RESUMEN
BACKGROUND: Plasmodium falciparum Apical Membrane Antigen 1 Diversity Covering (PfAMA1-DiCo) candidate vaccine is a formulation of three recombinant variants of AMA1 designed to provide broader protection against parasites with varying AMA1 sequences. METHODS: In this staggered phase Ia/Ib randomized, double blind trial, healthy French adults received AMA1-DiCo with either Alhydrogel® (n=15) or GLA-SE (n=15). Following a safety assessment in French volunteers, GLA-SE was chosen for the phase Ib trial where healthy Burkinabe adults received either AMA1-DiCo/GLA-SE (n=18) or placebo (n=18). AMA1-DiCo (50µg) was administered intramuscularly at baseline, Week 4 and 26. RESULTS: AMAI-DiCo was safe, well tolerated either with Alhydrogel® or GLA-SE. In European volunteers, the ratios of IgG increase from baseline were about 100 fold in Alhydrogel® group and 200-300 fold in GLA-SE group for the three antigens. In African volunteers, immunization resulted in IgG levels exceeding those observed for the European volunteers with a 4-fold increase. DiCo-specific IgG remained higher 26weeks after the third immunization than at baseline in both European and African volunteers. Induced antibodies were reactive against whole parasite derived from different strains. CONCLUSION: AMA1-DiCo vaccine was safe and immunogenic whatever the adjuvant although GLA-SE appeared more potent than Alhydrogel® at inducing IgG responses. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov NCT02014727; PACTR201402000719423.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Antígenos de Protozoos/inmunología , Inmunogenicidad Vacunal/inmunología , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adulto , África , Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos/inmunología , Método Doble Ciego , Europa (Continente) , Femenino , Humanos , Inmunización/efectos adversos , Inmunoglobulina G/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Masculino , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.
Asunto(s)
Interleucina-5/genética , Sigmodontinae/genética , Secuencia de Aminoácidos , Animales , Western Blotting , División Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Vectores Genéticos/genética , Interleucina-5/metabolismo , Interleucina-5/farmacología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.
Asunto(s)
Interferón gamma/genética , Sigmodontinae/genética , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Células Cultivadas , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Escherichia coli/genética , Expresión Génica , Interferón gamma/aislamiento & purificación , Interferón gamma/farmacología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrolloRESUMEN
The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.
Asunto(s)
Virus de la Parotiditis/efectos de los fármacos , Proteínas de la Nucleocápside/genética , ARN Catalítico/farmacología , ARN Mensajero/genética , Animales , Secuencia de Bases , Chlorocebus aethiops , Cartilla de ADN , Virus de la Parotiditis/genética , ARN Mensajero/metabolismo , Células VeroRESUMEN
Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.
Asunto(s)
Anticuerpos Antivirales/sangre , Hemaglutininas Virales/inmunología , Inmunoglobulina M/sangre , Virus del Sarampión/inmunología , Sarampión/diagnóstico , Adolescente , Adulto , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactante , Proteínas Recombinantes/inmunologíaRESUMEN
Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage.
Asunto(s)
ADN Nucleotidilexotransferasa/química , Reacción en Cadena de la Polimerasa/métodos , ARN Catalítico/metabolismo , Secuencia de Bases , Cartilla de ADN , ADN Complementario , HidrólisisRESUMEN
Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as "gold standards." In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.
Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Hemaglutininas Virales/inmunología , Virus del Sarampión/inmunología , Sarampión/inmunología , Adolescente , Adulto , Animales , Línea Celular , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Hemaglutininas Virales/genética , Humanos , Inmunoglobulina G/sangre , Lactante , Masculino , Sarampión/diagnóstico , Valor Predictivo de las Pruebas , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , TransfecciónRESUMEN
In an attempt to develop efficient antiviral agents against Mumps virus, we designed ribozymes targeting the nucleocapsid (NP) mRNA. Transacting catalytic RNAs of the hammerhead and hairpin types were synthesized; they contained specific motifs, shared similar flanking regions and were directed against a 5'GUC3' target immediately downstream to the initiation codon of NP mRNA. Both ribozymes were first assayed on a synthetic 16 bases target RNA and found to catalytically and efficiently cleave the substrate in a sequence specific way. No cleavage, however, occurred when mutated forms of the ribozymes were used. In addition, both ribozyme types, when tested on the full length NP mRNA, were also able to cleave the substrate although turnover could not be demonstrated. As a rule, the hammerhead ribozyme proved more efficient than its hairpin counterpart, as well on the synthetic RNA substrate as on the full length NP mRNA target.
Asunto(s)
Virus de la Parotiditis/metabolismo , Nucleocápside/genética , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Sitios de Unión , Conformación de Ácido Nucleico , ARN/síntesis química , ARN/metabolismo , ARN Catalítico/química , ARN Mensajero , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of mumps virus (MuV) have been produced in CV1 cells via vaccinia virus recombinants. Recombinant proteins accumulated in infected cells and were glycosylated. Upon reduction, the F protein product was completely converted into its subunits. Hamsters infected with vaccinia recombinants expressing either the F or HN proteins produced antibodies recognizing MuV antigens and neutralizing MuV infectivity in vitro. These antibodies provided protection against MuV-induced encephalitis in newborn hamsters.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteína HN/inmunología , Inmunización Pasiva , Virus de la Parotiditis/inmunología , Paperas/prevención & control , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Antivirales/análisis , Cricetinae , Proteínas Recombinantes/inmunología , Virus Vaccinia/genéticaRESUMEN
Chinese hamster ovary cells have been engineered to secrete an anchorless form of the varicella-zoster virus gpII protein. Purification of the recombinant product was achieved by a combination of hydrophobic and gel filtration chromatography giving rise to a protein more than 85% pure. Recombinant gpII was composed of several polypeptides which, on the basis of amino-terminal sequence analysis, corresponded to a 93-kDa precursor and to the N- and C-terminal subunits of the molecule (64 and 39/36 kDa, respectively). All polypeptides carried N-linked high-mannose and complex glycosylations, whereas O-glycosylations were carried by the precursor and the N-terminal subunits only. Surprisingly, purified recombinant gpII spontaneously formed large oligomeric structures of variable size. These complexes contained noncovalently linked lipids. Mice inoculated with the recombinant gpII absorbed onto the weak adjuvant, aluminium hydroxide, produced virus neutralizing antibodies. The recombinant gpII may thus constitute a good candidate for the development of a subunit vaccine against varicella-zoster virus.
Asunto(s)
Herpesvirus Humano 3/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Células CHO , Carbohidratos/análisis , Cromatografía en Gel , Clonación Molecular , Cricetinae , Vectores Genéticos/genética , Lípidos/análisis , Ratones , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Transfección , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
Oligonucleotide primers have been used to amplify DNA regions of the M. leprae genome by the polymerase chain reaction. A first set of primers, PLp1 and PLp2, identifies a specific 386 bp DNA fragment located in the gene coding for the 65 kDa antigen of M. leprae. A second pair of primers, targetted to the same gene, leads to the amplification of a 154 bp DNA piece conserved in mycobacteria. Primers PLp1 and PLp2 discriminate the pathogenic species from other mycobacteria, detect down to 40 bacilli, and constitute potentially useful tools for the identification of M. leprae in clinical specimens.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Amplificación de Genes , Lepra/diagnóstico , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Animales , Antígenos Bacterianos/genética , Secuencia de Bases , ADN Bacteriano/genética , Humanos , Lepra/microbiología , Datos de Secuencia Molecular , Mycobacterium/genética , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Valor Predictivo de las Pruebas , Especificidad de la EspecieRESUMEN
Oligonucleotide primers have been used to amplify DNA regions of the M. leprae genome by the polymerase chain reaction. A first set of primers, PLp1 and PLp2, identifies a specific 386 bp DNA fragment located in the gene coding for the 65 kDa antigen of M. leprae. A second pair of primers, targetted to the same gene, leads to the amplification of a 154 bp DNA piece conserved in mycobacteria. Primers PLp1 and PLp2 discriminate the pathogenic species from other mycobacteria, detect down to 40 bacilli, and constitute potentially useful tools for the identification of M. leprae in clinical specimens.
Asunto(s)
Humanos , Animales , Amplificación de Genes , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Lepra/diagnóstico , Lepra/microbiología , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Mycobacterium leprae/aislamiento & purificación , Mycobacterium/genéticaRESUMEN
Oligonucleotide primers were used to amplify specific DNA regions of the Bordetella pertussis genome by the polymerase chain reaction. One pair of primers, PTp1/PTp2, identified a 191-bp DNA fragment located in the regulatory region of the pertussis toxin operon; a second pair of primers led to amplification of a 121-bp DNA piece located in an insertion-like element specific to B. pertussis. Both sets of primers were able to discriminate between the pathogen and related Bordetella species; they detected down to 6 bacteria and appeared suitable for routine detection of B. pertussis in clinical specimens.
Asunto(s)
Bordetella pertussis/aislamiento & purificación , Amplificación de Genes , Biblioteca Genómica , Reacción en Cadena de la Polimerasa , Tos Ferina/microbiología , ADN , Humanos , Oligonucleótidos , Tos Ferina/diagnósticoRESUMEN
Specific oligodeoxynucleotide probes ranging from 20 to 35 nucleotides were defined to differentiate each of the HPV1a, 5, 6b, 8, 11, 16, 18 and 33. They were chosen using computer programs developed to compare simultaneously several 8000 bp long DNA sequences. Sequences common to all and to specific groups of the HPV DNA were also selected. Specificity of 32P-labelled probes for HPV6b, 11, 16, 18 and 33 was demonstrated and the sensitivity of the assays was evaluated by filter hybridization with viral clones and with DNA from cervical tumor biopsies.
Asunto(s)
Sondas de ADN de HPV/análisis , Sondas de ADN/análisis , ADN Viral/análisis , Papillomaviridae/genética , Secuencia de Bases , Femenino , Humanos , Hibridación de Ácido Nucleico , Neoplasias del Cuello Uterino/análisis , Neoplasias del Cuello Uterino/microbiologíaRESUMEN
The boundaries of gene conversion in variant-specific antigen genes have been determined in six clones of Trypanosoma brucei. In each clone, antigenic switching involved interaction between two telomeric members of the AnTat 1.1 multigene family, which share extensive homology throughout their coding regions. All conversion events occurred by substitution of faithful copies of donor sequences. Conversion endpoints were nonrandomly distributed. In four clones, the 5' conversion limit was near the antigen translation initiation codon, while in three clones, the 3' conversion limit was located at the "hinge" between the two major antigen domains. In one case, two segmental conversions were involved in antigen switching. These observations reveal that antigen gene conversion can occur without generating point mutations, and suggest that postrecombinational selection may impose a limit on the number of possible rearrangements within antigen genes.