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1.
Vaccines (Basel) ; 8(2)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517032

RESUMO

Abstract: Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV) [...].

2.
J Infect Dis ; 221(Supplement_4): S480-S492, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32037447

RESUMO

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.


Assuntos
Antivirais/farmacologia , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Chlorocebus aethiops , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Células HeLa , Infecções por Henipavirus/virologia , Humanos , Mesocricetus , Vírus Nipah/isolamento & purificação , Lectinas de Plantas/uso terapêutico , Células Vero
3.
iScience ; 21: 391-402, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31704650

RESUMO

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

4.
Antiviral Res ; 168: 156-167, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31153968

RESUMO

Chronic hepatitis B virus (HBV) infections cause more than 800,000 deaths per year and currently approved treatments do not cure the disease. Because a hallmark of acute infection resolution is the presence of functional CD8+ T cells to the virus, activation of the immune system with therapeutic vaccines represents a potential approach for treating chronic hepatitis B. In this study, we evaluated the immunogenicity and efficacy of two attenuated vesiculovirus-based platforms expressing HBV Core antigen, the highly attenuated vesicular stomatitis virus (VSV) N4CT1 and a unique vaccine platform [virus-like vesicles (VLV)] that is based on a Semliki Forest virus replicon expressing the VSV glycoprotein. We found that heterologous prime-boost immunization with VLV and N4CT1 induced Core-specific CD8+ T cell responses in naïve mice. When immunized mice were later challenged with AAV-HBV, functional Core-specific CD8+ T cells were present in the liver, and mice were protected from establishment of persistent infection. In contrast, when mice with pre-established persistent HBV replication received prime-boost immunization, functional Core-specific CD8+ T cells were found in the spleen but not in the liver. These results highlight the importance of investigating the therapeutic value of different HBV antigens alone and in combination using preclinical animal models, and understanding the correlation between anti-HBV efficacy in these models with human infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B/imunologia , Hepatite B/prevenção & controle , Vesiculovirus/genética , Animais , Vetores Genéticos , Hepatite B/imunologia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/genética , Imunização Secundária , Fígado/imunologia , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Floresta de Semliki/genética , Vacinação , Vacinas Atenuadas , Vacinas de Partículas Semelhantes a Vírus , Vírus da Estomatite Vesicular Indiana/genética , Replicação Viral
5.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541859

RESUMO

Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8+ T cell and antibody responses. The CD8+ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8+ T cell responses that corresponded with reductions in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in the frequency of intrahepatic HBV-specific CD8+ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multispecific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patient's own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV infection.


Assuntos
Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/terapia , Vacinas Atenuadas/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Alanina Transaminase/sangue , Animais , Linfócitos T CD8-Positivos/imunologia , Hepatite B Crônica/imunologia , Imunoterapia/métodos , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas Virais/imunologia , Replicação Viral/imunologia
6.
Vaccine ; 36(27): 3894-3900, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29807712

RESUMO

Chikungunya virus (CHIKV) and Zika virus (ZIKV) have recently expanded their range in the world and caused serious and widespread outbreaks of near pandemic proportions. There are no licensed vaccines that protect against these co-circulating viruses that are transmitted by invasive mosquito vectors. We report here on the development of a single-dose, bivalent experimental vaccine for CHIKV and ZIKV. This vaccine is based on a chimeric vesicular stomatitis virus (VSV) that expresses the CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G) and also expresses the membrane-envelope (ME) glycoproteins of ZIKV. This vaccine induced neutralizing antibody responses to both CHIKV and ZIKV in wild-type mice and in interferon receptor-deficient A129 mice, animal models for CHIKV and ZIKV infection. A single vaccination of A129 mice with the vector protected these mice against infection with both CHIKV and ZIKV. Our single-dose vaccine could provide durable, low-cost protection against both CHIKV and ZIKV for people traveling to or living in areas where both viruses are circulating, which include most tropical regions in the world.


Assuntos
Febre de Chikungunya/prevenção & controle , Vírus Chikungunya/imunologia , Vacinas Virais/administração & dosagem , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Febre de Chikungunya/terapia , Febre de Chikungunya/virologia , Cricetinae , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Vesiculovirus/genética , Proteínas da Matriz Viral/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/uso terapêutico , Infecção por Zika virus/terapia , Infecção por Zika virus/virologia
7.
EMBO J ; 36(5): 679-692, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28188244

RESUMO

Vesiculoviruses enter cells by membrane fusion, driven by a large, low-pH-induced, conformational change in the fusion glycoprotein G that involves transition from a trimeric pre-fusion toward a trimeric post-fusion state via monomeric intermediates. Here, we present the structure of the G fusion protein at intermediate pH for two vesiculoviruses, vesicular stomatitis virus (VSV) and Chandipura virus (CHAV), which is responsible for deadly encephalopathies. First, a CHAV G crystal structure shows two intermediate conformations forming a flat dimer of heterodimers. On virions, electron microscopy (EM) and tomography reveal monomeric spikes similar to one of the crystal conformations. In solution, mass spectrometry shows dimers of G. Finally, mutations at a dimer interface, involving fusion domains associated in an antiparallel manner to form an intermolecular ß-sheet, affect G fusion properties. The location of the compensatory mutations restoring fusion activity strongly suggests that this interface is functionally relevant. This work reveals the range of G structural changes and suggests that G monomers can re-associate, through antiparallel interactions between fusion domains, into dimers that play a role at some early stage of the fusion process.


Assuntos
Glicoproteínas/metabolismo , Vesiculovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Tomografia
8.
J Virol ; 91(6)2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28077641

RESUMO

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.


Assuntos
Encéfalo/patologia , Vesiculovirus/patogenicidade , Vacinas Virais/efeitos adversos , Animais , Vírus Chikungunya/genética , Vírus Chikungunya/imunologia , Interferon Tipo I/metabolismo , Camundongos , Vírus Nipah/genética , Vírus Nipah/imunologia , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/imunologia , Análise de Sobrevida , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia
9.
Microbes Infect ; 19(4-5): 277-287, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28025070

RESUMO

Anti-retroviral therapy is useful to treat human immunodeficiency virus type 1 (HIV-1)-infected individuals, but has some major problems, such as the generation of multidrug-resistant viruses. To develop a novel supplemental or alternative therapeutic for CCR5-tropic (R5) HIV-1 infection, we generated a recombinant vesicular stomatitis virus (rVSV) in which the gene encoding its envelope glycoprotein (G) was replaced with the genes encoding R5 HIV-1 receptors (human CD4 and CCR5), designated VSVΔG-CC5. Our present data demonstrate that this rVSV specifically infects cells that are transiently expressing R5 HIV-1 envelope glycoproteins, but does not infect those expressing CXCR4-tropic HIV-1 envelope glycoproteins. Notably, after a CD4+CCR5+ T cell line or primary cells initially infected with R5 HIV-1 were inoculated with G-complemented VSVΔG-CC5, the rVSV significantly reduced the number of HIV-1-infected cells, probably through direct targeting of the rVSV and VSV-mediated cytolysis and/or through syncytium formation- or cell-cell fusion-dependent killing, and markedly inhibited HIV-1 production. Furthermore, G-complemented VSVΔG-CC5 also efficiently inhibited HIV-1 infection in R5 HIV-1-infected humanized immunodeficient mice. Taken together, our findings indicate that a cytolytic rVSV that targets and eliminates R5 HIV-1-infected cells potentially has therapeutic value for controlling R5 HIV-1 infection.


Assuntos
Antígenos CD4/genética , Infecções por HIV/prevenção & controle , Terapia Viral Oncolítica/métodos , Receptores CCR5/genética , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Cricetinae , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Carga Viral
10.
Vaccine ; 34(51): 6597-6609, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27395563

RESUMO

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viral and other microbial pathogens in their genome (so-called "chimeric virus vaccines"). Many such viral vector vaccines are now at various stages of clinical evaluation. Here, we introduce an attenuated form of recombinant vesicular stomatitis virus (rVSV) as a potential chimeric virus vaccine for HIV-1, with implications for use as a vaccine vector for other pathogens. The rVSV/HIV-1 vaccine vector was attenuated by combining two major genome modifications. These modifications acted synergistically to greatly enhance vector attenuation and the resulting rVSV vector demonstrated safety in sensitive mouse and non-human primate neurovirulence models. This vector expressing HIV-1 gag protein has completed evaluation in two Phase I clinical trials. In one trial the rVSV/HIV-1 vector was administered in a homologous two-dose regimen, and in a second trial with pDNA in a heterologous prime boost regimen. No serious adverse events were reported nor was vector detected in blood, urine or saliva post vaccination in either trial. Gag specific immune responses were induced in both trials with highest frequency T cell responses detected in the prime boost regimen. The rVSV/HIV-1 vector also demonstrated safety in an ongoing Phase I trial in HIV-1 positive participants. Additionally, clinical trial material has been produced with the rVSV vector expressing HIV-1 env, and Phase I clinical evaluation will initiate in the beginning of 2016. In this paper, we use a standardized template describing key characteristics of the novel rVSV vaccine vectors, in comparison to wild type VSV. The template facilitates scientific discourse among key stakeholders by increasing transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.


Assuntos
Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Portadores de Fármacos , Vesiculovirus/genética , Vacinas contra a AIDS/genética , Animais , Ensaios Clínicos Fase I como Assunto , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Vetores Genéticos , Humanos , Primatas , Medição de Risco , Linfócitos T/imunologia , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
11.
J Virol ; 90(5): 2544-50, 2015 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-26676789

RESUMO

UNLABELLED: Seasonal influenza virus infections continue to cause significant disease each year, and there is a constant threat of the emergence of reassortant influenza strains causing a new pandemic. Available influenza vaccines are variably effective each season, are of limited scope at protecting against viruses that have undergone significant antigenic drift, and offer low protection against newly emergent pandemic strains. "Universal" influenza vaccine strategies that focus on the development of humoral immunity directed against the stalk domains of the viral hemagglutinin (HA) show promise for protecting against diverse influenza viruses. Here, we describe such a strategy that utilizes vesicular stomatitis virus (VSV) as a vector for chimeric hemagglutinin (cHA) antigens. This vaccination strategy is effective at generating HA stalk-specific, broadly cross-reactive serum antibodies by both intramuscular and intranasal routes of vaccination. We show that prime-boost vaccination strategies provide protection against both lethal homologous and heterosubtypic influenza challenge and that protection is significantly improved with intranasal vaccine administration. Additionally, we show that vaccination with VSV-cHAs generates greater stalk-specific and cross-reactive serum antibodies than does vaccination with VSV-vectored full-length HAs, confirming that cHA-based vaccination strategies are superior at generating stalk-specific humoral immunity. VSV-vectored influenza vaccines that express chimeric hemagglutinin antigens offer a novel means for protecting against widely diverging influenza viruses. IMPORTANCE: Universal influenza vaccination strategies should be capable of protecting against a wide array of influenza viruses, and we have developed such an approach utilizing a single viral vector system. The potent antibody responses that these vaccines generate are shown to protect mice against lethal influenza challenges with highly divergent viruses. Notably, intranasal vaccination offers significantly better protection than intramuscular vaccination in a lethal virus challenge model. The results described in this study offer insights into the mechanisms by which chimeric hemagglutinin (HA)-based vaccines confer immunity, namely, that the invariant stalk of cHA antigens is superior to full-length HA antigens at inducing cross-reactive humoral immune responses and that VSV-cHA vaccine-induced protection varies by site of inoculation, and contribute to the further development of universal influenza virus vaccines.


Assuntos
Portadores de Fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Orthomyxoviridae/imunologia , Vesiculovirus/genética , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Peso Corporal , Reações Cruzadas , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Intramusculares , Camundongos Endogâmicos BALB C , Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sobrevida , Vacinação/métodos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
12.
J Virol ; 89(20): 10407-15, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26246574

RESUMO

UNLABELLED: More than 500,000 people die each year from the liver diseases that result from chronic hepatitis B virus (HBV) infection. Therapeutic vaccines, which aim to elicit an immune response capable of controlling the virus, offer a potential new treatment strategy for chronic hepatitis B. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the vesicular stomatitis virus glycoprotein (VSV G) has been described. This platform generates virus-like vesicles (VLVs) that contain VSV G but no other viral structural proteins. We report here that the evolved VLV vector engineered to additionally express the HBV middle surface envelope glycoprotein (MHBs) induces functional CD8 T cell responses in mice. These responses were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. Additionally, a single immunization with VLV-MHBs protected mice from HBV hydrodynamic challenge, and this protection correlated with the elicitation of a CD8 T cell recall response. In contrast to MHBs, a VLV expressing HBV core protein (HBcAg) neither induced a CD8 T cell response in mice nor protected against challenge. Finally, combining DNA and VLV-MHBs immunization led to induction of HBV-specific CD8 T cell responses in a transgenic mouse model of chronic HBV infection. The ability of VLV-MHBs to induce a multispecific T cell response capable of controlling HBV replication, and to generate immune responses in a tolerogenic model of chronic infection, indicates that VLV vaccine platforms may offer a unique strategy for HBV therapeutic vaccination. IMPORTANCE: HBV infection is associated with significant morbidity and mortality. Furthermore, treatments for chronic infection are suboptimal and rarely result in complete elimination of the virus. Therapeutic vaccines represent a unique approach to HBV treatment and have the potential to induce long-term control of infection. Recently, a virus-based vector system that combines the nonstructural proteins of Semliki Forest virus with the VSV glycoprotein has been described. In this study, we used this system to construct a novel HBV vaccine and demonstrated that the vaccine is capable of inducing virus-specific immune responses in mouse models of acute and chronic HBV replication. These findings highlight the potential of this new vaccine system and support the idea that highly immunogenic vaccines, such as viral vectors, may be useful in the treatment of chronic hepatitis B.


Assuntos
Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/prevenção & controle , Imunidade Celular/efeitos dos fármacos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Cricetulus , ELISPOT , Células Epiteliais/imunologia , Células Epiteliais/virologia , Engenharia Genética , Vetores Genéticos/química , Vetores Genéticos/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Imunização , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Replicação Viral/efeitos dos fármacos
13.
PLoS Pathog ; 11(3): e1004756, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25803715

RESUMO

Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.


Assuntos
Evolução Molecular , Nucleocapsídeo/química , Vesiculovirus/química , Proteínas Virais de Fusão/química , Humanos , Concentração de Íons de Hidrogênio , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Estrutura Terciária de Proteína , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
14.
Jpn J Infect Dis ; 68(3): 203-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25672345

RESUMO

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.


Assuntos
Proteínas de Fluorescência Verde/genética , Hepacivirus/genética , Modelos Biológicos , Estomatite Vesicular/genética , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Cricetinae , Proteínas de Fluorescência Verde/metabolismo , Hepacivirus/metabolismo , Hepatite C/virologia , Humanos , Proteínas do Envelope Viral/metabolismo
15.
Virology ; 476: 405-412, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25591175

RESUMO

We reported previously on a vaccine approach that conferred apparent sterilizing immunity to SIVsmE660. The vaccine regimen employed a prime-boost using vectors based on recombinant vesicular stomatitis virus (VSV) and an alphavirus replicon expressing either SIV Gag or SIV Env. In the current study, we tested the ability of vectors expressing only the SIVsmE660 Env protein to protect macaques against the same high-dose mucosal challenge. Animals developed neutralizing antibody levels comparable to or greater than seen in the previous vaccine study. When the vaccinated animals were challenged with the same high-dose of SIVsmE660, all became infected. While average peak viral loads in animals were slightly lower than those of previous controls, the viral set points were not significantly different. These data indicate that Gag, or the combination of Gag and Env are required for the generation of apparent sterilizing immunity to the SIVsmE660 challenge.


Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Mucosa/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/genética , HIV/genética , HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Macaca mulatta , Mucosa/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética
16.
J Virol ; 90(6): 3268-73, 2015 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-26719251

RESUMO

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.


Assuntos
Transmissão de Doença Infecciosa/prevenção & controle , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/transmissão , Animais , Modelos Animais de Doenças , Furões , Vírus da Influenza A Subtipo H1N1/fisiologia , Vacinas contra Influenza/administração & dosagem , Masculino , Infecções por Orthomyxoviridae/virologia , Carga Viral , Replicação Viral/imunologia
17.
J Virol ; 89(5): 2820-30, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25540378

RESUMO

UNLABELLED: The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCE: Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a potent antibody response in mice that was effective against infection with a lethal influenza virus. The mice showed no adverse reactions to the vaccine, and they were protected against an otherwise lethal influenza infection after only 14 days postvaccination and after as many as 140 days postvaccination. The ability to rapidly produce this safe and effective vaccine in cell culture is additionally advantageous.


Assuntos
Portadores de Fármacos , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Vesiculovirus/genética , Proteínas Virais/imunologia , Administração Intranasal , Animais , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , 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 , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Intramusculares , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Orthomyxoviridae , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Análise de Sobrevida , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Replicação Viral
18.
Proc Natl Acad Sci U S A ; 111(47): 16866-71, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25385608

RESUMO

Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein. The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which high-titer VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity.


Assuntos
Alphavirus/genética , Evolução Biológica , Proteínas Estruturais Virais/química , Alphavirus/química , Motivos de Aminoácidos , Técnicas In Vitro , Microscopia Eletrônica de Transmissão
19.
PLoS One ; 9(10): e109678, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25360552

RESUMO

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID50. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.


Assuntos
Macaca mulatta/virologia , Filogenia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Proteínas do Envelope Viral/genética , Animais , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene gag/genética , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunização , Macaca mulatta/imunologia , Dados de Sequência Molecular , Vacinas contra a SAIDS/imunologia , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Estomatite Vesicular/virologia , Proteínas do Envelope Viral/imunologia , Carga Viral
20.
J Virol ; 88(6): 3432-42, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24403585

RESUMO

UNLABELLED: Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-based universal influenza virus vaccine strategies are being developed as promising countermeasures for influenza virus infections. The pan-H1-reactive monoclonal antibody 6F12 has been extensively characterized and shows broad efficacy against divergent H1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemagglutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal antibody and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbinates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, both strategies showed efficacy in reducing viral loads after an influenza virus challenge in the ferret model. IMPORTANCE: Influenza virus hemagglutinin stalk-reactive antibodies tend to be less potent yet are more broadly reactive and can neutralize seasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza virus infection. The novelty and significance of the findings described in this report support the development of vaccines stimulating stalk-specific antibody responses.


Assuntos
Modelos Animais de Doenças , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Masculino , Estrutura Terciária de Proteína
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