RESUMO
During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.
Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Propiolactona/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Desinfetantes/metabolismo , Furões , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Masculino , Infecções por Orthomyxoviridae/prevenção & controle , Análise de Sobrevida , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H5N2/imunologia , Vírus da Influenza A Subtipo H7N3/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Neuraminidase/imunologia , Animais , Anticorpos Antivirais/imunologia , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunidade , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H9N2/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Neuraminidase/administração & dosagem , Neuraminidase/genética , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologiaRESUMO
Avian-origin influenza represents a global public health concern. In 2013, the H10N8 virus caused documented human infections for the first time. Currently, there is no approved vaccine against H10 influenza. Recombinant virus-like particles (VLPs) represent a promising vaccine approach. In this study, we evaluated H10 VLPs containing hemagglutinin from H10N8 virus as an experimental vaccine in a ferret challenge model. In addition, we evaluated quadri-subtype VLPs co-localizing H5, H7, H9 and H10 subtypes. Both vaccines elicited serum antibody that reacted with the homologous H10 derived from H10N8 virus and cross-reacted with the heterologous H10N1 virus. Quadri-subtype vaccine also elicited serum antibody to the homologous H5, H7, and H9 antigens and cross-reacted with multiple clades of H5N1 virus. After heterologous challenge with the H10N1 virus, all vaccinated ferrets showed significantly reduced titers of replicating virus in the respiratory tract indicating protective effect of vaccination with either H10 VLPs or with quadri-subtype VLPs.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H10N8/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/sangue , Reações Cruzadas , Furões , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/química , Replicação ViralRESUMO
Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3' untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety.
Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Vacinas Virais/genética , Sequência de Bases , Vírus Chikungunya/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Vacinação/veterinária , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/sangue , Baculoviridae , Galinhas , Testes de Hemaglutinação , Virus da Influenza A Subtipo H5N1 , Eliminação de Partículas ViraisRESUMO
Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150-200nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation.
Assuntos
Produtos do Gene gag/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Imunodeficiência Bovina/imunologia , Neuraminidase/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Produtos do Gene gag/genética , Vírus da Imunodeficiência Bovina/genética , Vírus da Influenza A Subtipo H10N8/genética , Vírus da Influenza A Subtipo H10N8/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/imunologia , Insetos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Células Sf9 , SpodopteraRESUMO
A novel avian-origin influenza A H7N9 virus emerged in China in 2013 and continues to cause sporadic human infections with mortality rates approaching 35%. Currently there are no approved human vaccines for H7N9 virus. Recombinant approaches including hemagglutinin (HA) and virus-like particles (VLPs) have resulted in experimental vaccines with advantageous safety and manufacturing characteristics. While high immunogenicity of VLP vaccines has been attributed to the native conformation of HA arranged in the regular repeated patterns within virus-like structures, there is limited data regarding molecular organization of HA within recombinant HA vaccine preparations. In this study, the full-length recombinant H7 protein (rH7) of A/Anhui/1/2013 (H7N9) virus was expressed in Sf9 cells. We showed that purified full-length rH7 retained functional ability to agglutinate red blood cells and formed oligomeric pleomorphic subviral particles (SVPs) of â¼20nm in diameter composed of approximately 10 HA0 molecules. No significant quantities of free monomeric HA0 were observed in rH7 preparation by size exclusion chromatography. Immunogenicity and protective efficacy of rH7 SVPs was confirmed in the mouse and ferret challenge models suggesting that SVPs can be used for vaccination against H7N9 virus.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Modelos Animais de Doenças , Feminino , Furões , Hemaglutinação , Vacinas contra Influenza/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Multimerização Proteica , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/ß/γ. Finally, direct vaccination of BALB/c mice with a single 20 µg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF.
Assuntos
Plasmídeos/genética , Vacina contra Febre Amarela/imunologia , Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/imunologia , Animais , Anticorpos Antivirais/biossíntese , Chlorocebus aethiops , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/imunologia , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia , Células Vero , Vacinas Virais , Replicação ViralRESUMO
The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.
Assuntos
DNA Viral/administração & dosagem , Produtos do Gene gag/genética , Técnicas de Transferência de Genes , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Vírus da Imunodeficiência Símia/genética , Vagina/imunologia , Vírion/genética , Alphapapillomavirus/genética , Alphapapillomavirus/imunologia , Animais , DNA Viral/imunologia , Feminino , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/imunologia , Células HEK293 , Humanos , Imunidade nas Mucosas/genética , Proteínas Luminescentes/administração & dosagem , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Macaca fascicularis , Macaca mulatta , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vagina/metabolismo , Vagina/virologia , Vírion/imunologia , Proteína Vermelha FluorescenteRESUMO
An effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. Humoral immunologic memory is mediated by long-lived antibody-secreting plasma cells and differentiation of memory B cells into short-lived plasma blasts following re-exposure to immunizing antigen. Here we studied memory B cells in vaccinated rhesus macaques. PBMC were stimulated polyclonally using CD40 Ligand, IL-21 and CpG to induce B cell proliferation and differentiation into antibody secreting cells (ASCs). Flow cytometry was used for phenotyping and evaluating proliferation by CFSE dilution. B cell responses were quantified by ELISPOT. Methodology was established using PBMC of vaccinated elite-controller macaques that exhibited strong, multi-functional antibody activities. Subsequently, memory B cells elicited by two replicating Ad-recombinant prime/envelope boost regimens were retrospectively evaluated pre- and post-SIV and SHIV challenges. The vaccine regimens induced SIV and HIV Env-specific IgG and IgA memory B cells. Prior to challenge, IgA memory B cells were more numerous than IgG memory B cells, reflecting the mucosal priming immunizations. Pre- and post-challenge memory B cells were correlated with functional antibody responses including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI) and transcytosis inhibition. Post-challenge, Env-specific IgG and IgA memory B cells were correlated with reduced chronic viremia. We conclude that functional antibody responses elicited by our prime/boost regimen were effectively incorporated into the memory B cell pool where they contributed to control of viremia following re-exposure to the immunizing antigen.
Assuntos
Linfócitos B/imunologia , HIV/imunologia , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Vírus da Imunodeficiência Símia/imunologia , Proteínas do Envelope Viral/imunologia , Viremia/prevenção & controle , Animais , ELISPOT , Citometria de Fluxo , Memória Imunológica , Macaca mulattaRESUMO
We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)(89.6P) challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene env/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/imunologia , Animais , Humanos , Imunização , Imunização Secundária , Macaca mulatta , Masculino , Reto/imunologia , Reto/virologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Viremia/prevenção & controle , Viremia/virologia , Internalização do VírusRESUMO
An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector.
Assuntos
Vacinas contra a AIDS/imunologia , Adenoviridae/imunologia , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Vacinas contra a AIDS/biossíntese , Adenoviridae/genética , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Feminino , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Cell-mediated immunity and neutralizing antibodies contribute to control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but the role of nonneutralizing antibodies is not defined. Previously, we reported that sequential oral/oral or intranasal/oral (I/O) priming with replication-competent adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants, followed by intramuscular envelope protein boosting, elicited systemic and mucosal cellular immunity and exhibited equivalent, significant reductions of chronic viremia after rectal SIV(mac251) challenge. However, I/O priming gave significantly better control of acute viremia. Here, systemic and mucosal humoral immunity were investigated for potential correlates with the acute challenge outcome. Strong serum binding but nonneutralizing antibody responses against SIV(mac251) were induced in both groups. Antibody responses appeared earlier and overall were higher in the I/O group. Reduced acute viremia was significantly correlated with higher serum binding titer, stronger antibody-dependent cellular cytotoxicity activity, and peak prechallenge and 2-week-postchallenge antibody-dependent cell-mediated viral inhibition (ADCVI). The I/O group consistently displayed greater anti-envelope immunoglobulin A (IgA) antibody responses in bronchoalveolar lavage and a stronger rectal anti-envelope IgA anamnestic response 2 weeks postchallenge. Pre- and postchallenge rectal secretions inhibited SIV transcytosis across epithelial cells. The inhibition was significantly higher in the I/O group, although a significant correlation with reduced acute viremia was not reached. Overall, the replicating Ad5hr-SIV priming/envelope boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities. The pattern of elevated immune responses in the I/O group is consistent with its better control of acute viremia mediated, at least in part, by ADCVI activity and transcytosis inhibition.
Assuntos
Anticorpos Anti-HIV/análise , Anticorpos Anti-HIV/sangue , Imunidade nas Mucosas , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/prevenção & controle , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Imunoglobulina A/análise , Macaca mulatta , Masculino , Reto/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Oral, replication-competent Ad-HIV vaccines are advancing to human trials. Previous evaluation of protective efficacy in non-human primates has primarily followed upper respiratory tract administrations. Here we compared sequential oral (O/O) versus intranasal/oral (I/O) priming of rhesus macaques with Ad5 host range mutant-SIV recombinants expressing SIV env/rev, gag, and nef genes followed by boosting with SIV gp120 protein. Cellular immune responses in PBMC were stronger and more frequent after I/O administration. Both groups developed mucosal immunity, including memory cells in bronchial alveolar lavage, and gut-homing receptors on PBMC. Following intrarectal SIV(mac251) challenge, both groups exhibited equivalent, significant protection and robust post-challenge cellular immunity. Our results illustrate the promise of oral replication-competent Ad-recombinant vaccines. Pre-challenge PBMC ELISPOT and proliferative responses did not predict protection in the O/O group, highlighting the need for simple, non-invasive methods to reliably assess mucosal immunity.
Assuntos
Adenoviridae/genética , Replicação do DNA/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Administração Intranasal , Administração Oral , Animais , Células Cultivadas , Fezes/virologia , Humanos , Memória Imunológica/imunologia , Integrinas/metabolismo , Interferon gama/metabolismo , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Macaca mulatta , Masculino , Mutação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas contra a SAIDS/administração & dosagem , Comprimidos , Vacinas SintéticasRESUMO
The NS3 protein of hepatitis C virus (HCV) has a serine protease activity in its N-terminal region, which plays a crucial role in virus replication. This region has also been reported to interact not only with its viral cofactor NS4A, but also with a number of host-cell proteins, which suggests a multifunctional feature of NS3. By means of yeast two-hybrid screening using an N-terminal region of NS3 as bait, a human cDNA encoding a region of ELKS-delta, a member of a novel family of proteins involved in intracellular transport and secretory pathways, was molecularly cloned. Using co-immunoprecipitation, GST pull-down and confocal and immunoelectron microscopic analyses, it was shown that full-length NS3 interacted physically with full-length ELKS-delta and its splice variant, ELKS-alpha, both in the absence and presence of NS4A, in cultured human cells, including Huh-7 cells harbouring an HCV subgenomic RNA replicon. The degree of binding to ELKS-delta varied with different sequences of the N-terminal 180 residues of NS3. Interestingly, NS3, either full-length or N-terminal fragments, enhanced secretion of secreted alkaline phosphatase (SEAP) from the cells, and the increase in SEAP secretion correlated well with the degree of binding between NS3 and ELKS-delta. Taken together, these results suggest the possibility that NS3 plays a role in modulating host-cell functions such as intracellular transport and secretion through its binding to ELKS-delta and ELKS-alpha, which may facilitate the virus life cycle and/or mediate the pathogenesis of HCV.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfatase Alcalina/metabolismo , Transporte Biológico , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , DNA Complementar , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Replicon , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.
Assuntos
Hepacivirus/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Replicon/fisiologia , Proteínas não Estruturais Virais/metabolismo , Regiões 5' não Traduzidas , Sequência de Bases , Linhagem Celular Tumoral , Inativação Gênica , Vetores Genéticos , Genoma Viral , Hepacivirus/genética , Humanos , Japão , Lentivirus/genética , Lentivirus/metabolismo , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Viral/genética , RNA Viral/metabolismo , Replicon/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/genéticaRESUMO
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has versatile functions and has been implicated in viral pathogenesis, including interferon (IFN) resistance and hepatocarcinogenesis. It has been reported that NS5A is cleaved into a few fragments by a caspase(s) or caspase-like enzyme(s) under certain conditions. Two cleavage sites have been mapped to the Asp residues at positions 154 and 398 (D154 and D398). However, other possible cleavage sites were not determined yet so far. In this study, we demonstrated caspase-3-mediated NS5A cleavage upon apoptotic stimuli and identified a new site as the third cleavage target. This site was mapped to D251, which lies within IFN sensitivity-determining region (ISDR). Although D251 was conserved among all HCV subtype 1b (HCV-1b) strains tested, the consensus caspase-3 recognition sequence (D248-X-X-D251) was not conserved due to the sequence variation at position 248 in ISDR. Furthermore, A252 was found to be necessary for efficient cleavage of NS5A. The virological significance of the HCV strain-dependent NS5A cleavage at this site awaits further investigation.
Assuntos
Caspases/fisiologia , Hepacivirus/efeitos dos fármacos , Interferons/farmacologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Caspase 3 , Cicloeximida/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Poli I-C/farmacologia , Proteínas não Estruturais Virais/químicaRESUMO
By using a vaccinia virus-T7 expression system, possible effects of hepatitis C virus (HCV) core protein on synthesis and accumulation of host cellular proteins transiently expressed in cultured cells were analyzed. Immunoblot and immunofluorescence analyses revealed that synthesis and accumulation of certain nuclear proteins, such as p21/Waf1, p53, proliferating cell nuclear antigen and c-Fos, were strongly inhibited by HCV core protein. On the other hand, synthesis and accumulation of cytoplasmic proteins, such as 2'-5'-oligoadenylate synthetase (2'-5'-OAS), RNase L and MEK1, were barely affected by HCV core protein. Northern blot analysis showed that the degrees of mRNA expression for those proteins did not differ between HCV core protein-expressing cells and the control, suggesting that the inhibition occurred at the post-transcription level. Pulse-labeling analysis suggested that HCV core protein strongly inhibited synthesis of p21/Waf1 at the translation level. Once being accumulated in the nucleus, p21/Waf1 stability was not significantly affected by HCV core protein. Mutants of HCV core protein C-terminally deleted by 18 or 41 amino acids (aa), which were localized almost exclusively in the nucleus, lost their ability to inhibit synthesis/accumulation of p21/Waf1 whereas another mutant C-terminally deleted by 8 aa still maintained the same properties (subcellular localization and the inhibitory effect) as the full-length HCV core protein of 191 aa. Taken together, our present results suggest that expression of HCV core protein in the cytoplasm selectively inhibits synthesis of p21/Waf1 and some other nuclear proteins at the translation level.
Assuntos
Ciclinas/antagonistas & inibidores , Regulação Viral da Expressão Gênica , Hepacivirus/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas do Core Viral/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Cicloeximida/farmacologia , Vetores Genéticos/genética , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Células HeLa/virologia , Hepacivirus/genética , Humanos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Deleção de Sequência , Relação Estrutura-Atividade , Vaccinia virus/genética , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
The NS3 protein of hepatitis C virus subtype 1b (HCV-1b) isolates obtained from 89 patients with hepatocellular carcinoma (HCC) and 78 patients without HCC were analyzed. On the basis of the secondary structure of the amino-terminal 120 residues of NS3, HCV-1b isolates were classified into group A, group B, and an indeterminate group, each of which was further divided into a number of subgroups, such as A1-1, A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, B2-2, C-1, C-2, and C-3. HCV-1b isolates of subgroup B1-1 were found in 53 (59.6%) of 89 patients with HCC and 19 (24.4%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.00001). Although the number of isolates was small, subgroup B2-1 was also highly associated with HCC, with all five isolates in that subgroup being found in patients with HCC (P < 0.05). On the other hand, HCV-1b isolates of subgroup A1-1 were associated only weakly with HCC; they were found in 6 (6.7%) of 89 patients with HCC and in 25 (32.1%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.0001). The other subgroups, such as A1-2, A2-1, B1-2, C-1, C-2, and C-3, were moderately associated with HCC; their distribution patterns among patients with HCC did not differ significantly from those among patients without HCC. Taken together, our results suggest that HCV-1b isolates of subgroups B1-1 and B2-1 are highly associated with HCC and that this secondary structure analysis may be useful for predicting the relative risk of developing HCC.
Assuntos
Carcinoma Hepatocelular/virologia , Hepacivirus/classificação , Hepatite C/complicações , Neoplasias Hepáticas/virologia , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Feminino , Hepacivirus/química , Hepacivirus/genética , Hepatite C/virologia , Humanos , Masculino , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas não Estruturais Virais/genéticaRESUMO
By using a yeast two-hybrid assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP), also called activating transcription factor 6beta (ATF6beta), was identified as a cellular protein that interacts with the NS4B protein of hepatitis C virus. An N-terminal half of NS4B and a central portion of CREB-RP/ATF6beta containing the basic leucine zipper (bZIP) domain were involved in the interaction. The interaction between NS4B and CREB-RP/ATF6beta was demonstrated also in mammalian cells by co-immunoprecipitation and confocal microscopic analyses using specific antibodies. The bZIP domain of ATF6alpha, which shares high sequence similarity with CREB-RP/ATF6beta, was also shown to interact with NS4B in yeast although the interaction was weaker than that between NS4B and CREB-RP/ATF6beta. CREB-RP/ATF6beta and ATF6alpha are known as endoplasmic reticulum (ER) stress-induced transcription factors. Collectively, our results imply the possibility that NS4B modulates certain cellular responses upon ER stress through the physical interaction with CREB-RP/ATF6beta and ATF6alpha.