RESUMEN
Chronic liver disease and cancer are global health challenges. The role of the circadian clock as a regulator of liver physiology and disease is well established in rodents, however, the identity and epigenetic regulation of rhythmically expressed genes in human disease is less well studied. Here we unravel the rhythmic transcriptome and epigenome of human hepatocytes using male human liver chimeric mice. We identify a large number of rhythmically expressed protein coding genes in human hepatocytes of male chimeric mice, which includes key transcription factors, chromatin modifiers, and critical enzymes. We show that hepatitis C virus (HCV) infection, a major cause of liver disease and cancer, perturbs the transcriptome by altering the rhythmicity of the expression of more than 1000 genes, and affects the epigenome, leading to an activation of critical pathways mediating metabolic alterations, fibrosis, and cancer. HCV-perturbed rhythmic pathways remain dysregulated in patients with advanced liver disease. Collectively, these data support a role for virus-induced perturbation of the hepatic rhythmic transcriptome and pathways in cancer development and may provide opportunities for cancer prevention and biomarkers to predict HCC risk.
Asunto(s)
Ritmo Circadiano , Hepacivirus , Hepatitis C , Hepatocitos , Hígado , Transcriptoma , Humanos , Hígado/metabolismo , Hígado/virología , Animales , Masculino , Hepatocitos/metabolismo , Hepatocitos/virología , Ratones , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Ritmo Circadiano/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/metabolismo , Relojes Circadianos/genética , Epigénesis GenéticaRESUMEN
BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is an underrecognized and emerging infectious disease that may threaten the safety of donor blood supply in many parts of the world. We sought to elucidate whether our local community blood supply is at increased susceptibility for transmission of transfusion-associated HEV infections. MATERIALS AND METHODS: We screened 10,002 randomly selected donations over an 8-month period between 2017 and 2018 at the Stanford Blood Center for markers of HEV infection using commercial IgM/IgG serological tests and reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR). Donor demographic information, including gender, age, self-identified ethnicity, location of residence and recent travel, were obtained from the donor database and used to generate multivariate binary logistic regressions for risk factors of IgG seropositivity. RESULTS: A total of 10,002 blood donations from 7507 unique donors were screened, and there was no detectable HEV RNA by RT-qPCR. The overall seropositivity rate was 12.1% for IgG and 0.56% for IgM. Multivariate analysis of unique donors revealed a significantly higher risk of IgG seropositivity with increasing age, White/Asian ethnicities and residence in certain local counties. CONCLUSION: Although HEV IgG seroprevalence in the San Francisco Bay Area is consistent with ongoing infection, the screening of a large donor population did not identify any viraemic blood donors. While HEV is an underrecognized and emerging infection in other regions, there is no evidence to support routine blood screening for HEV in our local blood supply currently; however, periodic monitoring may still be required to assess the ongoing risk.
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Virus de la Hepatitis E , Hepatitis E , Humanos , Donantes de Sangre , Anticuerpos Antihepatitis , Hepatitis E/epidemiología , Virus de la Hepatitis E/genética , Inmunoglobulina G , Inmunoglobulina M , ARN Viral , Estudios Seroepidemiológicos , Masculino , FemeninoRESUMEN
SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.
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Anticuerpos ampliamente neutralizantes , Anticuerpos contra la Hepatitis C , Hepatitis C , Inmunogenicidad Vacunal , Proteínas del Envoltorio Viral , Vacunas contra Hepatitis Viral , Animales , Anticuerpos ampliamente neutralizantes/biosíntesis , Anticuerpos ampliamente neutralizantes/sangre , Hepatitis C/prevención & control , Anticuerpos contra la Hepatitis C/biosíntesis , Anticuerpos contra la Hepatitis C/sangre , Ratones , Multimerización de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/química , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.
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Variación Antigénica/inmunología , Antígenos Virales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Hepacivirus/inmunología , Pruebas de Neutralización/métodos , Proteínas del Envoltorio Viral/inmunología , Variación Antigénica/genética , Antígenos Virales/genética , Línea Celular Tumoral , Hepacivirus/genética , Hepatitis C/prevención & control , Humanos , Inmunogenicidad Vacunal , Reproducibilidad de los Resultados , Desarrollo de Vacunas , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.
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Hepacivirus/efectos de los fármacos , Anticuerpos contra la Hepatitis C/biosíntesis , Hepatitis C/prevención & control , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Femenino , Expresión Génica , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C/inmunología , Hepatitis C/patología , Hepatitis C/virología , Humanos , Inmunogenicidad Vacunal , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Receptores Virales/genética , Receptores Virales/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Solubilidad , Tetraspanina 28/genética , Tetraspanina 28/inmunología , Vacunación , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/administración & dosificación , Vacunas contra Hepatitis Viral/química , Vacunas contra Hepatitis Viral/genéticaRESUMEN
We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two VL/VH modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, VHSer113 (Kabat numbering), in the elbow region linking the VH and CH1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab-protein complexes for cryoEM.
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Anticuerpos Biespecíficos/química , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Biespecíficos/genética , Afinidad de Anticuerpos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/genética , Mutación , Multimerización de Proteína , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-Actividad , TermodinámicaRESUMEN
An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.
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Hepacivirus/genética , Hepacivirus/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Línea Celular , Epítopos/química , Epítopos/inmunología , Femenino , Células HEK293 , Hepatitis C/inmunología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/sangre , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Inmunogenicidad Vacunal , Ratones , Modelos Moleculares , Pruebas de Neutralización , Conformación Proteica , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the VH1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-VH1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.
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Hepatitis C , Vacunas contra Hepatitis Viral , Anticuerpos Neutralizantes , Epítopos , Hepacivirus , Anticuerpos contra la Hepatitis C , HumanosRESUMEN
Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.
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Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Hepatitis C/inmunología , Hepatitis C/virología , Evasión Inmune , Interferones/fisiología , Proteínas de la Membrana/inmunología , Enfermedad Aguda , Células Cultivadas , Hepatocitos , HumanosRESUMEN
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.
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Anticuerpos Neutralizantes/inmunología , Diseño de Fármacos , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C/prevención & control , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunología , Adulto , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Genotipo , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Masculino , Pruebas de Neutralización , Estudios Prospectivos , Homología de Secuencia , Adulto JovenRESUMEN
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.
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Formación de Anticuerpos/inmunología , Hepacivirus/inmunología , Insectos/inmunología , Mamíferos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Línea Celular , Epítopos/inmunología , Femenino , Glicosilación , Células HEK293 , Hepatitis C/inmunología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Insectos/virología , Mamíferos/virología , Ratones , Polisacáridos/inmunología , Células Sf9RESUMEN
Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.
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Anticuerpos Monoclonales/administración & dosificación , Claudina-1/inmunología , Anticuerpos contra la Hepatitis C/administración & dosificación , Hepatitis C/prevención & control , Proteínas del Envoltorio Viral/inmunología , Internalización del Virus/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Antivirales/administración & dosificación , Antivirales/farmacología , Combinación de Medicamentos , Sinergismo Farmacológico , Hepacivirus/efectos de los fármacos , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Ratones Endogámicos NOD , Prueba de Estudio ConceptualRESUMEN
Yeast surface display (YSD) enables efficient screening and selection of single chain variable fragments (scFvs) of heavy (VH) and light (VL) chains that bind to target antigen with different affinities. Assembly of a scFv library from cDNA usually involves adding different primers and linkers (Gly4/Ser)3 through multiple rounds of PCR amplification and purification. We describe here a simplified scFv assembly method by creating a modified YSD vector with a built-in linker that reduces the time of assembly and decreases accumulated base exchanges due to PCR errors. In addition, we describe a bias screening strategy toward maximizing novel antibodies of interest by a combination of memory B cell selection and depletion by binding to mutant antigens that do not bind to previously identified monoclonal antibodies.
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Anticuerpos Neutralizantes/aislamiento & purificación , Técnicas de Visualización de Superficie Celular/métodos , Anticuerpos contra la Hepatitis C/aislamiento & purificación , Hepatitis C/sangre , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Técnicas de Visualización de Superficie Celular/instrumentación , Clonación Molecular/métodos , Ensayo de Inmunoadsorción Enzimática/instrumentación , Ensayo de Inmunoadsorción Enzimática/métodos , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/genética , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Biblioteca de Péptidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Transformación GenéticaRESUMEN
Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.
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Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C/inmunología , Pruebas de Neutralización/métodos , Internalización del Virus , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Línea Celular Tumoral , Genes Reporteros/genética , Células HEK293 , Hepacivirus/inmunología , Hepatitis C/sangre , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/sangre , Humanos , Pruebas de Neutralización/instrumentación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Virión/inmunología , Liberación del Virus/inmunologíaRESUMEN
With more than 71 million people chronically infected, hepatitis C virus (HCV) is one of the leading causes of liver disease and hepatocellular carcinoma. While efficient antiviral therapies have entered clinical standard of care, the development of a protective vaccine is still elusive. Recent studies have shown that the HCV life cycle is closely linked to lipid metabolism. HCV virions associate with hepatocyte-derived lipoproteins to form infectious hybrid particles that have been termed lipo-viro-particles. The close association with lipoproteins is not only critical for virus entry and assembly but also plays an important role during viral pathogenesis and for viral evasion from neutralizing antibodies. In this review, we summarize recent findings on the functional role of apolipoproteins for HCV entry and assembly. Furthermore, we highlight the impact of HCV-apolipoprotein interactions for evasion from neutralizing antibodies and discuss the consequences for antiviral therapy and vaccine design. Understanding these interactions offers novel strategies for the development of an urgently needed protective vaccine.
RESUMEN
Hepatitis C virus (HCV) continues to spread worldwide with an annual increase of 1.75 million new infections. The number of HCV cases in the U.S. is now greater than the number of HIV cases and is increasing in young adults because of the opioid epidemic sweeping the country. HCV-related liver disease is the leading indication of liver transplantation. An effective vaccine is of paramount importance to control and prevent HCV infection. While this vaccine will need to induce both cellular and humoral immunity, this review is focused on the required antibody responses. For highly variable viruses, such as HCV, isolation and characterization of monoclonal antibodies mediating broad virus neutralization are an important guide for vaccine design. The viral envelope glycoproteins, E1 and E2, are the main targets of these antibodies. Epitopes on the E2 protein have been studied more extensively than epitopes on E1, due to higher antibody targeting that reflects these epitopes having higher degrees of immunogenicity. E2 epitopes are overall organized in discrete clusters of overlapping epitopes that ranged from high conservation to high variability. Other epitopes on E1 and E1E2 also are targets of neutralizing antibodies. Taken together, these regions are important for vaccine design. Another element in vaccine design is based on information on how the virus escapes from broadly neutralizing antibodies. Escape mutations can occur within the epitopes that are involved in antibody binding and in regions that are not involved in their epitopes, but nonetheless reduce the efficiency of neutralizing antibodies. An understanding on the specificities of a protective B cell response, the molecular locations of these epitopes on E1, E2, and E1E2, and the mechanisms, which enable the virus to negatively modulate neutralizing antibody responses to these regions will provide the necessary guidance for vaccine design.
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Anticuerpos Neutralizantes/metabolismo , Mapeo Epitopo , Epítopos de Linfocito B/metabolismo , Hepacivirus/fisiología , Hepatitis C/inmunología , Vacunas contra Hepatitis Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Diseño de Fármacos , Epítopos de Linfocito B/genética , Humanos , Evasión Inmune , Inmunidad Humoral , RacionalizaciónRESUMEN
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/análisis , Anticuerpos Neutralizantes/análisis , Mapeo Epitopo , Genotipo , Hepacivirus/química , Hepacivirus/genética , Hepatitis C/genética , Humanos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. CONCLUSION: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.