RESUMO
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.
Assuntos
Anticorpos Amplamente Neutralizantes , Anticorpos Anti-Hepatite C , Hepatite C , Imunogenicidade da Vacina , Proteínas do Envelope Viral , Vacinas contra Hepatite Viral , Animais , Anticorpos Amplamente Neutralizantes/biossíntese , Anticorpos Amplamente Neutralizantes/sangue , Hepatite C/prevenção & controle , Anticorpos Anti-Hepatite C/biossíntese , Anticorpos Anti-Hepatite C/sangue , Camundongos , Multimerização Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
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.
Assuntos
Hepacivirus/efeitos dos fármacos , Anticorpos Anti-Hepatite C/biossíntese , Hepatite C/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Feminino , Expressão Gênica , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/imunologia , Hepatite C/patologia , Hepatite C/virologia , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Multimerização Proteica , Receptores Virais/genética , Receptores Virais/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Solubilidade , Tetraspanina 28/genética , Tetraspanina 28/imunologia , Vacinação , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/genéticaRESUMO
BACKGROUND AND AIM: HBV DNA can be reduced using antiviral drugs in patients with chronic hepatitis B (CHB); however, the rate of HBeAg seroconversion remains low. A clinical trial was conducted to assess the efficacy and safety of a de novo designed liposome-based nanoparticle lipopeptide vaccine, εPA-44, for CHB. APPROACH AND RESULTS: A two-stage phase 2 trial, which included a 76-week, randomized, double-blind, placebo-controlled trial (stage 1) and a 68-week open-label extension (stage 2), was conducted in 15 centers across China (Clinicaltrials.gov No. NCT00869778). In stage 1, 360 human leukocyte antigen A2 (HLA-A2)-positive and HBeAg-positive patients were randomly and equally distributed to receive six subcutaneous injections of 600 µg or 900 µg εPA-44 or placebo at week 0, 4, 8, 12, 20, and 28. In stage 2, 183 patients received extended 900 µg εPA-44, and 26 patients were observed for relapse without further treatment. The primary endpoint was the percentage of patients with HBeAg seroconversion at week 76. At week 76, patients receiving 900 µg εPA-44 achieved significantly higher HBeAg seroconversion rate (38.8%) versus placebo (20.2%) (95% CI, 6.9-29.6%; p = 0.002). With a combined endpoint of HBeAg seroconversion, alanine aminotransferase normalization and HBV DNA < 2,000 IU/mL, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. In stage 2, none (0 of 20) of 900 µg εPA-44-treated patients experienced serologic relapse. The safety profile of εPA-44 was comparable to that of placebo. CONCLUSIONS: Among HLA-A2-positive patients with progressive CHB, a finite duration of 900 µg εPA-44 monotherapy resulted in significantly higher HBeAg seroconversion rate than placebo and sustained off-treatment effect. A phase 3 trial is ongoing (ChiCTR2100043708).
Assuntos
Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/imunologia , Hepatite B Crônica/terapia , Vacinas contra Hepatite Viral/administração & dosagem , Adolescente , Adulto , Método Duplo-Cego , Feminino , Antígenos E da Hepatite B/imunologia , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Injeções Subcutâneas , Lipossomos , Masculino , Sistemas de Liberação de Fármacos por Nanopartículas , Soroconversão , Resposta Viral Sustentada , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/química , Vacinas contra Hepatite Viral/efeitos adversos , Vacinas contra Hepatite Viral/química , Adulto JovemRESUMO
Elicitation of broadly neutralizing antibodies (bnAbs) is a leading strategy in rational vaccine design against antigenically diverse pathogens. Here, we studied a panel of monoclonal antibodies (mAbs) from mice immunized with the hepatitis C virus (HCV) envelope glycoproteins E1E2. Six of the mAbs recognize the conserved E2 antigenic site 412-423 (AS412) and cross-neutralize diverse HCV genotypes. Immunogenetic and structural analysis revealed that the antibodies originated from two different germline (GL) precursors and bind AS412 in a ß-hairpin conformation. Intriguingly, the anti-HCV activity of one antibody lineage is associated with maturation of the light chain (LC), whereas the other lineage is dependent on heavy-chain (HC) maturation. Crystal structures of GL precursors of the LC-dependent lineage in complex with AS412 offer critical insights into the maturation process of bnAbs to HCV, providing a scientific foundation for utilizing the mouse model to study AS412-targeting vaccine candidates.
Assuntos
Anticorpos Neutralizantes/química , Hepacivirus/química , Anticorpos Anti-Hepatite C/química , Cadeias Leves de Imunoglobulina/química , Anticorpos de Cadeia Única/química , Proteínas do Envelope Viral/química , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Humanos , Cadeias Leves de Imunoglobulina/imunologia , Camundongos , Anticorpos de Cadeia Única/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
Despite the emergence of new direct-acting antivirals, hepatitis C virus (HCV) chronic infection and its consequent fibrosis and hepatocarcinoma remain a significant burden for public health, thus requiring an effective preventive vaccine. Our group previously showed that a subunit vaccine based on recombinant soluble E2 (sE2) can induce broadly neutralizing antibodies. To improve the immunogenicity of sE2, we designed and produced a fusion protein (sE2-ferritin) comprising sE2 and a ferritin unit in Drosophila S2 cells, which self-assembled into a nanoparticle with sE2 displayed on the surface. The sE2 moiety on the sE2-ferritin nanoparticle not only had nearly natural conformation but also had better affinities than the unfused sE2 to neutralizing antibodies, receptor, and patient serum. Mouse immunization studies showed that sE2-ferritin was more potent than sE2 in inducing anti-HCV broadly neutralizing antibodies. Our results demonstrate that sE2-ferritin is a vaccine candidate superior to previously developed sE2, providing a new possibility for controlling HCV.
Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/microbiologia , Nanopartículas/química , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Drosophila/imunologia , Genótipo , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/virologia , Imunização/métodos , Camundongos , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/químicaRESUMO
Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines.IMPORTANCE Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Epitopos/imunologia , Hepacivirus/imunologia , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Desenho de Fármacos , Epitopos/química , Camundongos , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/administração & dosagemRESUMO
Hepatitis C virus (HCV) infects almost 150 million people and is a leading cause of liver disease worldwide. It has been classified into seven genotypes; the most common genotype affecting Indian population is genotype 3 (60-70%). Currently there is no vaccine for any genotype of HCV. In order to develop peptide based vaccine against HCV, it is important to identify the conservancy in the circulating genotypes, along with the Human Leucocyte Antigen (HLA) alleles in the target population. The present study aims to identify conserved CD4 and CD8 T cells and B cell epitopes against Indian HCV-genotype-3a using an in silico analysis. In the present study, 28 promiscuous CD4 T cell epitopes and some CD8 epitopes were identified. The NS4 region was predicted to be the most antigenic with maximum number of conserved and promiscuous CD4 T cell epitopes and CD8 T cell epitopes having strong and intermediate affinity towards a number of HLA alleles prevalent in Indian population. Additionally, some linear B cell epitopes were also identified, which could generate neutralizing antibodies. In order to ascertain the binding pattern of the identified epitopes with HLA alleles, molecular docking analysis was carried out. The authors suggest further experimental validation to investigate the immunogenicity of the identified epitopes.
Assuntos
Simulação por Computador , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Genótipo , Hepacivirus/química , Simulação de Acoplamento Molecular , Vacinas contra Hepatite Viral/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Hepacivirus/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/química , Anticorpos Anti-Hepatite C/imunologia , Humanos , Índia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologiaRESUMO
Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. IMPORTANCE: A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development.
Assuntos
Anticorpos Neutralizantes/biossíntese , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Vacinas contra Hepatite Viral/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular , Drosophila , Feminino , Glicosilação , Hepacivirus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Deleção de Sequência , Solubilidade , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/química , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
BACKGROUND: Virus-induced dendritic cells (DCs) functional deficiency leads to sub-optimal initiation of adaptive immune responses and consequently chronic infection establishment. The present study reports an advanced hepatitis C virus (HCV) therapeutic vaccine model based on In vivo enrichment of DCs with barberry ethanolic crude extract (BCE) then pulsing them with HCV core protein. METHODS: DCs were enriched by BCE intravenous injection in BALB/c mice. Vaccine efficiency was assessed by flow cytometric analysis of splenocytes of immunized mice, cytokine profiling, cytotoxic T lymphocyte assay, and humoral immune response assessment. RESULTS: There was no significant difference in surface phenotypic characterization of splenocytes from mice immunized with non-BCE-enriched-core-pulsed DCs (iDcs-core) compared to those from mice injected with RPMI-1640 medium. However, splenocytes from mice immunized with BCE-enriched-core-pulsed DCs showed 197 % increase in CD16+ population, 33 % increase in MHCII(+) population, and 43 % decrease in CD3(+) population. In iDCs-core group, 57.9 % greater anti-core cytotoxic T lymphocyte activity, up-regulation in interferon gamma and interleukin (IL) -12 expression, and down-regulation in IL-4 and IL-10 were recorded. Moreover, sustained specific anti-core antibodies were detected only in sera of the same group. CONCLUSIONS: results indicate that BCE-enriched-core-transduced DCs may serve as a new model for immunotherapy of HCV chronic infection.
Assuntos
Berberis/química , Imunidade Humoral/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Citocinas/análise , Citocinas/metabolismo , Células Dendríticas/imunologia , Feminino , Imunoglobulina G/sangue , Fatores Imunológicos/química , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/química , Vacinas contra Hepatite Viral/químicaRESUMO
Hepatitis C virus (HCV) is considered as a foremost cause affecting numerous human liver-related disorders. An effective immuno-prophylactic measure (like stable vaccine) is still unavailable for HCV. We perform an in silico analysis of nonstructural protein 5B (NS5B) based CD4 and CD8 epitopes that might be implicated in improvement of treatment strategies for efficient vaccine development programs against HCV. Here, we report on effective utilization of knowledge obtained from multiple sequence alignment and phylogenetic analysis for investigation and evaluation of candidate epitopes that have enormous potential to be used in formulating proficient vaccine, embracing multiple strains prevalent among major geographical locations. Mutational variability data discussed herein focus on discriminating the region under active evolutionary pressure from those having lower mutational potential in existing experimentally verified epitopes, thus, providing a concrete framework for designing an effective peptide-based vaccine against HCV. Additionally, we measured entropy distribution in NS5B residues and pinpoint the positions in epitopes that are more susceptible to mutations and, thus, account for virus strategy to evade the host immune system. Findings from this study are expected to add more details on the sequence and structural aspects of NS5B protein, ultimately facilitating our understanding about the pathophysiology of HCV and assisting advance studies on the function of NS5B antigen on the epitope level. We also report on the mutational crosstalk between functionally important coevolving residues, using correlated mutation analysis, and identify networks of coupled mutations that represent pathways of allosteric communication inside and among NS5B thumb, finger, and palm domains.
Assuntos
Biologia Computacional , Mapeamento de Epitopos , Epitopos/metabolismo , Variação Genética , Filogenia , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Entropia , Hepacivirus/imunologia , Humanos , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/metabolismo , Vacinas contra Hepatite Viral/uso terapêutico , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Infection with the hepatitis C virus (HCV) is one of the causes of liver cancer, which is the world's sixth most prevalent and third most lethal cancer. The current treatments do not prevent reinfection; because they are expensive, their usage is limited to developed nations. Therefore, a prophylactic vaccine is essential to control this virus. Hence, in this study, an immunoinformatics method was applied to design a multi-epitope vaccine against HCV. The best B- and T-cell epitopes from conserved regions of the E2 protein of seven HCV genotypes were joined with the appropriate linkers to design a multi-epitope vaccine. In addition, cholera enterotoxin subunit B (CtxB) was included as an adjuvant in the vaccine construct. This study is the first to present this epitopes-adjuvant combination. The vaccine had acceptable physicochemical characteristics. The vaccine's 3D structure was predicted and validated. The vaccine's binding stability with Toll-like receptor 2 (TLR2) and TLR4 was confirmed using molecular docking and molecular dynamics (MD) simulation. The immune simulation revealed the vaccine's efficacy by increasing the population of B and T cells in response to vaccination. In silico expression in Escherichia coli (E. coli) was also successful.
Assuntos
Epitopos de Linfócito B , Epitopos de Linfócito T , Hepatite C , Imunoinformática , Vacinas contra Hepatite Viral , Humanos , Simulação por Computador , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Hepatite C/imunologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/químicaRESUMO
We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund's adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8(+) T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D(b)-restricted and three HLA-A(*)0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.
Assuntos
Epitopos de Linfócito T/imunologia , Hepacivirus/imunologia , Antígenos da Hepatite C/imunologia , Fragmentos de Peptídeos/imunologia , Vacinas contra Hepatite Viral/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD4/genética , Epitopos de Linfócito T/química , Antígeno HLA-A2 , Antígenos da Hepatite C/química , Memória Imunológica/imunologia , Interferon gama/metabolismo , Lipossomos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/química , Linfócitos T Citotóxicos/imunologia , Vacinas contra Hepatite Viral/químicaRESUMO
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.
Assuntos
Hepacivirus , Hepatite C , Proteínas do Envelope Viral , Humanos , Antivirais/química , Anticorpos Amplamente Neutralizantes , Microscopia Crioeletrônica , Hepacivirus/química , Hepacivirus/imunologia , Hepatite C/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Multimerização Proteica , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Cabras/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/prevenção & controle , Vacinação , Proteínas do Envelope Viral/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/farmacologia , Especificidade de Anticorpos , Variação Antigênica , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Sequência Conservada/imunologia , Epitopos/imunologia , Cabras/virologia , Hepacivirus/química , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/isolamento & purificação , Anticorpos Anti-Hepatite C/farmacologia , Humanos , Testes de Neutralização , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Development of preventive vaccines against hepatitis C virus (HCV) remains one of the main strategies in achieving global elimination of the disease. The effort is focused on the quest for vaccines capable of inducing protective cross-neutralizing humoral and cellular immune responses, which in turn dictate the need for rationally designed cross-genotype vaccine antigens and potent immunoadjuvants systems. This review provides an assessment of the current state of knowledge on immunopotentiating compounds and vaccine delivery systems capable of enhancing HCV antigen-specific immune responses, while focusing on the synergy and interplay of two modalities. Structural, physico-chemical, and biophysical features of these systems are discussed in conjunction with the analysis of their in vivo performance. Extreme genetic diversity of HCV-a well-known hurdle in the development of an HCV vaccine, may also present a challenge in a search for an effective immunoadjuvant, as the effort necessitates systematic and comparative screening of rationally designed antigenic constructs. The progress may be accelerated if the preference is given to well-defined molecular immunoadjuvants with greater formulation flexibility and adaptability, including those capable of spontaneous self-assembly behavior, while maintaining their robust immunopotentiating and delivery capabilities.
Assuntos
Sistemas de Liberação de Medicamentos , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Imunogenicidade da Vacina , Vacinas contra Hepatite Viral/imunologia , Adjuvantes Imunológicos , Animais , Ensaios Clínicos como Assunto , Composição de Medicamentos , Hepatite C/imunologia , Humanos , Nanopartículas , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/químicaRESUMO
Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.
Assuntos
Epitopos/imunologia , Hepacivirus/imunologia , Antígenos da Hepatite C/química , Desenvolvimento de Vacinas , Vacinas contra Hepatite Viral/química , Animais , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Epitopos/química , Genótipo , Hepacivirus/genética , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Camundongos , Eficácia de Vacinas , Vacinas contra Hepatite Viral/análiseRESUMO
An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.
Assuntos
Hepacivirus/química , Hepatite C/prevenção & controle , Proteínas do Envelope Viral/química , Vacinas contra Hepatite Viral/química , Animais , Epitopos/imunologia , Células HEK293 , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/virologia , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
In a continuous effort to develop effective vaccines against hepatitis E (HE), oral vaccine nanoparticles using the truncated capsid protein p146 (aa460-605) are formulated and characterized. To improve the immunogenicity of p146, chitosan nanoparticles (CSNPs) are used as a mucosal delivery system. Next, the physical-chemical properties, cytotoxic effects in vitro, and immunogenicity in mice of the produced NPs are analyzed. The results show that the produced CS/p146 NPs are stable and well dispersive and display a near-spherical shape with a mean size of 200-300 nm. The findings also demonstrate high encapsulation efficiency (65-73.9%) and loading capacity (27.7-67.5%) of the formulated nanoparticles. Further, the CS/p146 NPs exhibit low cytotoxicity and an obvious sustained-release effect in vitro. Immunogenicity experiments in mice indicate that CS/p146 NPs can induce antigen-specific systemic and mucosal immune responses higher than the purified p146 do. Besides, the expression levels and mRNA transcription of Interleukin (IL)-4 in spleen cells of CS/p146 NPs-immunized mice are higher than those of p146, indicating that a Th2-mediated cellular immune response is activated by the CS/p146 NPs. Overall, the synthesized CS/p146 NPs display promising properties as a potential HE oral vaccine candidate.
Assuntos
Quitosana/química , Hepatite E/prevenção & controle , Nanopartículas/química , Vacinas contra Hepatite Viral/química , Proteínas Virais/química , Adjuvantes Imunológicos/química , Animais , Escherichia coli/metabolismo , Feminino , Imunidade Celular , Imunização , Imunoglobulina G/química , Técnicas In Vitro , Interleucina-4/química , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Peptídeos/química , RNA Mensageiro/metabolismo , Baço/metabolismo , Desenvolvimento de VacinasRESUMO
The live attenuated vaccine against duck viral hepatitis currently available in Korea requires special freezers for storage and transportation with extra costs involved. The development of a lyophilization stabilizer for live attenuated duck viral hepatitis virus (DHV) vaccines, therefore, has been highly recommended for the wider application of the vaccines. Four conventional vaccine stabilizer formulations containing a disaccharide, such as lactose, trehalose, or sucrose, and new formulations containing sorbitol were tested for their efficacy in stabilizing a new attenuated DHV type 3 vaccine candidate under different storage temperatures, 4 and 37 degrees C. The vaccine virus and each stabilizer formulation were combined and submitted to lyophilization and the viability of the virus was measured in 7-d-old specific-pathogen-free chicken embryos by determining the 50% egg lethal dose. Stabilizer formulations containing 2, 4, or 8% sorbitol preserved the viability of the vaccine virus much better than the other stabilizer formulations and 2% sorbitol was the optimal concentration in a standard stabilizing buffer, phosphate glutamate gelatin (0.0038 M KH2PO4, 0.0071 M K2HPO4, 0.0049 M monosodium L-glutamate, and 0.5% gelatin). The results demonstrate that the stabilizer formulation containing 2% sorbitol and 0.5% gelatin can be used for convenient storage and transportation of live DHV vaccines.
Assuntos
Patos , Excipientes , Liofilização , Hepatite Viral Animal/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Vacinas contra Hepatite Viral/imunologia , Animais , Embrião de Galinha , Armazenamento de Medicamentos , Doenças das Aves Domésticas/virologia , Organismos Livres de Patógenos Específicos , Vacinas contra Hepatite Viral/químicaRESUMO
Hepatitis E, which is caused by infection with hepatitis E virus (HEV), is a global health problem in both developed and developing countries. An efficacious hepatitis E vaccine was licensed (by China) in 2011 with a trade name of Hecolin®. The antigen contained in this vaccine is a truncated version of the sole capsid protein encoded by open reading frame 2, which is designated p239. In this study, the real-time and real-condition stability and accelerated stability of five lots of hepatitis E vaccine products at the end of the designated shelf life, were assessed by a well-established quality analysis platform. The protein integrity of p239 that was recovered from the vaccine lots was demonstrated using CE-SDS, LC-MS and MALDI-TOF MS. The particle characteristics of the recovered vaccine antigen were assessed by TEM and HPSEC. The immunogenicity of hepatitis E vaccines was assessed by a mouse potency assay, which is part of product release and stability testing. Several methods were employed to assess the antigenicity of vaccines with or without adjuvant dissolution. Specifically, the well-established methods of sandwich ELISA and surface plasma resonance (SPR)-based BIAcore were used with unique murine monoclonal antibodies. Most interesting, two 'dissolution-free' immunoassays were also used for in situ antigenicity assessment of the vaccines. In addition to the confirmation of vaccine stability at the end of expiry dating, i.e., after storage in recommended conditions (2-8⯰C) for 36â¯months, the mouse potency assay and sandwich ELISA were used to assess the accelerated stability of prefilled syringes to demonstrate the feasibility of out-of-cold-chain storage. In summary, molecular and functional characterization confirmed the shelf life stability of the vaccine at the end of expiry dating and the feasibility of transporting the hepatitis E vaccine for a given period of time out of cold chains.