程度最高的部分,参与病毒识别、黏附和入侵等过程。了解HVR的特点、变异规律及其生物学意义,不仅有利于阐明HCV持续感染及耐药机制,还可为未来HCV疫苗和新型治疗药物的设计提供理论依据。     

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.     

Evolution of Hepatitis C Virus Quasispecies in Hypervariable Region 1 and the Putative Interferon Sensitivity-Determining Region during Interferon Therapy and Natural Infection

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.     

Three Different Functional Microdomains in the Hepatitis C Virus Hypervariable Region 1 (HVR1) Mediate Entry and Immune Evasion

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25–27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16–24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1–13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.     

Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.     

丙型肝炎病毒中和抗体的研究进展

丙型肝炎病毒(HCV)感染在全球范围内流行,如何能够有效控制和阻断HCV的感染和传播成为研究热点。HCV借助其极高的变异率逃避机体的免疫监视,并通过多种机制得以侵入、繁殖,引发一系列病理改变。因此,在感染初期激发机体有效的体液免疫反应,产生强烈而又广泛的中和作用,对阻断入侵和感染至关重要。我们对HCV中和抗体的研究进展予以简要综述。     

Hypervariable Region 1 Deletion and Required Adaptive Envelope Mutations Confer Decreased Dependency on Scavenger Receptor Class B Type I and Low-Density Lipoprotein Receptor for Hepatitis C Virus

Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. We previously showed that the viability of HVR1-deleted JFH1-based recombinants with Core-NS2 of H77 (H77_ΔHVR1, genotype 1a) and S52 (S52_ΔHVR1, genotype 3a) in Huh7.5 cells was rescued by E2 substitutions N476D/S733F and an E1 substitution, A369V, respectively; HVR1-deleted J6 (J6_ΔHVR1, genotype 2a) was fully viable. In single-cycle production assays, where HCV RNA was transfected into entry-deficient Huh7-derived S29 cells with low CD81 expression, we found no effect of HVR1 deletion on replication or particle release for H77 and S52. HCV pseudoparticle assays in Huh7.5 cells showed that HVR1 deletion decreased entry by 20- to 100-fold for H77, J6, and S52; N476D/S733F restored entry for H77_ΔHVR1, while A369V further impaired S52_ΔHVR1 entry. We investigated receptor usage by antibody blocking and receptor silencing in Huh7.5 cells, followed by inoculation of parental and HVR1-deleted HCV recombinants. Compared to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77_{ΔHVR1/N476D/S733F}, H77_N476D/S733F, S52_ΔHVR1/A369V, and S52_A369V, but not for J6_ΔHVR1. Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77_N476D/S733F and S52_A369V. Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry.     

Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous,but Not Heterologous,Challenge

The importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro. Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.     

Hepatitis C Virus: Assembly and Release of Virus Particles

Hepatitis C virus is a blood-borne virus that typically establishes a chronic infection in the liver, which often results in cirrhosis and hepatocellular carcinoma. Progress in understanding the complete virus life cycle has been greatly enhanced by the recent availability of a tissue culture system that produces infectious virus progeny. Thus, it is now possible to gain insight into the roles played by viral components in assembly and egress and the cellular pathways that contribute to virion formation. This minireview describes the key determining viral and host factors that are needed to produce infectious virus.