RESUMEN
Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Gripe Humana/virología , Interferones/metabolismo , Orthomyxoviridae/patogenicidad , Proteínas Virales/fisiología , Células A549 , Animales , Perros , Femenino , Células HEK293 , Historia del Siglo XX , Humanos , Gripe Humana/epidemiología , Gripe Humana/historia , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/metabolismo , Pandemias , Proteolisis , Transducción de Señal , Células U937 , Proteínas Virales/metabolismo , Virulencia/fisiologíaRESUMEN
The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic "pace-keeping" role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.
Asunto(s)
Ferritinas , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Chaperonas Moleculares , Nanopartículas , ARN Viral , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Femenino , Ferritinas/química , Vectores Genéticos , Humanos , Inmunidad Celular , Inmunización , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestructura , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Chaperonas Moleculares/química , Nanopartículas/química , Nanopartículas/ultraestructura , Unión Proteica , Multimerización de Proteína , ARN Viral/química , Proteínas Recombinantes/química , Solubilidad , Análisis Espectral , Vacunas Sintéticas , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas ViralesRESUMEN
Influenza virus infections continually pose a major public health threat with seasonal epidemics and sporadic pandemics worldwide. While currently licensed influenza vaccines provide only strain-specific protection, antigenic drift and shift occasionally render the viruses resistant to the host immune responses, which highlight the need for a vaccine that provides broad protection against multiple subtypes. In this study, we suggest a vaccination strategy using cold-adapted, live attenuated influenza vaccines (CAIVs) to provide a broad, potent, and safe cross-protection covering antigenically distinct hemagglutinin (HA) groups 1 and 2 influenza viruses. Using a mouse model, we tested different prime-boost combinations of CAIVs for their ability to induce humoral and T-cell responses, and protective efficacy against H1 and H5 (HA group 1) as well as H3 and H7 (HA group 2) influenza viruses. Notably, even in the absence of antibody-mediated neutralizing activity or HA inhibitory activity in vitro, CAIVs provided a potent protection against heterologous and heterosubtypic lethal challenges in vivo. Heterologous combination of prime (H1)-boost (H5) vaccine strains showed the most potent cross-protection efficacy. In vivo depletion experiments demonstrated not only that T cells and natural killer cells contributed to the cross-protection, but also the involvement of antibody-dependent mechanisms for the cross-protection. Vaccination-induced antibodies did not enhance the infectivity of heterologous viruses, and prime vaccination did not interfere with neutralizing antibody generation by the boost vaccination, allaying vaccine safety concerns associated with heterogeneity between the vaccines and challenge strains. Our data show that CAIV-based strategy can serve as a simple but powerful option for developing a "truly" universal influenza vaccine providing pan-influenza A protection, which has not been achieved yet by other vaccine strategies. The promising results of potency, breadth, and safety demonstrated in the mouse model support further studies in higher animal models for clinical relevance.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza , Vacunas Atenuadas , Animales , Modelos Animales de Enfermedad , Perros , Células Asesinas Naturales/inmunología , Células de Riñón Canino Madin Darby , Ratones , Células RAW 264.7 , Linfocitos T/inmunologíaRESUMEN
A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.-Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Virus de la Influenza A/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Multimerización de Proteína , ARN de Transferencia/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunización , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Mutación , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/inmunología , ConejosRESUMEN
Cold-adapted live attenuated influenza vaccines (CAIVs) have been considered as a safe prophylactic measure to prevent influenza virus infections. The safety of a CAIV depends largely on genetic markers that confer specific attenuation phenotypes. Previous studies with other CAIVs reported that polymerase genes were primarily responsible for the attenuation. Here, we analyzed the genetic mutations and their phenotypic contribution in the X-31 ca strain, a recently developed alternative CAIV donor strain. During the cold-adaptation of its parental X-31 virus, various numbers of sequence changes were accumulated in all six internal genes. Phenotypic analysis with single-gene and multiple-gene reassortant viruses suggests that NP gene makes the largest contribution to the cold-adapted (ca) and temperature-sensitive (ts) characters, while the remaining other internal genes also impart attenuation characters with varying degrees. A balanced contribution of all internal genes to the attenuation suggests that X-31 ca could serve as an ideal master donor strain for CAIVs preventing influenza epidemics and pandemics.
Asunto(s)
Frío , Virus de la Influenza A/genética , Vacunas contra la Influenza , Virus Reordenados/genética , Adaptación Biológica/genética , Sustitución de Aminoácidos , Animales , Perros , Femenino , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Fenotipo , Alineación de Secuencia , Vacunas Atenuadas , Ensayo de Placa ViralRESUMEN
In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Gripe Humana/virología , Mutación Missense , Vacunas Virales/inmunología , Adaptación Fisiológica , Animales , Frío , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/efectos adversos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/efectos adversos , Vacunas Virales/genéticaRESUMEN
The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.
Asunto(s)
Adyuvantes Inmunológicos , Inmunidad Mucosa , Inmunidad , Vacunas contra la Influenza/inmunología , Soluciones Oftálmicas/administración & dosificación , Poli I-C , Vacunas de Productos Inactivados , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Antígenos Virales/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Ratones , Soluciones Oftálmicas/efectos adversos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
UNLABELLED: Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes. IMPORTANCE: Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of influenza virus hemagglutinin and the ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for the prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks, including pandemics.
Asunto(s)
Adenoviridae/genética , Anticuerpos Antivirales/sangre , Portadores de Fármacos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/genética , Proteínas de la Matriz Viral/inmunología , Administración Intranasal , Animales , Protección Cruzada , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas de la Matriz Viral/genéticaRESUMEN
Influenza virus continues to take a heavy toll on human health and vaccination remains the mainstay of efforts to reduce the clinical impact imposed by viral infections. Proven successful for establishing live attenuated vaccine donor strains, cold-adapted live attenuated influenza vaccines (CAIVs) have become an attractive modality for controlling the virus infection. Previously, we developed the cold-adapted strains A/X-31 and B/Lee/40 as novel donor strains of CAIVs against influenza A and B viruses. In this study, we investigated the protective immune responses of both mono- and trivalent vaccine formulations in the mouse model. Two type A vaccines and one type B vaccine against A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shangdong/7/97 in the background of the A/X-31 ca or B/Lee/40 ca were generated by a reassortment procedure and evaluated for their immunogenicity and protective efficacy. Each monovalent vaccine elicited high levels of serum antibodies and conferred complete protection against homologous wild type virus infection. As compared to the monovalent vaccines, trivalent formulation induced higher levels of type A-specific serum antibodies and slightly lower levels of type B-specific antibodies, suggesting an immunological synergism within type A viruses and an interference in the replication of type B virus. Relatively lower type B-specific immunogenicity in trivalent vaccine formulation could be effectively implemented by increasing the vaccine dose of influenza B virus. These results of immunogenicity, protection efficacy, and immunological synergism between type A vaccines provide an experimental basis for optimal composition of trivalent vaccines for subsequent developments of multivalent CAIVs against seasonal and pandemic influenza viruses.
Asunto(s)
Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Femenino , Pruebas de Inhibición de Hemaglutinación , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Vacunas contra la Influenza/clasificación , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Virus Reordenados , Vacunas Atenuadas/clasificación , Vacunas Atenuadas/inmunologíaRESUMEN
Live attenuated vaccine (LAV), mimicking natural infection, provides an excellent protection against microbial infection. The development of LAV, however, still remains highly empirical and the rational design of clinically useful LAV is scarcely available. Apoptosis and caspase activation are general host antiviral responses in virus-infected cells. Utilizing these tightly regulated host defense mechanisms, we present a novel apoptosis-triggered attenuation of viral virulence as a rational design of live attenuated vaccine with desired levels of safety, efficacy, and productivity. Mutant influenza viruses carrying caspase recognition motifs in viral NP and the interferon-antagonist NS1 proteins were highly attenuated both in vitro and in vivo by caspase-mediated cleavage of those proteins in infected cells. Both viral replication and interferon-resistance were substantially reduced, resulting in a marked attenuation of virulence of the virus. Despite pronounced attenuation, the viruses demonstrated high growth phenotype in embryonated eggs at lower temperature, ensuring its productivity. A single dose vaccination with the mutant virus elicited high levels of systemic and mucosal antibody responses and provided complete protection against both homologous and heterologous lethal challenges in mouse model. While providing a practical means to generate seasonal or pandemic influenza live vaccines, the sensitization of viral proteins to pathogen-triggered apoptotic signals presents a potentially universal, mechanism-based rational design of live vaccines against many viral infections.
Asunto(s)
Virus de la Influenza A/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caspasas/metabolismo , Línea Celular , Perros , Femenino , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Ratones , Mutación , Motivos de Nucleótidos/genética , Óvulo/virología , Proteolisis , Vacunas Atenuadas/inmunología , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Despite global efforts to control influenza viruses, they have taken a heavy toll on human public health worldwide. Among particular threats is highly pathogenic avian H5N1 influenza virus (HPAI) due to not only its high mortality in humans but also possible human-to-human transmission either through reassortment with other human influenza viruses such as 2009 pandemic H1N1 influenza virus, or by genetic mutations. With the aim of developing effective vaccines against the H5N1 viruses, we generated two live attenuated H5N1 vaccine candidates against A/Indonesia/05/2005 (clade 2.1) and A/chicken/Korea/ES/2003 (clade 2.5) strains, in the genetic background of the cold-adapted donor strain of X-31. In mice, a single dose of immunization with each of the two vaccines was highly immunogenic inducing high titers of serum viral-neutralizing and hemagglutinin-inhibiting antibodies against the homologous H5N1 strain. Furthermore, significant levels of cross-clade antibody responses were induced by the vaccines, suggesting a broad-spectrum cross-reactivity against the heterologous H5N1 strains. The immunizations provided solid protections against heterologous lethal challenges with H5N2 virus, significantly reducing the morbidity and challenge virus replications in the respiratory tracts. The robustness of the antibody responses against both the homologous and heterologous strains, together with efficient protection against the lethal H5N2 challenge, strongly support the protection against wild type H5N1 infections. These results could serve as an experimental basis for the development of safe and effective H5N1 pre-pandemic vaccines while further addressing the biosecurity concerns associated with H5N1 HPAI.
Asunto(s)
Formación de Anticuerpos , Protección Cruzada , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/sangre , Pollos , Frío , Perros , Huevos/virología , Femenino , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas , Subtipo H5N1 del Virus de la Influenza A , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/inmunología , Vacunas Atenuadas/inmunología , Cultivo de VirusRESUMEN
The 2009 pandemic influenza H1N1 (pdmH1N1) is characterized by rapid transmission among humans and disproportionate infection to children and young adults. Although the pdmH1N1 demonstrated less lethality than initially expected and has now moved into its post-pandemic period, it remains highly possible that through antigenic shift or antigenic drift the pdmH1N1 might re-emerge in the future as a more virulent strain than before, underscoring the need for vaccination prior to an outbreak. Using X-31 ca as a backbone strain, we generated a live attenuated pdmH1N1 vaccine and evaluated its potential as a safe and effective vaccine using mouse and ferret models. Despite an acceptable level of attenuation phenotypes, single dose of immunization with the vaccine efficiently stimulated both systemic and mucosal antibody responses and provided complete protection against lethal challenge with wild type pdmH1N1 virus, even at the lowest immunization dose of 10(3)PFU. The promising results of safety, immunogenicity, and protective efficacy of the vaccine not only contribute to expanding the repertoire of live vaccines as a judicious choice for pandemic H1N1 preparedness, but also suggest the great potential of X-31 ca donor strain to serve as reliable platform for generating diverse live vaccine constructs against seasonal influenza viruses and other pandemic strains.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Adaptación Biológica , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Frío , Modelos Animales de Enfermedad , Femenino , Hurones , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Análisis de Supervivencia , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/aislamiento & purificaciónRESUMEN
BACKGROUND: Hemodialysis (HD) patients have multiple causes of immune dysfunction and poor immune response to influenza vaccination. We investigated the antibody response rate to a pandemic H1N1/2009 influenza vaccination and clinical parameters influencing the induction of antibody responses in HD patients. METHODS: A total of 114 HD patients were vaccinated with a monovalent adjuvanted H1N1 inactivated influenza vaccine. Titers of neutralizing antibodies were evaluated by hemagglutination inhibition (HI) assay at pre- and 4 weeks after vaccination. Seroconversion was defined as either a pre-vaccination HI titer < 1:10 and a post vaccination HI titer > 1:40 or a pre-vaccination HI titer ≥ 1:10 and a minimum four-fold rise in post-vaccination HI antibody titer. Seventeen out of 114 HD patients (14.9%) tested positive for antibodies against influenza A/H1N1/2009 before vaccination. The remaining 97 baseline sero-negative patients were included in the analysis. RESULTS: Only 30 (30.9%) HD patients had seroconversion 4 weeks after vaccination. The elderly patients, those over 65 years of age, showed significantly lower seroconversion rate compared to younger HD patients (20.5% vs. 39.6%, p = 0.042). Furthermore, patients with hemoglobin values less than 10 g/dL had a significantly lower seroconversion rate compared to those with higher hemoglobin values (20.0 vs. 38.6%, p = 0.049). By multivariate logistic regression analysis, only age ≥65 years (OR = 0.336, 95% confidence interval (CI) 0.116-0.971, p = 0.044) and hemoglobin levels <10 g/dL (OR = 0.315, 95% CI 0.106-0.932, p = 0.037) were independently associated with seroconversion after vaccination. CONCLUSIONS: Our data show that HD patients, especially who are elderly with low hemoglobin levels, are at increased risk for lower seroconversion rate after influenza A/H1N1 vaccination. Further studies are needed to improve the efficacy of vaccination in these high risk patients.
Asunto(s)
Anticuerpos Antivirales/sangre , Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/prevención & control , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/rehabilitación , Diálisis Renal/estadística & datos numéricos , Comorbilidad , Femenino , Humanos , Gripe Humana/epidemiología , Gripe Humana/inmunología , Fallo Renal Crónico/epidemiología , Masculino , Vacunación Masiva/estadística & datos numéricos , Persona de Mediana Edad , Prevalencia , República de Corea/epidemiología , Factores de Riesgo , Resultado del TratamientoRESUMEN
The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.
Asunto(s)
Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Proteínas no Estructurales Virales/inmunología , Administración Sublingual , Animales , Anticuerpos Antivirales/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Mucosa , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.
Asunto(s)
Reacciones Cruzadas , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Adaptación Biológica , Animales , Frío , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Pandemias , República de Corea/epidemiología , Estaciones del Año , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Flagelina/administración & dosificación , Inmunidad Mucosa , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Mucosa Respiratoria/inmunología , Análisis de Supervivencia , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
BACKGROUND: The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. METHODS AND RESULTS: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. CONCLUSIONS: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.
Asunto(s)
Inmunidad/inmunología , Inmunización , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Proteínas de la Matriz Viral/inmunología , Administración Intranasal , Administración Sublingual , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Protección Cruzada/inmunología , Femenino , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Plásmidos/genética , Proteínas Recombinantes/inmunología , Solubilidad , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/aislamiento & purificaciónRESUMEN
Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1â»/â») mice develop significant defects in the infiltration of Ly6C(hi) monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C(hi) monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C(int) monocytes of Ifnar1â»/â» mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1â»/â» mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C(hi) monocytes. By using BM chimeric mice (WT BM into Ifnar1â»/â» and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C(hi) monocytes. Of note, WT BM reconstituted Ifnar1â»/â» chimeric mice with increased numbers of Ly6C(hi) monocytes survived longer than influenza-infected Ifnar1â»/â» mice. In contrast, WT mice that received Ifnar1â»/â» BM cells with alternative Ly6C(int) monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.
Asunto(s)
Monocitos/metabolismo , Monocitos/patología , Neutrófilos/metabolismo , Neutrófilos/patología , Neumonía Viral/patología , Receptor de Interferón alfa y beta/fisiología , Animales , Biomarcadores/metabolismo , Western Blotting , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Femenino , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Neumonía/etiología , Neumonía/patología , Neumonía Viral/inmunología , Neumonía Viral/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Recent studies have revealed that innate immunity is involved in the development of adaptive immune responses; however, its role in protection is not clear. In order to elucidate the exact role of Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling on immunogenicity and protective efficacy against influenza A virus infection (A/PR/8/34 [PR8]; H1N1), we adapted several innate signal-deficient mice (e.g., TRIF(-/-), MyD88(-/-), MyD88(-/-) TRIF(-/-), TLR3(-/-) TLR7(-/-), and IPS-1(-/-)). In this study, we found that MyD88 signaling was required for recruitment of CD11b(+) granulocytes, production of early inflammatory cytokines, optimal proliferation of CD4 T cells, and production of Th1 cytokines by T cells. However, PR8 virus-specific IgG and IgA antibody levels in both systemic and mucosal compartments were normal in TLR- and RLR-deficient mice. To further assess the susceptibility of these mice to influenza virus infection, protective efficacy was determined after primary or secondary lethal challenge. We found that MyD88(-/-) and MyD88(-/-) TRIF(-/-) mice were more susceptible to primary influenza virus infection than the B6 mice but were fully protected against homologous (H1N1) and heterosubtypic (H5N2) secondary infection when primed with a nonlethal dose of PR8 virus. Taken together, these results show that MyD88 signaling plays an important role for resisting primary influenza virus infection but is dispensable for protection against a secondary lethal challenge.