RESUMEN
The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.
Asunto(s)
Péptidos Cíclicos/inmunología , Administración Oral , Animales , Formación de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Sueros Inmunes/inmunología , Inmunización , Toxinas Marinas , Ratones , Ratones Endogámicos BALB C , Microcistinas , Péptidos Cíclicos/aislamiento & purificación , ConejosRESUMEN
To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Epítopos de Linfocito T/metabolismo , Lipoproteínas/metabolismo , Péptidos/inmunología , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Pollos , Dipéptidos/inmunología , Inhibidores Enzimáticos/inmunología , Epítopos de Linfocito T/inmunología , Humanos , Inmunización , Leucocitos Mononucleares/inmunología , Lipoproteínas/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Microcistinas , Péptidos Cíclicos/inmunología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , ConejosRESUMEN
Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (Th)-cell epitope into the lipopeptide-hapten conjugate. The Th-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1-16)] and [Mel(17-26)-CONH2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Formación de Anticuerpos , Epítopos/inmunología , Haptenos/farmacología , Meliteno/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Ratones , Ratones Endogámicos BALB CRESUMEN
To evaluate the ability of the lipotripeptide P3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P3CSS-[RT-(522-543)] and P3CSS-[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant Th epitope [i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific Th responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2b, H-2d and H-2k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these Th and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528-549) a promising candidate for vaccine design. The use of the P3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.
Asunto(s)
Linfocitos B/inmunología , Transcriptasa Inversa del VIH/inmunología , Lipoproteínas/inmunología , Péptidos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Mapeo Epitopo , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/química , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Various lipopeptides representing the N-terminal part of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas virdis were prepared by solid-phase peptide synthesis. These lipopeptides consisted of a S-[2,3-dihydroxypropyl]-cysteinyl (Dhc) residue N-terminally coupled to the nonapeptide FEPPPATTT. Different numbers of palmitoyl (Pam) chains were attached to Dhc via ester and/or amide bonds. The lipopeptide Dhc(Pam)2-FEPPPATTT containing two ester-bonded palmitoyl residues and a free N-terminus was a potent polyclonal activator of murine (BALB/c) spleen cells at subnanomolar concentrations. The lipopeptide Pam-Dhc(Pam)2-FEPPPATTT containing three palmitoyl residues, the two-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residue, and the N-terminally elongated lipopeptide SLVAG-Dhc(Pam)2-FEPPPATTT were less active. The nonapeptide FEPPPATTT and the decapeptide Dhc-FEPPPATTT were only marginal splenocyte activators, even at concentrations as high as 1 microM. Thus, lipopeptide Dhc(Pam)2-FEPPPATTT constitutes the first potent splenocyte stimulation Dhc-lipopeptide described so far that contains only two fatty acid residues.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Péptidos/síntesis química , Péptidos/farmacología , Proteínas del Complejo del Centro de Reacción Fotosintética/síntesis química , Proteínas del Complejo del Centro de Reacción Fotosintética/farmacología , Adyuvantes Inmunológicos/química , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Técnicas In Vitro , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/farmacología , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Péptidos/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodopseudomonas , Bazo/citología , Bazo/efectos de los fármacosRESUMEN
The lipoprotein (LP) from Escherichia coli and other Enterobacteriaceae as well as lipoprotein derived lipopeptides constitute potent B lymphocyte mitogens. Several synthetic peptide segments of the lipoprotein were tested for mitogenic activity towards splenocytes of BALB/c mice. Mitogenic effects were observed for the lipoprotein fragments LP-(2-15), LP-(2-20), LP-(2-25), and LP-(33-47), and a less pronounced effect was determined for LP-(2-10). The mitogenicity of the peptides could be further enhanced by their attachment to the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)- cysteine (Pam3Cys). The additional insertion of 6-aminohexanoic acid (Ahx) between the peptide segments and Pam3Cys further increased mitogenicity. Thus, in addition to the known mitogenic N-terminal lipopeptide of lipoprotein, additional peptide segments of the molecule were shown to exhibit a biological activity.
Asunto(s)
Escherichia coli/metabolismo , Lipoproteínas/farmacología , Activación de Linfocitos/efectos de los fármacos , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Técnicas In Vitro , Lipoproteínas/química , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Conformación Molecular , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Bazo/citología , Timidina/metabolismoRESUMEN
The immunogenic and immunomodulatory properties of a lysed fraction from selected E. coli strains (OM-89, Uro-Vaxom) were determined in vivo and in vitro. It could be demonstrated that OM-89 constitutes an active immunogen in mice. Maximum OM-89-specific antibody titers were obtained after 4-5 i.p. immunizations; the titers could be further enhanced by the simultaneous injection of lipopeptide adjuvants. It was shown by ELISA that the antibodies obtained bound to the bacterial strains used for the preparation of the OM-89 extract. Immunogenicity was observed both after intraperitoneal and oral application of the extract. Besides being active as an immunogen. OM-89 was able to act in vitro as a polyclonal lymphocyte activator, as determined in splenocyte cultures of different inbred murine strains, and in cultures of human peripheral blood lymphocytes. Our results show that the B lymphocyte stimulating activity of the bacterial extract OM-89 was comparable to that of lipopeptide adjuvants. In conclusion, the bacterial extract both an active immunogen in vivo, and a polyclonal B cell activator in vitro. These findings may be of importance for the understanding of the therapeutic effect of OM-89.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos Bacterianos , Escherichia coli/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Linfocitos B/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Cinética , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Bazo/citología , Bazo/inmunologíaRESUMEN
Lipopeptide analogues of bacterial lipoprotein activate macrophages and B lymphocytes. The products formed by coupling these lipopeptides to low molecular mass antigens can be used to induce antigen-specific antibodies in mice. In the present work, it is shown that HIV-1 gp160-derived synthetic oligopeptides coupled to the synthetic lipodipeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-s eryl- serine (P3CSS) induce peptide-specific antibodies in mice without adding further adjuvants. Depending on the peptides applied, the conjugates exhibited different lymphocyte stimulatory activity, immunoglobulin isotype patterns, and boost reactions; lipopeptide conjugates inducing a pronounced secondary immune response are considered to possess both B- and T-cell epitopes. Antibodies induced by the lipopeptide-HIV-1-peptide conjugates were also reactive against the recombinant gp160 of HIV-1.
Asunto(s)
Formación de Anticuerpos/efectos de los fármacos , Productos del Gen env/farmacología , Productos del Gen gag/farmacología , Antígenos VIH/farmacología , Lipoproteínas/farmacología , Activación de Linfocitos/efectos de los fármacos , Péptidos/farmacología , Precursores de Proteínas/farmacología , Proteínas Virales , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Linfocitos B/inmunología , Dipéptidos/metabolismo , Dipéptidos/farmacología , Epítopos/inmunología , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Productos del Gen gag/inmunología , Productos del Gen gag/metabolismo , Antígenos VIH/inmunología , Antígenos VIH/metabolismo , Proteínas gp160 de Envoltorio del VIH , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/inmunología , Precursores de Proteínas/inmunología , Precursores de Proteínas/metabolismo , Linfocitos T/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
Lipopeptide analogues of bacterial lipoprotein constitute polyclonal B lymphocyte activators. Combined with or covalently coupled to antigens, they act as potent adjuvants. We could show that antigens (BSA-DNP, TNP-SRBC, saxitoxin, HIV-1 gp160(BH10303-329, EGFR516-523) combined with or coupled to the synthetic lipodipeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-s erine (P3CS) constitute active immunogens in vivo in mice. They were also able to induce an in vitro humoral immune response in the murine and human systems, and B lymphocytes thus activated were suitable for fusion. Thus, the antigens chaperonin/phytochrome, BSA-saxitoxin, histamine, HIV-1 gp160 (BH10(303-329)), HIV-1 gp160 (RF316-341)), and HIV-2 p17 (ROD111-121) combined with or conjugated to P3CS could be used for in vitro immunization followed by the preparation of murine and human monoclonal antibodies. Our novel immunization procedure offers reproducibility, high antibody titers often after one immunization, lack of toxicity of the adjuvants, easy chemical preparation of the conjugates in mg amounts, and the applicability of the conjugates for screening for the antibodies obtained.