RESUMEN
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.
Asunto(s)
Glicoproteínas/metabolismo , Vesiculovirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Microscopía Electrónica , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , TomografíaRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Infecciones por Henipavirus/tratamiento farmacológico , Virus Nipah/efectos de los fármacos , Lectinas de Plantas/farmacología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Células HEK293 , Células HeLa , Infecciones por Henipavirus/virología , Humanos , Mesocricetus , Virus Nipah/aislamiento & purificación , Lectinas de Plantas/uso terapéutico , Células VeroRESUMEN
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.
Asunto(s)
Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/prevención & control , Hepatitis B Crónica/terapia , Vacunas Atenuadas/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Alanina Transaminasa/sangre , Animales , Linfocitos T CD8-positivos/inmunología , Hepatitis B Crónica/inmunología , Inmunoterapia/métodos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Vacunas Virales/inmunología , Replicación Viral/inmunologíaRESUMEN
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.
Asunto(s)
Encéfalo/patología , Vesiculovirus/patogenicidad , Vacunas Virales/efectos adversos , Animales , Virus Chikungunya/genética , Virus Chikungunya/inmunología , Interferón Tipo I/metabolismo , Ratones , Virus Nipah/genética , Virus Nipah/inmunología , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/inmunología , Análisis de Supervivencia , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vesiculovirus/genética , Vesiculovirus/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Evolución Molecular , Nucleocápside/química , Vesiculovirus/química , Proteínas Virales de Fusión/química , Humanos , Concentración de Iones de Hidrógeno , Nucleocápside/genética , Nucleocápside/metabolismo , Estructura Terciaria de Proteína , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
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.
Asunto(s)
Alphavirus/genética , Evolución Biológica , Proteínas Estructurales Virales/química , Alphavirus/química , Secuencias de Aminoácidos , Técnicas In Vitro , Microscopía Electrónica de TransmisiónRESUMEN
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.
Asunto(s)
Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/prevención & control , Inmunidad Celular/efectos de los fármacos , Vacunas de Partículas Similares a Virus/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Línea Celular , Cricetulus , Ensayo de Immunospot Ligado a Enzimas , Células Epiteliales/inmunología , Células Epiteliales/virología , Ingeniería Genética , Vectores Genéticos/química , Vectores Genéticos/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Inmunización , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Portadores de Fármacos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Orthomyxoviridae/inmunología , Vesiculovirus/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Peso Corporal , Reacciones Cruzadas , Modelos Animales de Enfermedad , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Inyecciones Intramusculares , Ratones Endogámicos BALB C , Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Portadores de Fármacos , Vectores Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Neuraminidasa/inmunología , Vesiculovirus/genética , Proteínas Virales/inmunología , Administración Intranasal , Animales , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Inyecciones Intramusculares , Ratones Endogámicos BALB C , Neuraminidasa/genética , Orthomyxoviridae , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Análisis de Supervivencia , Vacunación/métodos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Replicación ViralRESUMEN
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.
Asunto(s)
Transmisión de Enfermedad Infecciosa/prevención & control , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/transmisión , Animales , Modelos Animales de Enfermedad , Hurones , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/administración & dosificación , Masculino , Infecciones por Orthomyxoviridae/virología , Carga Viral , Replicación Viral/inmunologíaRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hemaglutininas , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Masculino , Estructura Terciaria de ProteínaRESUMEN
While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Virus Chikungunya/inmunología , Vectores Genéticos , Vesiculovirus/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Infecciones por Alphavirus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Virus Chikungunya/genética , Modelos Animales de Enfermedad , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos C57BL , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.
Asunto(s)
Virus Nipah/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Línea Celular , Análisis Mutacional de ADN , Humanos , Virus de la Enfermedad de Newcastle/genética , Virus Nipah/genética , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Replication-defective vaccine vectors based on vesicular stomatitis virus (VSV) lacking its envelope glycoprotein gene (G) are highly effective in animal models. However, such ΔG vectors are difficult to grow because they require complementation with the VSV G protein. In addition, the complementing G protein induces neutralizing antibodies in animals and thus limits multiple vector applications. In the process of generating an experimental Nipah virus (a paramyxovirus) vaccine, we generated two defective VSVΔG vectors, each expressing one of the two Nipah virus (NiV) glycoproteins (G and F) that are both required for virus entry to host cells. These replication-defective VSV vectors were effective at generating NiV neutralizing antibody in mice. Most interestingly, we found that these two defective viruses could be grown together and passaged in tissue culture cells in the absence of VSV G complementation. This mixture of complementing defective viruses was also highly effective at generating NiV neutralizing antibody in animals. This novel approach to growing and producing a vaccine from two defective viruses could be generally applicable to vaccine production for other paramyxoviruses or for other viruses where the expression of at least two different proteins is required for viral entry. Such an approach minimizes biosafety concerns that could apply to single, replication-competent VSV recombinants expressing all proteins required for infection.
Asunto(s)
Virus Defectuosos/genética , Expresión Génica , Vectores Genéticos/genética , Virus Nipah/inmunología , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Línea Celular , Virus Defectuosos/inmunología , Virus Defectuosos/fisiología , Femenino , Prueba de Complementación Genética , Vectores Genéticos/inmunología , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/prevención & control , Infecciones por Henipavirus/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Virus Nipah/genética , Vesiculovirus/genética , Vesiculovirus/fisiología , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , Replicación ViralRESUMEN
We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.
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Anticuerpos Neutralizantes/sangre , Portadores de Fármacos , Vectores Genéticos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Vesiculovirus/genética , Animales , Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Macaca mulattaRESUMEN
We constructed vaccine vectors based on live recombinant vesicular stomatitis virus (VSV) and a Semliki Forest virus (SFV) replicon (SFVG) that propagates through expression of the VSV glycoprotein (G). These vectors expressing simian immunodeficiency virus (SIV) Gag and Env proteins were used to vaccinate rhesus macaques with a new heterologous prime-boost regimen designed to optimize induction of antibody. Six vaccinated animals and six controls were then given a high-dose mucosal challenge with the diverse SIVsmE660 quasispecies. All control animals became infected and had peak viral RNA loads of 10(6) to 10(8) copies/ml. In contrast, four of the vaccinees showed significant (P = 0.03) apparent sterilizing immunity and no detectable viral loads. Subsequent CD8(+) T cell depletion confirmed the absence of SIV infection in these animals. The two other vaccinees had peak viral loads of 7 × 10(5) and 8 × 10(3) copies/ml, levels below those of all of the controls, and showed undetectable virus loads by day 42 postchallenge. The vaccine regimen induced high-titer prechallenge serum neutralizing antibodies (nAbs) to some cloned SIVsmE660 Env proteins, but antibodies able to neutralize the challenge virus swarm were not detected. The cellular immune responses induced by the vaccine were generally weak and did not correlate with protection. Although the immune correlates of protection are not yet clear, the heterologous VSV/SFVG prime-boost is clearly a potent vaccine regimen for inducing virus nAbs and protection against a heterogeneous viral swarm.
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Anticuerpos Antivirales/sangre , Vectores Genéticos/inmunología , Esquemas de Inmunización , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Productos del Gen env/genética , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Productos del Gen gag/metabolismo , Vectores Genéticos/administración & dosificación , Inmunización , Inmunización Secundaria , Macaca mulatta , Pruebas de Neutralización , Vacunas contra el SIDAS/genética , Vacunas contra el SIDAS/inmunología , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Carga ViralRESUMEN
Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G(th)) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G(th) obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal.
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Glicoproteínas/química , Vesiculovirus/química , Proteínas Virales/química , Cristalización , Cristalografía por Rayos XRESUMEN
Our previous studies using intranasal inoculation of mice with vesicular stomatitis virus (VSV) vaccine vectors showed persistence of vector genomic RNA (gRNA) for at least 60 days in lymph nodes in the absence of detectable infectious virus. Here we show high-level concentration of virus and gRNA in lymph nodes after intramuscular inoculation of mice with attenuated or single-cycle VSV vectors as well as long-term persistence of gRNA in the lymph nodes. To determine if the persistence of gRNA was due to ongoing viral replication, we developed a tagged-primer approach that was critical for detection of VSV mRNA specifically. Our results show that VSV gRNA persists long-term in the lymph nodes while VSV mRNA is present only transiently. Because VSV transcription is required for replication, our results indicate that persistence of gRNA does not result from continuing viral replication. We also performed macrophage depletion studies that are consistent with initial trapping of VSV gRNA largely in lymph node macrophages and subsequent persistence elsewhere in the lymph node.
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Genoma Viral , ARN Viral/análisis , Virus de la Estomatitis Vesicular Indiana/genética , Replicación Viral , Animales , Ganglios Linfáticos/virología , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/virología , Reacción en Cadena de la Polimerasa , Transcripción Genética , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
As one of the world's most common infectious diseases, hepatitis B virus (HBV) is a serious worldwide public health problem, with HBV-associated liver disease accounting for more than half a million deaths each year. Although there is an effective prophylactic vaccine currently available to prevent infection, it has a number of characteristics that are suboptimal: multiple doses are needed to induce long-lasting immunity, immunity declines over time, it does not elicit protection in some individuals, and it is not effective therapeutically. We produced a recombinant vesicular stomatitis virus (VSV)-based vaccine vector expressing the HBV middle envelope surface protein (MS) and found that this vector was able to efficiently generate a strong HBs-specific antibody response following a single immunization in mice. A single immunization with the VSV-MS vector also induced robust CD8 T-cell activation. The CD8 T-cell response was greater in magnitude and broader in specificity than the response generated by a vaccinia virus-based vaccine vector or by recombinant protein immunization. Furthermore, a single VSV-MS immunization provided protection against virus challenge in mice. Given the similar antibody titers and superior T-cell responses elicited from a single immunization, a VSV-based HBV vaccine may have advantages over the current recombinant protein vaccine.
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Vectores Genéticos , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/prevención & control , Vacunación/métodos , Vesiculovirus/genética , Animales , Peso Corporal , Linfocitos T CD8-positivos/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Hepatitis B/patología , Anticuerpos contra la Hepatitis B/sangre , Vacunas contra Hepatitis B/genética , Virus de la Hepatitis B/genética , Interferón gamma/metabolismo , Ratones , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genéticaRESUMEN
Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation.