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INTRODUCTION: The use of novel adjuvants in human vaccines continues to expand as their contribution to preventing disease in challenging populations and caused by complex pathogens is increasingly understood. AS01 is a family of liposome-based vaccine Adjuvant Systems containing two immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01-containing vaccines have been approved and administered to millions of individuals worldwide. AREAS COVERED: Here, we report advances in our understanding of the mode of action of AS01 that contributed to the development of efficacious vaccines preventing disease due to malaria, herpes zoster, and respiratory syncytial virus. AS01 induces early innate immune activation that induces T cell-mediated and antibody-mediated responses with optimized functional characteristics and induction of immune memory. AS01-containing vaccines appear relatively impervious to baseline immune status translating into high efficacy across populations. Currently licensed AS01-containing vaccines have shown acceptable safety profiles in clinical trials and post-marketing settings. EXPERT OPINION: Initial expectations that adjuvantation with AS01 could support effective vaccine responses and contribute to disease control have been realized. Investigation of the utility of AS01 in vaccines to prevent other challenging diseases, such as tuberculosis, is ongoing, together with efforts to fully define its mechanisms of action in different vaccine settings.
Adjuvants are added to vaccines to increase the immune response produced after vaccination. Adjuvant Systems contain two or more molecules that stimulate the immune system. AS01 is an Adjuvant System that contains two components, MPL and QS-21, that stimulate the immune system. AS01 is included in three approved vaccines: a malaria vaccine for children, a herpes zoster vaccine for older adults, and a respiratory syncytial virus vaccine also for older adults. Vaccines containing AS01 have been extensively evaluated in clinical trials and administered to millions of individuals during market use. These vaccines are effective in preventing disease and have acceptable safety in different age groups. Experiments have been done to investigate how AS01 works in vaccines to produce an efficient immune response that helps to protect against the disease being targeted. A key effect of AS01 is to encourage specific immune cells to produce chemicals that stimulate the immune system. We now know that this effect is due to co-operation between MPL and QS-21. Experiments have shown that AS01 induces a sophisticated immune 'gene signature' in blood within 24 h after vaccination, and people who developed this 'gene signature' had a stronger response to vaccination. AS01 seems to be able to stimulate the immune system of most people even if they are older or have a weakened immune system. This means that AS01 could be included in other vaccines against other challenging diseases, such as tuberculosis, or could be used in the treatment of some disease, such as chronic hepatitis B.
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Adjuvantes Imunológicos , Adjuvantes de Vacinas , Saponinas , Humanos , Saponinas/imunologia , Saponinas/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeo A/farmacologia , Animais , Imunidade Inata/efeitos dos fármacos , Vacinas contra Vírus Sincicial Respiratório/imunologia , Lipossomos , Malária/prevenção & controle , Malária/imunologia , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/administração & dosagem , Combinação de MedicamentosRESUMO
Respiratory syncytial virus (RSV) causes a high disease burden in older adults. An effective vaccine for this RSV-primed population may need to boost/elicit robust RSV-neutralizing antibody responses and recall/induce RSV-specific T cell responses. To inform the selection of the vaccine formulation for older adults, RSVPreF3 (RSV fusion glycoprotein engineered to maintain the prefusion conformation) with/without AS01 adjuvant was evaluated in mice and bovine RSV infection-primed cattle. In mice, RSVPreF3/AS01 elicited robust RSV-A/B-specific neutralization titers and RSV F-specific polyfunctional CD4+ T cell responses exceeding those induced by non-adjuvanted RSVPreF3. In primed bovines, RSVPreF3/AS01 tended to induce higher pre-/post-vaccination fold-increases in RSV-A/B-specific neutralization titers relative to non-adjuvanted and Alum-adjuvanted RSVPreF3 formulations, and elicited higher RSV F-specific CD4+ T cell frequencies relative to the non-adjuvanted vaccine. Though AS01 adjuvanticity varied by animal species and priming status, RSVPreF3/AS01 elicited/boosted RSV-A/B-specific neutralization titers and RSV F-specific CD4+ T cell responses in both animal models, which supported its further clinical evaluation as prophylactic candidate vaccine for older adults.
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The RSVPreF3-AS01 vaccine, containing the respiratory syncytial virus (RSV) prefusion F protein and the AS01 adjuvant, was previously shown to boost neutralization responses against historical RSV strains and to be efficacious in preventing RSV-associated lower respiratory tract diseases in older adults. Although RSV F is highly conserved, variation does exist between strains. Here, we characterized variations in the major viral antigenic sites among contemporary RSV sequences when compared with RSVPreF3 and showed that, in older adults, RSVPreF3-AS01 broadly boosts neutralization responses against currently dominant and antigenically distant RSV strains. RSV-neutralizing responses are thought to play a central role in preventing RSV infection. Therefore, the breadth of RSVPreF3-AS01-elicited neutralization responses may contribute to vaccine efficacy against contemporary RSV strains and those that may emerge in the future.
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Infecções por Vírus Respiratório Sincicial , Vacinas , Humanos , Idoso , Vírus Sinciciais Respiratórios , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Antígenos ViraisRESUMO
Mouse models are useful for the early down-selection of malaria vaccine candidates. The Walter Reed Army Institute of Research has optimized a transgenic Plasmodium berghei sporozoite challenge model to compare the efficacy of Plasmodium falciparum circumsporozoite protein (CSP) vaccines. GSK's RTS,S vaccine formulated in the adjuvant AS01 can protect malaria-naïve individuals against malaria. We report that the RTS,S/AS01 vaccine induces high level sterile protection in our mouse model. Down titration of the antigen at a constant AS01 dose revealed a potent antigen dose-sparing effect and the superiority of RTS,S/AS01 over a soluble CSP antigen. RTS,S-mediated protective immunity was associated with a threshold of major repeat antibody titer. Combined titration of the antigen and adjuvant showed that reducing the adjuvant could improve antibody boosting post-3rd vaccination and reduce the threshold antibody concentration required for protection. Mouse models can provide a pathway for preclinical assessment of strategies to improve CSP vaccines against malaria.
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Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dengue/imunologia , Animais , Dengue/virologia , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Feminino , Cinética , Macaca mulatta/imunologia , Mosquitos Vetores/virologia , RNA Viral/sangue , Glândulas Salivares/virologia , Vacinação , Carga Viral , Viremia/prevenção & controle , Replicação ViralRESUMO
The need for improved dengue vaccines remains since the only licensed vaccine, Dengvaxia, shows variable efficacy depending on the infecting dengue virus (DENV) type, and increases the risk of hospitalization for severe dengue in children not exposed to DENV before vaccination. Here, we developed a tetravalent dengue purified and inactivated vaccine (DPIV) candidate and characterized, in rhesus macaques, its immunogenicity and efficacy to control DENV infection by analyzing, after challenge, both viral replication and changes in biological markers associated with dengue in humans. Although DPIV elicited cross-type and long-lasting DENV-neutralizing antibody responses, it failed to control DENV infection. Increased levels of viremia/RNAemia (correlating with serum capacity at enhancing DENV infection in vitro), AST, IL-10, IL-18 and IFN-γ, and decreased levels of IL-12 were detected in some vaccinated compared to non-vaccinated monkeys, indicating the vaccination may have triggered antibody-dependent enhancement of DENV infection. The dengue macaque model has been considered imperfect due to the lack of DENV-associated clinical signs. However, here we show that post-vaccination enhanced DENV infection can be detected in this model when integrating several parameters, including characterization of DENV-enhancing antibodies, viremia/RNAemia, and biomarkers relevant to dengue in humans. This improved dengue macaque model may be crucial for early assessment of efficacy and safety of future dengue vaccines.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Vacinas de Produtos Inativados/imunologia , Viremia/imunologia , Animais , Anticorpos Facilitadores , Dengue/prevenção & controle , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Modelos Animais de Doenças , Feminino , Macaca mulatta , Masculino , Vacinação , Viremia/virologiaRESUMO
The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model's predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.
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Vírus da Dengue/imunologia , Dengue/patologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Brasil , Quimiocina CCL2/metabolismo , Chlorocebus aethiops , Dengue/veterinária , Vírus da Dengue/isolamento & purificação , Regulação para Baixo , Interferon gama/metabolismo , Interleucina-8 , Macaca mulatta , Sorogrupo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células VeroRESUMO
UNLABELLED: Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.
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Vírus GB B/fisiologia , Hepacivirus/fisiologia , Estágios do Ciclo de Vida/fisiologia , Modelos Animais , Primatas/virologia , Internalização do Vírus , Animais , Sequência de Bases , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Células HEK293 , Hepacivirus/genética , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Immunoblotting , Dados de Sequência Molecular , Plasmídeos/genética , Análise de Sequência de DNA , ViremiaRESUMO
The immunogenicity and protective efficacy of a candidate tetravalent dengue virus purified inactivated vaccine (TDENV PIV) formulated with alum or an Adjuvant System (AS01, AS03 tested at three different dose levels, or AS04) was evaluated in a 0, 1-month vaccination schedule in rhesus macaques. One month after dose 2, all adjuvanted formulations elicited robust and persisting neutralizing antibody titers against all four dengue virus serotypes. Most of the formulations tested prevented viremia after challenge, with the dengue serotype 1 and 2 virus strains administered at 40 and 32 weeks post-dose 2, respectively. This study shows that inactivated dengue vaccines, when formulated with alum or an Adjuvant System, are candidates for further development.
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Anticorpos Antivirais/biossíntese , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Vacinação , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/biossíntese , Chlorocebus aethiops , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Modelos Animais de Doenças , Feminino , Macaca mulatta , Masculino , RNA Viral/sangue , Distribuição Aleatória , Fatores de Tempo , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Células Vero , Viremia/prevenção & controle , Viremia/virologiaRESUMO
Chikungunya virus (CHIKV) is an alphavirus prevalent in tropical regions. It causes an acute febrile disease that, in elderly individuals and newborns, is often associated with severe complications. We previously reported the isolation and characterization of 2 human monoclonal antibodies neutralizing CHIKV in vitro: 5F10 and 8B10. Here, we tested their efficacy in vivo as prophylactic and therapeutic treatments of CHIKV infection in AGR129 mice. In both settings, 5F10 and 8B10 were able to significantly delay CHIKV-driven lethality. Our results support the development of prophylactic and therapeutic treatments for CHIKV infection, using a combination of 5F10 and 8B10.
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Infecções por Alphavirus/terapia , Anticorpos Monoclonais/uso terapêutico , Vírus Chikungunya/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/mortalidade , Infecções por Alphavirus/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Febre de Chikungunya , Modelos Animais de Doenças , Humanos , Camundongos , Resultado do TratamentoRESUMO
Dengue virus immune protection is specific to the serotype encountered and is thought to persist throughout one's lifetime. Many serotype cross-reactive memory B cells isolated from humans with previous dengue infection are specific for the nonstructural and the prM structural viral proteins, and they can enhance infection in vitro. However, plasmablasts circulating in enormous numbers during acute secondary infection have not been studied. In this study, we analyzed single plasmablasts from two patients by sorting the cells for Ig sequence analysis and for recombinant expression of Abs. In contrast to memory B cells, most plasmablast-derived Abs bound to the structural E protein of dengue, and protection experiments in mice revealed that virus serotypes encountered during past infections were neutralized more efficiently than were the serotypes of the current infection. Together with genetic analyses, we show evidence that plasmablasts in dengue patients are a polyclonal pool of activated E protein-specific memory B cells and that their specificity is not representative of the serum Abs secreted by long-lived plasma cells in the memory phase. These results contribute to the understanding of the phenomenon of original antigenic sin in dengue.
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Sítios de Ligação de Anticorpos , Diferenciação Celular/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/virologia , Epitopos de Linfócito B/metabolismo , Plasmócitos/imunologia , Plasmócitos/virologia , Adulto , Animais , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Dengue/classificação , Vírus da Dengue/classificação , Epitopos de Linfócito B/imunologia , Humanos , Memória Imunológica , Camundongos , Plasmócitos/metabolismo , Ligação Proteica/imunologia , Recidiva , Sorotipagem , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia , Vírion/metabolismo , Adulto JovemRESUMO
Anti-viral immune responses have been studied extensively in order to inform rational vaccine design. Following viral infection, the balance of pathologic and protective antibody responses in the host can critically influence clinical outcomes. Comparisons of the different classes of antibodies produced after acute or chronic viral infections have uncovered common features of anti-viral responses, but these analyses have also revealed temporal differences in neutralizing antibody production, variable neutralization potency and differential induction of cross-reactive antibodies. Cross-reactive antibodies are known to play crucial protective roles in host responses to chronic viral infections; recent studies in human immunodeficiency virus long-term controllers have identified a novel class of broadly neutralizing antibodies generated from highly mutated and selected memory B cells. Here, we summarize the various roles played by cross- and poly-reactive antibodies in acute and persistent viral infections, with a focus on the potential contribution of these antibodies to dengue virus (DENV) immunopathology and host protection. Since host antibodies profoundly alter the course of viral infections, effective DENV vaccine design will require a better understanding of the origin, affinity maturation and protective potential of the poly-reactive and cross-reactive antibodies induced by different interventions.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Imunidade Humoral , Doença Aguda , Animais , Antígenos Virais/imunologia , Doença Crônica , Reações Cruzadas , Dengue/prevenção & controle , HIV/imunologia , Infecções por HIV/imunologia , Humanos , Memória ImunológicaRESUMO
Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.
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Infecções por Alphavirus/imunologia , Infecções por Alphavirus/transmissão , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Evasão da Resposta Imune , Mutação , Proteínas Virais/imunologia , Infecções por Alphavirus/genética , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/genética , Vírus Chikungunya/genética , Doença Crônica , Células HEK293 , Humanos , Imunoglobulina M/imunologia , Camundongos , Camundongos Knockout , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais/genéticaRESUMO
Currently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.
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Ensaios de Triagem em Larga Escala/métodos , Malária Vivax/diagnóstico , Plasmodium vivax/isolamento & purificação , Plasmodium vivax/fisiologia , Reticulócitos/parasitologia , Células Cultivadas , Testes Hematológicos/métodos , Ensaios de Triagem em Larga Escala/normas , Interações Hospedeiro-Patógeno , Humanos , Malária Vivax/sangue , Malária Vivax/parasitologia , Malária Vivax/patologia , Plasmodium vivax/citologia , Reprodutibilidade dos TestesRESUMO
Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC(50) of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O'nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host-pathogen interactions and the rational design of vaccines against CHIKV.
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Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Anticorpos Monoclonais/fisiologia , Anticorpos Neutralizantes/fisiologia , Vírus Chikungunya/imunologia , Testes de Neutralização/métodos , Proteínas do Envelope Viral/imunologia , Infecções por Alphavirus/prevenção & controle , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular Transformada , Vírus Chikungunya/patogenicidade , Chlorocebus aethiops , Células HEK293 , Humanos , Imunização Passiva/métodos , Imunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , Células VeroRESUMO
The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.
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Replicação do DNA , Vírus GB B/enzimologia , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Infecções por Flaviviridae/virologia , Vírus GB B/química , Vírus GB B/genética , Vírus GB B/metabolismo , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/virologia , Hepatite Viral Humana/virologia , Humanos , Dados de Sequência Molecular , Ligação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) genome (within the 3'NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC) RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.
Assuntos
Vírus GB B/fisiologia , Hepacivirus/fisiologia , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Vírus GB B/genética , Vírus GB B/patogenicidade , Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/patogenicidade , Humanos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA não Traduzido/química , RNA não Traduzido/genética , RNA Viral/química , RNA Viral/genética , Replicon , Saguinus/virologia , Análise de Sequência de RNA , Replicação ViralRESUMO
GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of beta-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.
Assuntos
Vírus GB B/patogenicidade , Hepacivirus/patogenicidade , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vírus GB B/genética , Vírus GB B/metabolismo , Regulação da Expressão Gênica , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/genética , Frações Subcelulares/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteínas não Estruturais Virais/genética , beta Catenina/genética , beta Catenina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.