RESUMEN
To clarify the level of excretion of a recombinant Marek's disease virus type 1 (rMDV1) that confers good protection in chickens against both Marek's and Newcastle diseases, even in the presence of maternal antibodies, contact transmission tests were conducted. Naïve chickens kept in the same cage or room with chickens inoculated with rMDV1 did not produce antibodies against MDV1 or the fusion protein of Newcastle disease virus. Moreover, the rMDV1 was not isolated from the dander of chickens inoculated with rMDV1. Even under the stressful conditions of forced molting and a high temperature environment, rMDV1 was not isolated from the dander of inoculated birds. The viral DNA, however, was detected from the dander of chickens inoculated with rMDV1 as well as a commercial vaccine. These findings indicate that dander from chickens inoculated with rMDV1 includes viral DNA, but does not contain infectious virus.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Virus de la Enfermedad de Newcastle/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Embrión de Pollo , Pollos , ADN Viral/análisis , Heces/virología , Vectores Genéticos , Herpesvirus Gallináceo 2/inmunología , Herpesvirus Gallináceo 2/aislamiento & purificación , Esparcimiento de VirusRESUMEN
A full length cDNA of feline interleukin(IL)-12 p35 and p40 subunits was cloned. By transferring the plasmids containing both the subunit genes to mammalian cells, we expressed biologically active feline IL-12. The expressed feline IL-12 has interferon-gamma-inducing activity against both human and feline peripheral blood mononuclear cells (PBMC) and stimulates cytotoxic T lymphocyte activity against herpes simplex virus-infected human PBMCs. There were two kinds of molecules (p75, p80) in the purified recombinant feline IL-12, and both molecules exhibited biological activity. The difference between p75 and p80 was the degree of the glycosylation of the p35 chain. Moreover, when we modified the cDNA of p35 by changing some codons and deleted the 5' and 3' non-coding regions, the expression level of IL-12 increased about 100 fold.
Asunto(s)
Gatos/genética , Interleucina-12/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Clonación Molecular , Codón/farmacología , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida/veterinaria , Glicosilación , Herpesvirus Humano 1/inmunología , Humanos , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Interleucina-12/química , Interleucina-12/farmacología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Datos de Secuencia Molecular , Plásmidos , Conformación Proteica , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.
Asunto(s)
Virus del Moquillo Canino/inmunología , Moquillo/inmunología , Hemaglutininas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/clasificación , Antígenos Virales/inmunología , Chlorocebus aethiops , Efecto Citopatogénico Viral , Moquillo/virología , Virus del Moquillo Canino/clasificación , Perros , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Hemaglutininas Virales/clasificación , Japón , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización/veterinaria , Ensayo de Radioinmunoprecipitación/veterinaria , Células Vero , Vacunas Virales/inmunologíaRESUMEN
An earlier report (M. Sakaguchi et al., Vaccine 16:472-479, 1998) showed that recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion (F) protein of Newcastle disease virus (NDV-F) under the control of the simian virus 40 late promoter [rMDV1-US10L(F)] protected specific pathogen-free chickens from NDV challenge, but not commercial chickens with maternal antibodies against NDV and MDV1. In the present study, we constructed an improved polyvalent vaccine based on MDV1 against MDV and NDV in commercial chickens with maternal antibodies. The study can be summarized as follows. (i) We constructed rMDV1 expressing NDV-F under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10P(F)]. (ii) Much less NDV-F protein was expressed in cells infected with rMDV1-US10P(F) than in those infected with rMDV1-US10L(F). (iii) The antibody response against NDV-F and MDV1 antigens of commercial chickens vaccinated with rMDV1-US10P(F) was much stronger and faster than with rMDV1-US10L(F), and a high level of antibody against NDV-F persisted for over 80 weeks postvaccination. (iv) rMDV1-US10P(F) was readily reisolated from the vaccinated chickens, and the recovered viruses were found to express NDV-F. (v) Vaccination of commercial chickens having maternal antibodies to rMDV1-US10P(F) completely protected them from NDV challenge. (vi) rMDV1-US10P(F) offered the same degree of protection against very virulent MDV1 as the parental MDV1 and commercial vaccines. These results indicate that rMDV1-US10P(F) is an effective and stable polyvalent vaccine against both Marek's and Newcastle diseases even in the presence of maternal antibodies.
Asunto(s)
Anticuerpos Antivirales/inmunología , Herpesvirus Gallináceo 2/inmunología , Enfermedad de Marek/prevención & control , Enfermedad de Newcastle/prevención & control , Vacunas Virales/genética , Animales , Secuencia de Bases , Pollos , Herpesvirus Gallináceo 2/genética , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/aislamiento & purificación , ARN Mensajero/genética , Recombinación Genética , Virus 40 de los Simios/genética , Tráquea/virología , Proteínas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.
Asunto(s)
Anticuerpos Antivirales/sangre , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/inmunología , Respirovirus/genética , Secuencia de Bases , Western Blotting , Clonación Molecular , Cartilla de ADN , Citometría de Flujo , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , Humanos , Técnicas para Inmunoenzimas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
We investigated two forms (designated as yGI and yGII) of rabies virus glycoprotein (G) analogues produced in the G cDNA-transfected yeast cells. Molecular weights of yGI and yGII were estimated as 66 and 56 kDa, respectively, according to their relative mobility in SDS-PAGE. Although being produced in large amounts, yGI was present mostly in insoluble forms and hardly extractable with non-ionic detergents. The yGI reacted with polyclonal anti-G antibodies, but did not react with our conformational epitope-specific anti-G monoclonal antibodies (G-MAbs). No protective immunity was induced by yGI in guinea pigs nor in mice. On the other hand, yGII was Triton-soluble, but was only small in amount (at most 1% of total G proteins) and was shown to lack the cytoplasmic domain. The yGII, however, reacted with the G-MAbs and induced protective immunity in guinea pigs as well. When the G-cDNA was expressed in animal cells in culture, a single form (about 66 kDa) of G protein was produced, which displayed similar behaviors as seen in its reactivity with the MAbs and intracellular distribution as seen in the virus-infected cells. These results suggest that most G protein molecules were not processed normally in yeast cells, resulting in abnormal folding and multimer formation, while only a small fraction were occasionally folded normally to have conformational epitopes but were mostly deprived of the C-terminal portion.
Asunto(s)
Antígenos Virales/biosíntesis , Glicoproteínas/inmunología , Virus de la Rabia/inmunología , Saccharomyces cerevisiae/metabolismo , Animales , Detergentes , Evaluación Preclínica de Medicamentos , Regulación Viral de la Expresión Génica/fisiología , Genes Virales , Vectores Genéticos , Glicosilación , Cobayas , Immunoblotting , Ratones , Octoxinol , Solubilidad , Relación Estructura-ActividadRESUMEN
PURPOSE: To prepare poly(lactide-co-glycolide)(PLGA) microspheres containing recombinant hepatitis B core antigen (HBcAg; Mw = 3,600,000) by a w/o/w emulsion/solvent evaporation method and evaluate the possibility of this system as a potent long-acting carrier for hepatitis B core antigen in mice. METHODS: Various additives had been incorporated in the internal aqueous phase during the process of microencapsulating HBcAg, HBcAg antigenicity in the medium extracted from the prepared microspheres were measured by ELISA. Shape confirmation of the HBcAg antigen was performed by a sucrose gradient velocity centrifugal technique. For in vivo study, prepared microspheres were administered subcutaneously to Balb/C mice, and the serum IgG level was determined by ELISA. RESULTS: The inactivation of HBcAg by methylene chloride was dramatically reduced by the addition of gelatin (4-8% (w/v)) to the internal aqueous phase during the preparation. Further improvement of the loading efficiency to almost 61% resulted with cooling (4 degrees C). The prepared microspheres (4.27 microm+/-1.23 microm) containing 0.15% HBcAg displayed burst release (50-60% within 2 days). In subcutaneous inoculation, the adjuvant effect of PLGA microspheres was almost the same as that of the complete Freund's adjuvant. Whereas oral inoculation using the microspheres was not effective. CONCLUSIONS: The pH of the added gelatin seemed to be the key to the stabilization of HBcAg from various stability tests and CD spectrum study. Finally, the possibility of using this system as a potent long-acting hepatitis B vaccine was demonstrated.
Asunto(s)
Composición de Medicamentos , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/administración & dosificación , Animales , Estabilidad de Medicamentos , Femenino , Ratones , Ratones Endogámicos BALB C , Microesferas , Peso MolecularRESUMEN
The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type-1 from a father-to-mother-to-infant trimmer were studied and the horizontal and vertical transmissions compared. The father's virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence. In contrast, the mother's viral sequences were much less diverse and reacted with a virus neutralizing antibody. The infant's viral sequences were also less diverse than those of the father, and N-glycosylation sites were conserved. By phylogenetic analysis, the major clone, of which V3-peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody. These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/transmisión , VIH-1/genética , Fragmentos de Péptidos/genética , Secuencia de Aminoácidos , Southern Blotting , ADN Viral/análisis , Transmisión de Enfermedad Infecciosa , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Escherichia coli/genética , Femenino , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Immunoblotting , Lactante , Transmisión Vertical de Enfermedad Infecciosa , Leucocitos Mononucleares/inmunología , Masculino , Datos de Secuencia Molecular , Mutación , Pruebas de Neutralización , Filogenia , Reacción en Cadena de la Polimerasa , Embarazo , Recombinación Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , beta-Galactosidasa/inmunologíaRESUMEN
The Nishigahara strain of rabies virus, a current seed strain used for animal vaccine production in Japan, is believed to derived from the original Pasteur strain obtained from Paris in or before 1915. In Japan, the virus was serially passaged through several kinds of animals and cell cultures. Reactions with anti-nucleocapsid protein monoclonal antibodies (MAb-N) indicated the Nishigahara strain had maintained the antigenic profile of the Pasteur virus. Reactions with monoclonal antibodies to the glycoprotein (MAb-G) revealed differences between the Nishigahara strain and the Pasteur strain; however, the Nishigahara strain maintained a closer resemblance to the Pasteur virus than to other Pasteur-related viruses or to rabies strains unrelated to the Pasteur strain. Comparative amino acid sequence analysis of cloned cDNA encoding the G gene confirmed the antigenic differences among these strains and the resemblance of the Nishigahara strain to the original Pasteur strain. Comparative nucleotide sequence analysis of the noncoding pseudogene region (Tordo et al., Proc Natl Acad Sci USA 83, 3914-3918, 1986) revealed different relationships. Unlike the Pasteur strain, which encodes a transcription-terminating signal at the end of the G gene (marking the beginning of the pseudogene), a long G-L intergenic sequence in the Nishigahara strain was connected to the 3' end of the cDNA, and the transcription-terminating signal was present only at the end of, but not before, the pseudogene. These results are not inconsistent with the documented origin of the Nishigahara strain, but the genome structure around the pseudogene region suggests divergence from the Pasteur strain and a closer resemblance to other strains of rabies virus.
Asunto(s)
Vacunas Antirrábicas/aislamiento & purificación , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos Virales/genética , Secuencia de Bases , ADN Complementario/genética , ADN Viral/genética , Enfermedades de los Perros/prevención & control , Perros , Genes Virales , Glicoproteínas/genética , Glicoproteínas/inmunología , Japón , Datos de Secuencia Molecular , Pruebas de Neutralización , Rabia/prevención & control , Rabia/veterinaria , Virus de la Rabia/clasificación , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Cultivo de VirusRESUMEN
A chimeric mouse-human antibody (C beta 1) was constructed that recognized the principal neutralizing domain (PND) of human immunodeficiency virus type 1 (HIV-1) gp120. The constant (C) immunoglobulin regions (C gamma 1 and C kappa) of a mouse monoclonal antibody, 0.5 beta, were substituted for the human C gamma 1 and C kappa by recombining the DNA modules encoding variable or C regions. The DNA constructs were then transfected into X63 Ag8.653 myeloma cells. A clone with a high production of the chimeric antibody (C beta 1) was selected. This antibody was tested for its biological activity against HIV-1. It bound to the surface of HTLV-IIIB-infected cells and reacted with gp120/160 with equal affinity and specificity to that of the parental 0.5 beta murine monoclonal antibody in a Western blot assay. Neutralization and/or enhancement of HIV infection were evaluated with C beta 1 and 0.5 beta. Both C beta 1 and 0.5 beta neutralized cell-to-cell infection and cell-free virus infection by HTLV-IIIB. Antibody-dependence enhancement of HIV infection was not observed with either C beta 1 or 0.5 beta in the presence or absence of human complement. Antibody-dependent cell-mediated cytolysis (ADCC) and antibody-dependent complement-mediated cytolysis (ACC) were observed with C beta 1 but not with the parental 0.5 beta. These findings suggest that the neutralizing antibodies to PND may neutralize but not enhance HIV infection. Furthermore, the high levels of ACC and ADCC shown against HIV-infected cells by C beta 1 indicate that the clinical application of such monoclonal antibodies may be possible.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , Proteínas del Sistema Complemento/inmunología , ADN Viral , Epítopos/inmunología , VIH-1/fisiología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Homología de Secuencia de Ácido Nucleico , Replicación ViralRESUMEN
The acquired immunodeficiency syndrome (AIDS) is the late-stage clinical manifestation of long-term persistent infection with the human immunodeficiency virus type 1 (HIV-1). Immune responses directed against the virus and against virus-infected cells during the persistent infection fail to mediate resolution of the infection. As a result, a successful AIDS vaccine must elicit an immune state that will prevent the establishment of the persistent infection following introduction of the virus into the host. The third hypervariable (V3) domain of the HIV-1 gp120 envelope glycoprotein is a disulphide-linked closed loop of about 30 amino acids which binds and elicits anti-HIV-1 type-specific virus-neutralizing antibodies. The in vitro characteristics of anti-V3 domain antibody suggest that this antibody could by itself prevent HIV-1 infection in vivo, an idea supported by chimpanzee challenge studies in which protection against the HIV-1 persistent infection seemed to correlate with the presence of anti-V3 domain antibody. Here we directly demonstrate the protective efficacy of anti-V3 domain antibody in vivo and propose that this antibody is potentially useful as both a pre- and post-exposure prophylactic agent.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Anti-VIH/uso terapéutico , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Animales , ADN Viral/sangre , Anticuerpos Anti-VIH/sangre , VIH-1/genética , VIH-1/aislamiento & purificación , Leucocitos Mononucleares/microbiología , Pan troglodytes , Reacción en Cadena de la PolimerasaRESUMEN
The principal neutralizing determinants (PNDs) of 29 human immunodeficiency virus type 1 (HIV-1) isolates in Japan were analyzed using polymerase chain reactions. The viruses were isolated from 16 hemophiliacs, 11 individuals infected by their sexual transmission and 1 patient infected by blood transfusion (total 28 patients). Two virus isolates which were obtained from the same individual at different periods were also analyzed. All individuals were Japanese except one. The results produced 32 different PND sequences. A highly conserved central core sequence (GPG) was present in 27 of 32 patients, similar to the number reported in the United States, despite the marked heterogeneity in flanking regions of PNDs. The PNDs of all the 16 HIV-1 isolates obtained from patients with coagulation disorders had GPG sequences. Secondary structure prediction of PNDs by a joint method suggested that they were composed of coil-beta strand-coil-beta strand-alpha helix. It is suggested that the conserved core sequence has a type I turn. These findings may be useful in planning further clinical trials for passive vaccination.
Asunto(s)
Antígenos VIH/genética , VIH-1/genética , VIH-1/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , ADN/genética , Epítopos/química , Epítopos/genética , Antígenos VIH/química , VIH-1/aislamiento & purificación , Humanos , Japón , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Pruebas de Neutralización , Conformación Proteica , Homología de Secuencia de Ácido NucleicoRESUMEN
Mouse monoclonal antibody (mAb) 0.5 beta binds to the envelope protein gp120 of human immunodeficiency virus (HIV) and neutralizes infection by HIV in vitro. Mouse mAb 0.5 beta, therefore, has potential as a therapeutic agent for the prevention and treatment of acquired immunodeficiency syndrome (AIDS). Since mouse mAbs are highly immunogenic in humans, efforts are being made to humanize mouse mAbs that are being considered for use in humans. This report describes the design, construction, and expression of reshaped human 0.5 beta antibodies. In these antibodies, the entire constant (C) regions were derived from human sequences. The variable (V) regions were derived from human framework regions (FRs) and mouse 0.5 beta complementarity determining regions (CDRs). One version of reshaped human 0.5 beta light (L) chain and six versions of reshaped human 0.5 beta heavy (H) chain were made and tested. Following transient expression in cos cells, all of the constructions were capable of producing humanlike antibody. Three of the H chain constructions (RHc, RHe, and RHf), when co-expressed with the L chain construction (RL), produced reshaped human antibody capable of binding to the epitope on gp120 recognized by mouse 0.5 beta mAb. The best version (RL + RHe) of reshaped human 0.5 beta antibody had both binding affinity and neutralizing activity that were within twofold that of the mouse or chimeric 0.5 beta antibody.