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1.
Front Immunol ; 10: 1145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178869

RESUMO

Hepatitis C virus (HCV) persistently infects approximately 71 million people globally. To prevent infection a vaccine which elicits neutralizing antibodies against the virus envelope proteins (E1/E2) which are required for entry into host cells is desirable. DNA vaccines are cost-effective to manufacture globally and despite recent landmark studies highlighting the therapeutic efficacy of DNA vaccines in humans against cervical cancer, DNA vaccines encoding E1/E2 developed thus far are poorly immunogenic. We now report a novel and highly immunogenic DNA vaccination strategy that incorporates secreted E1 and E2 (sE1 and sE2) into oligomers by fusion with the oligomerization domain of the C4b-binding protein, IMX313P. The FDA approved plasmid, pVax, was used to encode sE1, sE2, or sE1E2 with or without IMX313P, and intradermal prime-boost vaccination studies in BALB/c mice showed that vaccines encoding IMX313P were the most effective in eliciting humoral and cell-mediated immunity against the envelope proteins. Further boosting with recombinant E1E2 proteins but not DNA nor virus-like particles (VLPs) expressing E1E2 increased the immunogenicity of the DNA prime-boost regimen. Nevertheless, the antibodies generated by the homologous DNA prime-boost vaccinations more effectively inhibited the binding of VLPs to target cells and neutralized transduction with HCV pseudoparticles (HCVpp) derived from different genotypes including genotypes 1, 2, 3, 4, 5, and 6. This report provides the first evidence that IMX313P can be used as an adjuvant for E1/E2-based DNA vaccines and represents a translatable approach for the development of a HCV DNA vaccine.


Assuntos
Anticorpos Neutralizantes/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Imunogenicidade da Vacina , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Formação de Anticorpos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite C/prevenção & controle , Hepatite C/virologia , Humanos , Imunização , Imunoglobulina G/imunologia , Camundongos , Testes de Neutralização , Peptídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas de DNA/genética , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/genética
2.
Vaccine ; 37(31): 4364-4369, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31227355

RESUMO

Duck hepatitis A virus (DHAV) is the major pathogen of duck viral hepatitis, which has caused great economic losses to duck breeding industry. As an effective delivery tool for protein antigens, Lactococcus lactis (L. lactis) has been successfully used to stimulate mucosal and systemic immune response. In this study, a recombinant L. lactis named NZ3900-VP1 was constructed, which could express VP1 protein of DHAV type 3 (DHAV-3) by using a nisin-controlled expression (NICE) system. The animal experiment in both mice and ducklings were performed to detect the immune response and protection effect of oral vaccination by the recombinant L. lactis. The results showed that oral vaccination with L. lactis NZ3900-VP1 significantly induced specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) of DHAV-3 in mice and ducklings, and cytokines including interleukin-2 (IL-2), interferon gamma (IFN-γ), interleukin-10 (IL-10) and interleukin-4 (IL-4). Notably, the ducklings vaccinated with L. lactis NZ3900-VP1 were effectively protected when facing natural infestation of DHAV-3, which indicated that the recombinant L. lactis could serve as an effective vaccine to prevent DHAV-3 infection in ducklings.


Assuntos
Vírus da Hepatite do Pato/imunologia , Imunogenicidade da Vacina , Lactococcus lactis/genética , Vacinas de DNA , Vacinas contra Hepatite Viral , Proteínas Estruturais Virais/imunologia , Imunidade Adaptativa , Administração Oral , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vírus da Hepatite do Pato/genética , Imunidade nas Mucosas , Imunização , Camundongos , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Proteínas Estruturais Virais/genética
3.
Sci Adv ; 5(1): eaav1882, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30613781

RESUMO

An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family V H 1-69. We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line-reverted versions of VH1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the V H 1-69 gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/imunologia , Afinidade de Anticorpos/imunologia , Sítios de Ligação , Doadores de Sangue , Linhagem Celular Tumoral , Reações Cruzadas/imunologia , Epitopos/química , Hepatite C/virologia , Humanos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia
4.
Curr Comput Aided Drug Des ; 15(2): 120-135, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30280672

RESUMO

BACKGROUND: Hepatitis C virus (HCV) infection is a global burden. There is no peptide vaccine found as modality to cure the disease is available due to the weak cellular immune response and the limitation to induce humoral immune response. METHODS: Five predominated HCV subtypes in Indonesia (1a, 1b, 1c, 3a, and 3k) were aligned and the conserved regions were selected. Twenty alleles of class I MHC including HLA-A, HLA-B, and HLAC types were used to predict the potential epitopes by using NetMHCPan and IEDB. Eight alleles of HLA-DRB1, together with a combination of 3 alleles of HLA-DQA1 and 5 alleles of HLA-DQB1 were utilized for Class II MHC epitopes prediction using NetMHCIIPan and IEDB. LBtope and Ig- Pred were used to predict B cells epitopes. Moreover, proteasome analysis was performed by NetCTL and the stability of the epitopes in HLA was calculated using NetMHCStabPan for Class I. All predicted epitopes were analyzed for its antigenicity, toxicity, and stability. Population coverage, molecular docking and molecular dynamics were performed for several best epitopes. RESULTS: The results showed that two best epitopes from envelop protein, GHRMAWDMMMNWSP (E1) and PALSTGLIHLHQN (E2) were selected as promising B cell and CD8+ T cell inducers. Other two peptides, LGIGTVLDQAETAG and VLVLNPSVAATLGF, taken from NS3 protein were selected as CD4+ T cell inducer. CONCLUSION: This study suggested the utilization of all four peptides to make a combinational peptide vaccine for in vivo study to prove its ability in inducing secondary response toward HCV.


Assuntos
Hepacivirus/genética , Hepacivirus/imunologia , Peptídeos/imunologia , Vacinas contra Hepatite Viral/imunologia , Sequência de Aminoácidos , Sequência Conservada , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Hepatite C/imunologia , Hepatite C/prevenção & controle , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Vacinas de Subunidades/química , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/farmacologia , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/farmacologia
5.
Mol Med Rep ; 19(2): 1016-1023, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569131

RESUMO

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct­acting 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 adeno­associated 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 E2­specific antigen was produced, and long­lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen­specific 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.


Assuntos
Anticorpos Neutralizantes/biossíntese , Dependovirus/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Hepatite C Crônica/prevenção & controle , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Dependovirus/genética , Feminino , Vetores Genéticos/química , Vetores Genéticos/imunologia , Células HEK293 , Hepacivirus/genética , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Soros Imunes/química , Imunidade Humoral/efeitos dos fármacos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/biossíntese , Vacinas de DNA/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética
6.
Antiviral Res ; 160: 25-37, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30217650

RESUMO

Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly-neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing of vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/sangue , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Sequência Consenso , Reações Cruzadas , Genótipo , Cobaias , Hepacivirus/classificação , Hepacivirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética
7.
World J Gastroenterol ; 24(30): 3374-3383, 2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30122877

RESUMO

At the 3' end of genomic hepatitis C virus (HCV) RNA there is a highly conserved untranslated region, the 3'X-tail, which forms part of the 3'UTR. This region plays key functions in regulation of critical processes of the viral life cycle. The 3'X region is essential for viral replication and infectivity. It is also responsible for regulation of switching between translation and transcription of the viral RNA. There is some evidence indicating the contribution of the 3'X region to the translation efficiency of the viral polyprotein and to the encapsidation process. Several different secondary structure models of the 3'X region, based on computer predictions and experimental structure probing, have been proposed. It is likely that the 3'X region adopts more than one structural form in infected cells and that a specific equilibrium between the various forms regulates several aspects of the viral life cycle. The most intriguing explanations of the structural heterogeneity problem of the 3'X region came with the discovery of its involvement in long-range RNA-RNA interactions and the potential for homodimer formation. This article summarizes current knowledge on the structure and function of the 3'X region of hepatitis C genomic RNA, reviews previous opinions, presents new hypotheses and summarizes the questions that still remain unanswered.


Assuntos
Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Hepatite C/terapia , Conformação de Ácido Nucleico , RNA Viral/química , Antivirais/farmacologia , Antivirais/uso terapêutico , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/epidemiologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Modelos Moleculares , Interferência de RNA , RNA Viral/antagonistas & inibidores , RNA Viral/genética , Relação Estrutura-Atividade , Resultado do Tratamento , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
8.
Viruses ; 10(8)2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30096846

RESUMO

Hepatitis C virus (HCV) represents a major global health problem for which a vaccine is not available. Modified vaccinia virus Ankara (MVA)-HCV is a unique HCV vaccine candidate based in the modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV genotype 1a that elicits CD8⁺ T-cell responses in mice. With the aim to improve the immune response of MVA-HCV and because of the importance of interferon (IFN) in HCV infection, we deleted in MVA-HCV the vaccinia virus (VACV) C6L gene, encoding an inhibitor of IFN-ß that prevents activation of the interferon regulatory factors 3 and 7 (IRF3 and IRF7). The resulting vaccine candidate (MVA-HCV ΔC6L) expresses all HCV antigens and deletion of C6L had no effect on viral growth in permissive chicken cells. In human monocyte-derived dendritic cells, infection with MVA-HCV ΔC6L triggered severe down-regulation of IFN-ß, IFN-ß-induced genes, and cytokines in a manner similar to MVA-HCV, as defined by real-time polymerase chain reaction (PCR) and microarray analysis. In infected mice, both vectors had a similar profile of recruited immune cells and induced comparable levels of adaptive and memory HCV-specific CD8⁺ T-cells, mainly against p7 + NS2 and NS3 HCV proteins, with a T cell effector memory (TEM) phenotype. Furthermore, antibodies against E2 were also induced. Overall, our findings showed that while these vectors had a profound inhibitory effect on gene expression of the host, they strongly elicited CD8⁺ T cell and humoral responses against HCV antigens and to the virus vector. These observations add support to the consideration of these vectors as potential vaccine candidates against HCV.


Assuntos
Expressão Gênica , Hepatite C/prevenção & controle , Imunogenicidade da Vacina , Interferon beta/antagonistas & inibidores , Vírus Vaccinia/genética , Vacinas contra Hepatite Viral/imunologia , Imunidade Adaptativa , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/virologia , Vetores Genéticos , Genoma Viral , Hepacivirus , Humanos , Memória Imunológica , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Interferon beta/genética , Camundongos , Vacinas contra Hepatite Viral/genética , Proteínas não Estruturais Virais/imunologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-30073153

RESUMO

Live attenuated vaccines are widely used to protect humans or animals from pathogen infections. We have previously developed a chicken embryo-attenuated Duck Hepatitis A Virus genotype 1 (DHAV-1) vaccine (CH60 strain). This study aims to understand the mechanisms that drive a virulent strain to an attenuated virus. Here, we systematically compared five DHAV-1 chicken embryo attenuated strains and 68 virulent strains. Phylogenetic analysis indicated that duck virulent strains isolated from different geographic regions of China undergo a convergent evolution in the chicken embryos. Comparative analysis indicated that the codon usage bias of the attenuated strains were shaped by chicken codons usage bias, which essentially contributed to viral adaption in the unsuitable host driven by incompatible translation. Of note, the missense mutations in coding region and mutations in untranslated regions may also contribute to viral attenuation of DHAV-1 to some extent. Importantly, we have experimentally confirmed that the expression levels of four viral proteins ( 2A 3 pro , 2A 3 pro , 3Cpro, and 3Dpro) in the liver and kidney of ducks infected with an attenuated strain are significantly lower than that infected with a virulent strain, despite with similar virus load. Thus, the key mechanisms of viral attenuation revealed by this study may lead to innovative and easy approaches in designing live attenuated vaccines.


Assuntos
Evolução Molecular Direcionada , Vírus da Hepatite do Pato/crescimento & desenvolvimento , Vírus da Hepatite do Pato/patogenicidade , Biossíntese de Proteínas , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/isolamento & purificação , Animais , Embrião de Galinha , China , Genótipo , Rim/patologia , Rim/virologia , Fígado/patologia , Fígado/virologia , Mutação de Sentido Incorreto , Filogenia , Análise de Sequência de DNA , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/isolamento & purificação , Vacinas contra Hepatite Viral/efeitos adversos , Vacinas contra Hepatite Viral/genética , Virulência
10.
Acta Virol ; 62(2): 157-163, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29895156

RESUMO

Hepatitis B virus (HBV) infection is a major public health problem and immune tolerance is responsible for persistent HBV infection. HBV therapeutic vaccines targeting HBV e antigen (HBeAg) may have an excellent effect in overcoming HBV immune tolerance. Thus, there is urgency for designing therapeutic vaccine candidates that target HBeAg. In this research, we fused the C (472-507) gene sequence of HBV with the extracellular domain of human CD40 ligand sequence and ligated this fused sequence into the pEGFP-N1 vector to construct the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L. Then, the dendritic cells (DCs) generated from human peripheral blood were transfected with this recombinant plasmid. After this, the phenotype and function of DCs were assessed. Compared with the three control groups of pEGFP-N1-C (472-507), pEGFP-N1 and phosphate buffered saline (PBS), we found that DCs transfected with the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L enhanced the expression of costimulatory molecules (CD80, CD86 and HLA-DR) and secretion of cytokine IL-12p70. Furthermore, the capacity of inducing the proliferation of allogeneic lymphocytes was also improved. Our study validated that transfecting DCs with recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L could activate DCs and enhance their functions. Therefore, C (472-507)-ecdCD40L fusion sequence may be a promising vaccine candidate for chronic hepatitis B therapythat targets HBeAg.


Assuntos
Antígenos CD40/imunologia , Células Dendríticas/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Vacinas contra Hepatite Viral/imunologia , Antígenos CD40/química , Antígenos CD40/genética , Células Dendríticas/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite B/prevenção & controle , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Domínios Proteicos , Transfecção , Vacinas contra Hepatite Viral/genética
11.
Biologicals ; 53: 63-71, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29519752

RESUMO

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/imunologia
12.
IUBMB Life ; 70(3): 207-214, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29369472

RESUMO

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4+ and CD8+ T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.


Assuntos
Vírus da Hepatite E/imunologia , Hepatite E/imunologia , Imunidade Humoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas Virais/administração & dosagem , Imunidade Adaptativa/imunologia , Animais , Autofagia/imunologia , Proteína Beclina-1/administração & dosagem , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Escherichia coli/genética , Genoma Viral/imunologia , Hepatite E/prevenção & controle , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia
13.
Appl Microbiol Biotechnol ; 102(1): 185-198, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29143081

RESUMO

Hepatitis E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products and with blood components through transfusions. There are no approved or golden standard serologic systems for HEV diagnostics. Commercially available HEV tests often provide inconsistent results which may differ among the assays. In this study, we describe generation in yeast and characterization of HEV genotype 3 (HEV-3) and rat HEV capsid proteins self-assembled into virus-like particles (VLPs) and the development of HEV-specific monoclonal antibodies (MAbs). Full-length HEV-3 and rat HEV capsid proteins and their truncated variants comprising amino acids (aa) 112-608 were produced in yeast S. cerevisiae. The yeast-expressed rat HEV capsid protein was found to be glycosylated. The full-length HEV-3 capsid protein and both full-length and truncated rat HEV capsid proteins were capable to self-assemble into VLPs. All recombinant proteins contained HEV genotype-specific linear epitopes and cross-reactive conformational epitopes recognized by serum antibodies from HEV-infected reservoir animals. Two panels of MAbs against HEV-3 and rat HEV capsid proteins were generated. Their cross-reactivity pattern was investigated by Western blot, ELISA, and immunofluorescence assay on HEV-3-infected cell cultures. The analysis revealed cross-reactive, genotype-specific, and virus-reactive MAbs. MAb epitopes were localized within S, M, and P domains of HEV-3 and rat HEV capsid proteins. Yeast-generated recombinant VLPs of HEV-3 and rat HEV capsid proteins and HEV-specific MAbs might be employed to develop novel HEV detection systems.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Vírus da Hepatite E/imunologia , Saccharomyces cerevisiae/genética , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Western Blotting , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Genótipo , Glicosilação , Hepatite E/diagnóstico , Hepatite E/prevenção & controle , Hepatite E/virologia , Vírus da Hepatite E/química , Vírus da Hepatite E/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas contra Hepatite Viral/genética
14.
PLoS Pathog ; 13(12): e1006735, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29253863

RESUMO

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.


Assuntos
Hepacivirus/genética , Hepacivirus/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/genética , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Hepacivirus/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Mutagênese , Engenharia de Proteínas , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tetraspanina 28/metabolismo , Proteínas do Envelope Viral/química , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Internalização do Vírus
15.
Viruses ; 9(9)2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28914805

RESUMO

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are causative agents of acute viral hepatitis transmitted via the fecal-oral route. Both viruses place a heavy burden on the public health and economy of developing countries. To test the possibility that HAV could be used as an expression vector for the development of a combination vaccine against hepatitis A and E infections, recombinant HAV-HEp148 was created as a vector to express an HEV neutralization epitope (HEp148) located at aa 459-606 of the HEV capsid protein. The recombinant virus expressed the HEp148 protein in a partially dimerized state in HAV-susceptible cells. Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice. Thus, the present study demonstrates a novel approach to the development of a combined hepatitis A and E vaccine.


Assuntos
Epitopos/imunologia , Vírus da Hepatite A/genética , Vírus da Hepatite A/imunologia , Anticorpos Anti-Hepatite/biossíntese , Vírus da Hepatite E/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Vetores Genéticos , Hepatite A/imunologia , Hepatite A/virologia , Anticorpos Anti-Hepatite/imunologia , Hepatite E/imunologia , Hepatite E/virologia , Vírus da Hepatite E/genética , Camundongos , Testes de Neutralização , Vacinação , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética
16.
Sci Rep ; 7(1): 12326, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28951612

RESUMO

While about a quarter of individuals clear their primary hepatitis C (HCV) infections spontaneously, clearance (spontaneous or treatment-induced) does not confer sterilizing immunity against a future infection. Since successful treatment does not prevent future infections either, an effective vaccine is highly desirable in preventing HCV (re)infection. However, development of an effective vaccine has been complicated by the diversity of HCV genotypes, and complexities in HCV immunological responses. Smaller studies on humans and chimpanzees reported seemingly opposing results regarding cross-neutralizing antibodies. We report a lack of cross-genotype immunity in the largest cohort of people to date. In the adjusted Cox proportional hazards model, reinfection with a heterologous HCV genotype (adjusted Hazard Ratio [aHR]: 0.45, 95% CI: 0.25-0.84) was associated with a 55% lower likelihood of re-clearance. Among those who cleared their first infection spontaneously, the likelihood of re-clearance was 49% lower (aHR: 0.51, 95% CI: 0.27-0.94) when reinfected with a heterologous HCV genotype. These findings indicate that immunity against a particular HCV genotype does not offer expanded immunity to protect against subsequent infections with a different HCV genotype. A prophylactic HCV vaccine boosted with multiple HCV genotype may offer a broader and more effective protection.


Assuntos
Proteção Cruzada/genética , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Vacinas contra Hepatite Viral/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Estudos de Coortes , Proteção Cruzada/imunologia , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/sangue , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Anticorpos Anti-Hepatite C/imunologia , Humanos , Imunização Secundária/métodos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Vacinação/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/uso terapêutico
17.
Antiviral Res ; 145: 168-174, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28778831

RESUMO

Hepatitis C virus (HCV) has a devastating impact on human health, and infections can progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no effective HCV vaccine. In this study, we rescued a recombinant PR8 influenza viral vector, called rgFLU-HCVCE1E2, carrying the core and envelope glycoprotein (C/E1/E2) epitopes of HCV inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of rgFLU-HCVCE1E2 and the expression of the C/E1/E2 epitopes of HCV were examined. rgFLU-HCVCE1E2 replicated in various cell lines, including MDCK, A549, and Huh7.5 cells. More importantly, in BALB/c mice immunized intranasally twice at a 21-day interval with 104, 105, or 106 TCID50 rgFLU-HCVCE1E2, the viral vector induced a robust antibody response to influenza and HCV and potent IFN-γ and IL-4 secretion in response to HCV antigens in a dose-dependent manner. The rgFLU-HCVCE1E2 virus also stimulated IFN-γ production by virus-specific peripheral blood mononuclear cells in patients with chronic HCV infection. The study demonstrated that rgFLU-HCVCE1E2 carrying HCV antigens is immunogenic in vivo and has potential for the development of a HCV vaccine.


Assuntos
Epitopos/imunologia , Hepacivirus/imunologia , Imunogenicidade da Vacina , Vacinas contra Hepatite Viral/imunologia , Células A549 , Administração Intranasal , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Cães , Vetores Genéticos , Hepacivirus/genética , Humanos , Imunização , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/genética
18.
Plant Biotechnol J ; 15(12): 1611-1621, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28419665

RESUMO

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium-mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.


Assuntos
Alface/genética , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia , Administração Oral , Animais , Feminino , Células HEK293 , Humanos , Imunidade Humoral , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/genética
19.
Sci Rep ; 7: 43531, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28266565

RESUMO

Direct-acting antiviral treatment for hepatitis C virus (HCV) infection is costly and does not protect from re-infection. For human and chimpanzees, recovery from acute HCV infection correlates with host CD4+ and CD8+ T cell responses. DNA plasmids targeting the HCV non-structural antigens NS3, NS4, and NS5, were previously reported to induce robust and sustained T cell responses in mice and primates. These plasmids were combined with a plasmid encoding cytokine IL-28B, together named as VGX-6150. The dose-dependent T cell response and safety of VGX-6150 administered intramuscularly and followed by electroporation was assessed in mice. Immune responses plateaued at 20 µg/dose with IL-28B demonstrating significant immunoadjuvant activity. Mice administered VGX-6150 at 40, 400, and 800 µg given either as a single injection or as 14 injections given bi-weekly over 26 weeks showed no vaccine related changes in any clinical parameter compared to placebo recipients. There was no evidence of VGX-6150 accumulation at the injection site or in any organ 1 month following the 14th vaccination. Based on these studies, the approximate lethal dose (ALD) exceeds 800 µg/dose and the NOAEL was 800 µg/dose in mouse. In conclusion, VGX-6150 appears safe and a promising preventive vaccine candidate for HCV infection.


Assuntos
Hepacivirus/genética , Hepacivirus/imunologia , Antígenos da Hepatite C/imunologia , Hepatite C/imunologia , Hepatite C/prevenção & controle , Vacinas de DNA/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Feminino , Antígenos da Hepatite C/genética , Humanos , Imunidade Celular , Imunização , Esquemas de Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/efeitos adversos , Vacinas contra Hepatite Viral/genética
20.
Gene Ther ; 23(10): 753-759, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27416077

RESUMO

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.


Assuntos
Linfócitos B/imunologia , Epitopos/genética , Hepacivirus/imunologia , Linfócitos T/imunologia , Vacinas contra Hepatite Viral/imunologia , Adenoviridae/genética , Animais , Linhagem Celular , Cricetinae , Epitopos/imunologia , Vetores Genéticos/genética , Imunogenicidade da Vacina , Interferon gama/sangue , Interleucina-4/sangue , Macaca mulatta , Masculino , Vírus Vaccinia/genética , Vacinas contra Hepatite Viral/genética
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