RESUMEN
In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process-from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.
Asunto(s)
Descubrimiento de Drogas/métodos , Virus de la Influenza A/patogenicidad , Vacunas contra la Influenza/aislamiento & purificación , Gripe Aviar/virología , Gripe Humana/prevención & control , Pandemias/prevención & control , Zoonosis/prevención & control , Animales , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Vacunas contra la Influenza/inmunología , Gripe Aviar/transmisión , Gripe Humana/epidemiología , Gripe Humana/inmunología , Gripe Humana/virología , Aves de Corral , Tecnología Farmacéutica/métodos , Zoonosis/epidemiología , Zoonosis/inmunología , Zoonosis/virologíaRESUMEN
Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.
Asunto(s)
Antígenos Nucleares , Núcleo Celular/enzimología , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP , Interferones/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteína SUMO-1/metabolismo , Factores de Transcripción/metabolismo , Virus/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/metabolismo , Animales , Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Compartimento Celular/efectos de los fármacos , Compartimento Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/virología , Proteínas Co-Represoras , ADN Helicasas/metabolismo , GTP Fosfohidrolasas/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Células HeLa/citología , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Humanos , Inmunohistoquímica , Interferones/farmacología , Ratones , Chaperonas Moleculares , Proteínas de Resistencia a Mixovirus , Proteína de la Leucemia Promielocítica , Proteínas/efectos de los fármacos , RecQ Helicasas , Transfección , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos HíbridosRESUMEN
Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.
Asunto(s)
ADN Complementario/genética , Thogotovirus/genética , Thogotovirus/aislamiento & purificación , Vacunas Virales/genética , Animales , Línea Celular , ADN Complementario/aislamiento & purificación , Humanos , Plásmidos , Recombinación GenéticaRESUMEN
The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.
Asunto(s)
Ingeniería Genética , Virus ARN/genética , Vacunas Sintéticas , Vacunas Virales , Virosis/virología , Animales , Quimera , Diseño de Fármacos , Genoma Viral , Humanos , Vacunas contra la Influenza , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Virus ARN/patogenicidad , Virulencia , Virosis/inmunología , Virosis/prevención & controlRESUMEN
PURPOSE: Evaluation of a growth model for the biometric parameters of early pregnancy: Chorionic cavity, yolk sac, amniotic cavity, crown-rump length and biparietal diameter. METHOD: 618 clinically well-dated singleton pregnancies of the first trimester were measured in a prospective cross-sectional study with high-frequency transvaginal ultrasound between 28 and 94 days post menstruation. RESULTS: Normal curves (5th, 50th, 95th percentiles) for all parameters were established using our growth model. The growth curves of chorionic cavity amniotic cavity, and biparietal diameter showed a linear growth whereas the growth curve of the crown-rump-length represented an exponential curve. Linear growth of the yolk sac was found until 8 weeks of gestation and there was no growth alteration after this time. CONCLUSION: With our growth curves of the first trimester it is possible to monitor the embryonic development with several parameters.
Asunto(s)
Antropometría , Desarrollo Embrionario y Fetal/fisiología , Endosonografía , Ultrasonografía Prenatal , Cefalometría , Largo Cráneo-Cadera , Femenino , Edad Gestacional , Humanos , Recién Nacido , Embarazo , Valores de ReferenciaRESUMEN
A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.
Asunto(s)
Virus de la Influenza A/genética , Plásmidos , ARN Viral/genética , Ribonucleoproteínas/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , ARN Viral/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.
Asunto(s)
ADN Recombinante/genética , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Animales , Línea Celular , Técnicas Genéticas , Humanos , Plásmidos/genética , ARN Viral/metabolismo , Transcripción Genética , Transfección , Proteínas Virales/metabolismoRESUMEN
Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.
Asunto(s)
Vacunas Bacterianas , Epítopos/inmunología , Virus de la Influenza A/inmunología , Enfermedades Pulmonares/microbiología , Porinas/inmunología , Infecciones por Pseudomonas/inmunología , Vacunas Sintéticas , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Enfermedad Crónica , Perros , Epítopos/química , Humanos , Vacunas contra la Influenza , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/prevención & control , Ratones , Porinas/química , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Virus de la Influenza A/inmunología , Linfocitos/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Quimera , Secuencia Conservada , Ensayo de Inmunoadsorción Enzimática , Femenino , Anticuerpos Anti-VIH/biosíntesis , VIH-1/genética , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Virus de la Influenza A/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Pulmón/inmunología , Pulmón/virología , Linfocitos/virología , Ratones , Datos de Secuencia Molecular , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Pruebas de Neutralización , Bazo/inmunología , Bazo/virología , Vagina/inmunología , Vagina/virologíaRESUMEN
The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F of Pseudomonas aeruginosa to serve as a protective vaccine against P. aeruginosa was tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains of P. aeruginosa but failed to react with a protein F-deficient strain of P. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity for P. aeruginosa than did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells of P. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 x 10(3) CFU of P. aeruginosa in their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused by P. aeruginosa.