Envelope Glycoprotein E2 Research Articles

A Novel Chimeric Anti-HCV Peptide Derived from Camel Lactoferrin and Molecular Level Insight on Its Interaction with E2

In the present study, a novel chimeric peptide was derived from camel lactoferrin designed with a considerable anti-HCV activity and its neutralization mechanism was predicted by molecular modelling tools. A novel anti-HCV peptide derived from camel lactoferrin (cLF36) was designed and expressed it recombinantly in HEK-293-T cells. Anti-viral activity of this peptide was evaluated against hepatitis C virus by Real-time PCR assay in vitro. Finally, to have a better insight into the mode of action of peptide on HCV entry inhibition, we examined the interaction of cLF36 with envelope glycoprotein E2 by molecular dynamic simulation. This chimeric peptide had significant inhibitory effects on both HCV entry (44 µg/mL) and viral replication (88 µg/mL) under in vitro (p > 0.01). Moreover, cLF36 peptide was not toxic to HEK cells as a normal cell at twofold of its anti-viral concentrations for HCV entry and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis (2.5%) against human RBCs (red blood cells). The results of in silico analysis showed that cLF36 interacted with β-sandwich and front layer of E2 protein as two potential CD81 binding sites. We generated and characterized a new camel lactoferrin derived HCV inhibitors. This peptide blocked HCV entry and also intracellular HCV replication in cell culture experiment.

Localized Outbreaks of Epidemic Polyarthritis among Military Personnel Caused by Different Sublineages of Ross River Virus, Northeastern Australia, 2016-2017.

Two outbreaks of epidemic polyarthritis occurred among Australian Defence Force personnel during and following short military exercises in the Shoalwater Bay Training Area, northeastern Australia, in 2016 and 2017. Ross River virus (RRV) IgM was detected in acute-phase serum samples from most patients (28/28 in 2016 and 25/31 in 2017), and RRV was recovered from 4/38 serum samples assayed (1/21 in 2016 and 3/17 in 2017). Phylogenetic analyses of RRV envelope glycoprotein E2 and nonstructural protein nsP3 nucleotide sequences segregated the RRV isolates obtained in 2016 and 2017 outbreaks into 2 distinct sublineages, suggesting that each outbreak was caused by a different strain of RRV. The spatiotemporal characteristics of the 2016 outbreak suggested that some of the infections involved human-mosquito-human transmission without any intermediate host. These outbreaks highlight the importance of personal protective measures in preventing vectorborne diseases for which no vaccine or specific prophylaxis exists.

Rapid decrease in titer and breadth of neutralizing anti-HCV antibodies in HIV/HCV-coinfected patients who achieved SVR

The main targets for neutralizing anti-hepatitis C virus (HCV) antibodies (HCV-nAbs) are the E1 and E2 envelope glycoproteins. We have studied the characteristics of HCV-nAbs through a retrospective study involving 29 HIV/HCV-coinfected patients who achieved sustained virological response (SVR) with peg-IFNα + ribavirin anti-HCV therapy. Plasma samples at baseline and week 24 after SVR were used to perform neutralization assays against five JFH1-based HCV recombinant viruses coding for E1 and E2 from genotypes 1a (H77), 1b (J4), 2a (JFH1), 3a (S52) and 4a (ED43). At baseline, the majority of plasma samples neutralized 1a, 1b, 2a, and 4a, but not 3a, genotypes. Twenty-four weeks following SVR, most neutralizing titers declined substantially. Furthermore, titers against 3a and 2a were not detected in many patients. Plasma samples with high HCV-nAb titers neutralized all genotypes, and the highest titers at the starting point correlated with the highest titers at week 24 after SVR. In conclusion, high titers of broad-spectrum HCV-nAbs were detected in HIV/HCV-coinfected individuals, however, those titers declined soon after SVR.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1–6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1–6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

Syntenin regulates hepatitis C virus sensitivity to neutralizing antibody by promoting E2 secretion through exosomes

Assembly of infectious hepatitis C virus (HCV) particles is known to involve host lipoproteins, giving rise to unique lipo-viro-particles (LVPs), but proteome studies now suggest that additional cellular proteins are associated with HCV virions or other particles containing the viral envelope glycoprotein E2. Many of these host cell proteins are common markers of exosomes, most notably the intracellular adaptor protein syntenin, which is required for exosome biogenesis. We aimed to elucidate the role of syntenin/E2 in HCV infection. Using cell culture-derived HCV, we studied the biogenesis and function of E2-coated exosomes in both hepatoma cells and primary human hepatocytes (PHHs). Knockout of syntenin had a negligible impact on HCV replication and virus production, whereas ectopic expression of syntenin at physiological levels reduced intracellular E2 abundance, while concomitantly increasing the secretion of E2-coated exosomes. Importantly, cells expressing syntenin and HCV structural proteins efficiently released exosomes containing E2 but lacking the core protein. Furthermore, infectivity of HCV released from syntenin-expressing hepatoma cells and PHHs was more resistant to neutralization by E2-specific antibodies and chronic-phase patient serum. We also found that high E2/syntenin levels in sera correlate with lower serum neutralization capability. E2- and syntenin-containing exosomes are a major type of particle released from cells expressing high levels of syntenin. Efficient production of E2-coated exosomes renders HCV infectivity less susceptible to antibody neutralization in hepatoma cells and PHHs. This study identifies a key role for syntenin in the regulation of E2 secretion via exosomes. Efficient production of E2-coated exosomes was shown to make hepatitis C virus less sensitive to antibody neutralization. These results may have implications for the development of a hepatitis C virus vaccine.

Structural Modification in Hepatitis C Virus Envelope Protein; Potential Viral Strategy Against Interferon Therapy

Interferons (IFNs) are the main part of the immune system as a defensive response to viral infection. The phosphorylation domain of Hepatitis C virus (HCV) E2 envelope glycoprotein is homologous to the host key enzymes in IFN-mediated response. So, if host kinases show no preference on E2 protein phosphorylation over IFN triggering factors, the IFN pathway would be competently inhibited. The aim of this study was to see if the resistance of HCV infected patients to IFN-therapy has something to do with HCV E2 envelope glycoprotein. For this purpose, the partially sequenced HCV E2 proteins obtained from 15 patients that were confirmed to be sustainable, semi-resistant (patients with relapsing infection) and resistant to IFN-therapy. The peptide sequences from 15 patients were analysed as a matter of homology, physicochemical properties and predicted structures using MEGA4 software, ProtParam, PSIPRED and I-TASSER tools, respectively. Then, certain point mutations introduced to sequences that acquired from patients resistant to IFN-therapy. Replacement of Asp 653 with Asn 653 in R1 at C-terminal of the E2 protein changes secondary and tertiary structures of downstream regions of E2 protein. Although the sequence of PKR-eIF2alpha phosphorylation homology domain in E2 protein remains intact during IFN therapy, its structure modifies in patients resistant to IFN-therapy, which then puts its phosphorylation potential in jeopardy. Therefore, structural modification in HCV E2 proteins, acquired form patients resistant to IFN-therapy, suggests a mechanism that shows how HCV gets resistant toward IFN-therapy.

First report of the genomic characterization of rubella viruses circulating in Cameroon.

Rubella is an acute and contagious viral infection whose gravidity resides in infection during pregnancy, which can result in miscarriage, fetal death, stillbirth, or infants with congenital malformations. This study aimed to describe the genome of rubella viruses (RUBVs) circulating in Cameroon. Throat swabs were collected from health districts as part of the measles surveillance program from 2010 to 2016 and sent to the Centre Pasteur of Cameroon. Samples were amplified by genotyping reverse transcription polymerase chain reaction (RT-PCR) in the search of two overlapping fragments of the gene that encodes the E1 envelope glycoprotein of RUBV. PCR products were sequenced and phylogenetic analysis was performed with MEGA 6 software. Overall, 9 of 43 samples (20.93%) were successfully amplified and sequenced but only eight sequences could be exploited for phylogenetic analysis with respect to the required fragment length of 739 nucleotides. Analysis of viral sequences from Cameroon with other epidemiologically relevant sequences from around the world showed that all RUBVs belonged to lineage L1 of genotype 1G. Cameroon sequences clustered with viruses from West Africa including Nigeria, Ivory Coast, and Ghana with a percentage similarity of 95.4% to 99.2%. This study will enable an update on the molecular epidemiology of RUBV in Cameroon and help in monitoring circulating RUBV for a better implementation of elimination strategies.

Function of the HCV E1 Envelope Glycoprotein in Viral Entry and Assembly

HCV envelope glycoproteins, E1 and E2, are multifunctional proteins. Until recently, E2 glycoprotein was thought to be the fusion protein and was the focus of investigations. However, the recently obtained partial structures of E2 and E1 rather support a role for E1 alone or in association with E2 in HCV fusion. Moreover, they suggest that HCV harbors a new fusion mechanism, distinct from that of other members of the Flaviviridae family. In this context, E1 aroused a renewed interest. Recent functional characterizations of E1 revealed a more important role than previously thought in entry and assembly. Thus, E1 is involved in the viral genome encapsidation step and influences the association of the virus with lipoprotein components. Moreover, E1 modulates HCV–receptor interaction and participates in a late entry step potentially fusion. In this review, we outline our current knowledge on E1 functions in HCV assembly and entry.

Abstract B109: Broadly neutralizing antibodies against HCV use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design

Abstract Hepatocellular carcinoma is the most common type of liver cancer and is responsible for approximately one million deaths each year. In the United States, persistent infection with hepatitis C virus (HCV) is among the leading causes of hepatocellular carcinoma. HCV vaccine efforts are hampered by genetic diversity of HCV envelope glycoproteins E1 and E2. Structural studies of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3 and HEPC74) isolated from individuals who spontaneously cleared HCV infection will facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores.New expression and purification techniques allowed us to produce bNAb complexes with full-length E2 ectodomains for structural studies. We solved crystal structures of HEPC3 and HEPC74 alone and complexed with E2 ectodomains. We measured the binding and neutralization potency of HEPC3/HEPC74-like germline precursors. Despite derivation from different individuals, HEPC3 and HEPC74 use the same germline heavy chain gene segments and possess a limited number of somatic mutations. We describe a shared CDRH3 disulfide motif that exhibits common interactions with a conserved epitope in the front layer of E2. The germline-encoded CDRH3 motif plays a critical role in initial recognition of HCV by naïve B cells. The structures of E2 ectodomain reveal regions (including a target of immunogen design) that were truncated or disordered in E2 core. We also identify candidate immunogens: E2 variants that bind to HEPC3/HEPC74-like germline precursors. These data reveal unusual structural features relevant to the fundamental understanding of antibody-antigen interactions. The identification of common genetic signatures of HCV bNAbs will facilitate lineage-targeted immunogen design to raise bNAbs against the conserved epitope in the E2 front layer. Citation Format: Andrew I. Flyak, Stormy Ruiz, Michelle Colbert, Tiffany Luong, James E. Crowe, Jr., Justin R. Bailey, Pamela J. Bjorkman. Broadly neutralizing antibodies against HCV use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B109.

Differential interaction strategies of hepatitis c virus genotypes during entry – An in silico investigation of envelope glycoprotein E2 - CD81 interaction

Hepatitis C Virus is a blood borne pathogen responsible for chronic hepatitis in more than 71 million people. Wide variations across strains and genotypes are one of the major hurdles in therapeutic development. While genotype 1 remains the most extensively studied and abundant strain, genotype 3 is more virulent and second most prevalent. This study aimed to compare differences in the glycoprotein E2 across HCV genotypes at nucleotide, protein and structural levels. Nucleotide sequences of E2 from 29 strains across genotypes 1a, 1b, 3a and 3b revealed a stark preference for C-richness which was attributed to a distinct bias for C-rich codons in genotype 1. Genotype 3 exhibited a similar preference to a lesser extent. Amino acid level comparison revealed majority of the changes at the C-terminal half of the proteins leaving the N-terminal region conspicuously conserved apart from the two hyper variable regions. Amino acid changes across genotypes were mostly polar-nonpolar alterations. In silico models of E2 glycoproteins and docking analysis with the energy minimized PDB-CD81 model revealed unique interacting residues in both E2 and CD81. While several CD81 binding residues were common for all four genotypes, number and composition of interacting residues varied. The interacting residues of E2 were however unique for each genotype. E2 of genotype 3a and CD81 had the strongest interaction. In conclusion this is the first comprehensive study comparing E2 sequences across genotypes 1a, 1b, 3a and 3b revealing stark genotype-specific differences which requires more extensive investigation.

Production of Antibodies against Hepatitis C Virus Envelope Glycoprotein E2- A Potential Vaccine Against HCV Infection

Production of Antibodies against Hepatitis C Virus Envelope Glycoprotein E2- A Potential Vaccine Against HCV Infection

Research Article An improved Bac-to-Bac/BmNPV technology expressing envelope E2 glycoprotein of classical swine fever virus (CSFV) in the silkworm, <i>Bombyx mori</i>

Research Article An improved Bac-to-Bac/BmNPV technology expressing envelope E2 glycoprotein of classical swine fever virus (CSFV) in the silkworm, <i>Bombyx mori</i>

Cloning and Analysis of Authentic Patient-Derived HCV E1/E2 Glycoproteins.

Experimental characterization of the properties of authentic viruses circulating in infected individuals presents a problem when investigating RNA viruses with error-prone polymerases. The hepatitis C virus provides an extreme example of RNA virus genetic variability, as the nucleotide composition of HCV genomes can vary by more than 30% between strains. The envelope glycoproteins E1 and E2 in particular are able to tolerate a particularly high level of variation. They are under continual selection pressure from the host antibody response during chronic infection and can tolerate adaptive mutations, leading to great diversity in a single host. The diversity of E1/E2 in circulating viruses has hindered investigations of their function and development of a vaccine that will generate antibodies able to potently neutralize entry of genetically distinct strains.Here we describe methods used in our laboratory to overcome the limitations of investigating the properties of the envelope glycoproteins representing only small numbers of HCV variants. Using a high-fidelity, limiting dilution ("endpoint") PCR approach to amplify single E1/E2 cDNA templates, which can then generate recombinant model viral particles using retrovirus packaging/reporter constructs. These retroviral pseudoparticles (pseudotypes) facilitate investigation of the properties of authentic E1/E2 glycoproteins in a single-round infection assay. We also describe optimized methods for generation of infectious pseudoparticles from patient-isolated E1/E2 and methods for performing neutralization assays with both anti-virus and anti-host antibodies.

Probing the Antigenicity of HCV Envelope Glycoproteins by Phage Display Antibody Technology.

The envelope glycoproteins E1 and E2 of hepatitis C virus form a heterodimeric complex on the viral surface. They are the targets of neutralizing antibodies and are being investigated as potential vaccine antigens. Because of the high level of cysteine residues and N-glycosylation sites in the polypeptide sequences, it is technically challenging to produce pure, folded recombinant E1, E2, and E1E2 complex for downstream analysis. In this chapter, the methods we used to isolate a panel of human antibodies specific to diverse antigenic regions on the glycoproteins are discussed. The antibodies have been found to be valuable reagents for the study of HCV envelope glycoproteins, including the determination of the first E2 core domain structure.

Simultaneous Quantification of Hepatitis C Virus Envelope Glycoproteins E1 and E2 by Dual-Color Fluorescence Immunoblot Analysis.

The hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are crucial for HCV assembly and entry, and are promising vaccine antigens. However, they are challenging to study because of technical difficulties in protein production and in quality control for protein folding and glycosylation. To study E1 and E2 in different experimental systems, e.g. infected cells, virus culture, virus-like particles, and clinical samples, a standardized method to accurately quantify the glycoproteins will be essential for most research projects. Here we outline a sensitive assay based on dual-color fluorescence immunoblot and the Odyssey imaging system to detect and quantify HCV E1 and E2 glycoproteins either using a purified E1E2 complex, or an engineered protein standard containing E1 and E2 at equal molar ratio. The method is capable of simultaneously detecting and quantifying as little as 7ng of E1 and 5ng of E2 in HCV pseudoparticles, and will be useful to quantify E1 and E2 from a wide variety of samples.

THE FUNCTIONAL E1–E2 HETERODIMER HCV GLYCOPROTEINS

Introduction: The two HCV envelope glycoproteins E1 and E2 are released fromHCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembraneproteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobicanchor. Methods and pathways: After their synthesis, HCV glycoproteins E1 and E2 associateas a non covalent heterodimer. The transmembrane domains of HCV envelope glycoproteinsplay a major role in E1–E2 heterodimer assembly and subcellular localization. The envelopeglycoprotein complex E1–E2 has been proposed to be essential for HCV entry. Results andconclusions: However, for a long time, HCV entry studies have been limited by the lack of arobust cell culture system for HCV replication and viral particle production. Recently, a modelmimicking the entry process of HCV lifecycle has been developed by pseudo typing retroviralparticles with native HCV envelope glycoproteins, allowing the characterization of functionalE1–E2 envelope glycoproteins., we review our understanding to date on the assembly of thefunctional HCV glycoprotein heterodimer.

Novel adeno‑associated virus‑based genetic vaccines encoding hepatitis C virus E2 glycoprotein elicit humoral immune responses in mice.

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct-acting antiviral agents (DAAs), with some 185 million individuals infected with HCV worldwide. There is an urgent need for an effective prophylactic HCV vaccine. In the present study, we constructed genetic vaccines based on novel recombinant adeno-associated viral (rAAV) vectors (AAV2/8 or AAV2/rh32.33) that express the envelope glycoprotein E2 from the HCV genotype 1b. Expression of HCV E2 protein in 293 cells was confirmed by western blot analysis. rAAV2/8.HCV E2 vaccine or rAAV2/rh32.33.HCV E2 vaccine was intramuscularly injected into C57BL/6 mice. HCV E2-specific antigen was produced, and long-lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen-specific antibody levels than the rAAV2/8 vaccine or AAV plasmid. Moreover, both AAV vaccines induced neutralizing antibodies against HCV genotypes 1a and 1b. Finally, it is worth mentioning that neutralizing antibody levels directed against AAV2/rh32.33 were lower than those against AAV2/8 in both mouse and human serum. These results demonstrate that AAV vectors, especially the AAVrh32.33, have particularly favorable immunogenicity for development into an effective HCV vaccine.

Identification of structural glycoprotein E2 domain critical to mediate replication of Classical Swine Fever Virus in SK6 cells

Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93–168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136–156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells.