RESUMEN
A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.
Asunto(s)
Pichia/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Protozoos/farmacología , División Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pichia/química , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Conejos , Tasa de Supervivencia , Células TH1/inmunología , Toxoplasmosis/diagnóstico , Toxoplasmosis/tratamiento farmacológico , Toxoplasmosis/inmunología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/mortalidad , Toxoplasmosis Animal/parasitología , Transformación GenéticaRESUMEN
The ROP2 protein of Toxoplasma gondii possesses immunological and biological properties which have led to its proposal as a vaccine candidate. To identify epitopes recognized by human T cells in the ROP2 antigen, we submitted the sequence of this protein to three reported T-cell epitope prediction algorithms. Three sequences that were predicted by all three methods were selected (sequences 197 to 216, 393 to 410, and 501 to 524), and the corresponding peptides were synthesized. The peptides were first tested in a proliferation assay with a DPw4-restricted, ROP2-specific human T-cell clone, and the peptide corresponding to residues 197 to 216 was shown to stimulate the T-cell clone. The three peptides were further tested in proliferation assays with peripheral blood mononuclear cells from a panel of T. gondii-seropositive and -seronegative individuals. We found that cells from a high proportion of the seropositive donors (64%) recognized at least one of the three peptides. The most frequently recognized ones were peptides 197 to 216 (45%) and 501 to 524 (36%). None of the seronegative donors responded to any peptide. These results show that the ROP2 antigen of T. gondii contains T-cell epitopes recognized by a high percentage of the immune population and further strengthen its potential as a vaccine candidate.
Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Mapeo Epitopo , Humanos , Activación de Linfocitos , Datos de Secuencia MolecularRESUMEN
Two splice variants of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PACAP receptor and PACAP/HOP receptor isoform) were stably expressed in Chinese hamster ovary (CHO) cells that did not express constitutively receptors for this family of peptides. The PACAP/HOP receptor protein had a 28 amino acid extension in the C-terminal part of the third intracellular loop. The two cell lines studied, CHO 2-10 (PACAP receptor) and CHO 4-12 (PACAP/HOP receptor) expressed a receptor density of 4.6 +/- 0.3 and 2.6 +/- 0.2 pmol/mg protein, respectively, with corresponding Kd values of 14.2 +/- 2.0 and 8.2 +/- 1.0 nM for [Ac-His1]PACAP-27 used as a tracer. Tracer binding was slightly decreased by GTP in both clones. The Kd values of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP), PACAP-27 fragments and analogues evaluated by binding competition curves, were higher in CHO 2-10 than in CHO 4-12, whereas the Kd for PACAP-38 fragments did not differ. The receptors were coupled to adenylate cyclase and the EC50 values were lower than the Kd values in both cell lines, suggesting an amplification process due to the existence of spare receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenilil Ciclasas/metabolismo , Neuropéptidos/farmacología , Receptores de la Hormona Hipofisaria/metabolismo , Empalme Alternativo , Animales , Células CHO , Cricetinae , Cinética , Neurotransmisores/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/genética , Recombinación Genética , TransfecciónRESUMEN
The binding properties, coupling to adenylate cyclase, and desensitization of secretin receptors stably expressed in transfected Chinese hamster ovary (CHO) cells were compared in two clones expressing high (CHO-SnR-c5, 450 +/- 80 fmol/mg of protein) and low (CHO-SnR-c1, 40 +/- 25 fmol/mg of protein) receptor densities. The Kd values for receptor occupancy by secretin, selected analogues, and fragments were identical in CHO-SnR-c1 and -c5 cells and identical to those described for native receptors from NG 108-15 cells. The Kact values for adenylate cyclase stimulation were identical to the Kd values in CHO-SnR-c1 cells but 5-10-fold higher than those in CHO-SnR-c5 cells. The Kact values in both CHO-SnR-c1 and -c5 cell lines were reduced in the presence of the nonhydrolyzable GTP derivative guanosine-5'-(beta, gamma-imido)triphosphate and after pretreatment of the cells with cholera toxin. Preincubation of both CHO-SnR-c1 and -c5 cell lines with secretin for 24 hr reduced their binding capacity and reduced secretin efficacy in CHO-SnR-c1 cells and secretin potency in CHO-SnR-c5 cells. These results suggest efficient coupling of the secretin receptor to the adenylate cyclase machinery and the existence of spare receptors in the clone expressing higher receptor density. Pretreatment of the two cell lines with the reducing agent dithiothreitol reduced the binding capacity and induced the appearance of a low affinity binding component. In both cell lines, dithiothreitol pretreatment decreased secretin potency but not secretin efficacy, suggesting the necessity of integrity of the disulfide bridges for optimal receptor recognition.
Asunto(s)
Adenilil Ciclasas/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/metabolismo , Animales , Células CHO , Toxina del Cólera/farmacología , Colforsina/farmacología , Cricetinae , Cricetulus , Ditiotreitol/farmacología , Guanilil Imidodifosfato/farmacología , Receptores Acoplados a Proteínas G , Receptores de la Hormona Gastrointestinal/efectos de los fármacos , Receptores de la Hormona Gastrointestinal/genética , Proteínas Recombinantes/metabolismo , Secretina/fisiología , TransfecciónRESUMEN
Multicomponent synthetic vaccines containing both B and T cell epitopes belonging to two different pre-erythrocytic Ag of Plasmodium falciparum are presented. In a di-component hybrid, a circumsporozoite T cell epitope and a peptide representing a liver stage-specific Ag were connected to obtain a reciprocal reinforcement of helical potentials. In a tri-component hybrid, a sequence corresponding to the circumsporozoite repeat tetrapeptide (NPNA) was tandemly synthesized on the N-terminal end of the di-component hybrid. Both hybrid molecules were able to adopt a partial helical conformation in water as determined by circular dichroism studies. To analyze if the different components were immunologically functional in these vaccines, mich bearing genetic backgrounds known to respond or not to the individual components were immunized with the hybrids. The tri-hybrid peptide showed high immunogenic capacity as it elicited, in both H-2b and H-2k mice, high antibody responses against every separate individual sequence. Moreover, the antibodies induced by these conformationally restricted peptides were able to recognize the corresponding native proteins in the liver schizont and the sporozoite surface. H-2d mice, in which the immune response to the individual components was genetically restricted, did respond against the di-hybrid peptide. The tri-hybrid peptide, in which NPNA repeats were present, lacked this H-2d-priming capacity but it triggered antibody production in H-2d mice previously primed with the di-hybrid peptide. These results indicate that multivalent vaccines can provide positive (potentiating) effects by carefully combining structurally well defined epitopes; however, negative (suppressive) effects are also possible suggesting that selection of multivalent vaccine components will require testing of combined molecules to optimize specific immune responses and avoid undesirable effects which may result from negative molecular interactions.
Asunto(s)
Antígenos de Protozoos/genética , Plasmodium falciparum/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Dicroismo Circular , Técnica del Anticuerpo Fluorescente , Haplotipos , Memoria Inmunológica , Ratones , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Protozoarias/ultraestructura , Proteínas Recombinantes de Fusión , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.
Asunto(s)
Antígenos de Protozoos , Malaria/prevención & control , Péptidos/inmunología , Plasmodium falciparum/inmunología , Vacunas Sintéticas , Vacunas , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos , Oligopéptidos/metabolismoRESUMEN
The liver phase of development of malaria parasites has been studied only recently and remains poorly understood compared to the other stages such as sporozoïtes, merozoïtes and gametes. Access to liver forms of Plasmodium falciparum has been improved by the development of in vivo and in vitro propagation methods, but the yield of mature schizonts remains limited and does not allow a detailed antigenic analysis. To date, only immunofluorescence assays (IFA) have permitted a description of a species and liver-stage-specific antigen(s) (LSA; ref. 3). Monospecific antibodies to these antigens have not been obtained due either to difficulty in immunizing mice (against LSA), or to poor stability of human monoclonal antibodies. Therefore, as a means of characterizing the LSA, we used an alternative immunological approach to identify clones of the corresponding LSA genes. We describe here the isolation of a DNA sequence coding for a P. falciparum liver-stage-specific antigen composed of repeats of 17 amino-acids, which is immunogenic in man.