RESUMEN
beta-Glucans are polysaccharides that act as nonspecific immune system stimulants. However, many beta-Glucans are sparingly soluble in water. This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties. Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants. Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-[6-(N,N'-dimethylamino)purin-9-yl]pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages. In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess.
Asunto(s)
Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Glucanos/química , Glucanos/farmacología , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Secuencia de Carbohidratos , Femenino , Glucanos/aislamiento & purificación , Humanos , Técnicas In Vitro , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidación-Reducción , Saccharomyces cerevisiae/química , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The tetrapeptide I (D-lysine-L-asparaginyl-L-prolyl-L-tyrosine or D-LysAsnProTyr), and analogue sequences, were synthesized and evaluated for the ability to stimulate immune cell subsets. These sequences were selected based on their perceived ability to readily adopt a beta-turn structure. In vitro immunological assays revealed a robust stimulation of mitogen activated B-cell proliferation and a modest to significant stimulation of cytotoxic T lymphocytes (CTLs). Further, this in vitro stimulation of B-cells was accompanied by an in vivo expansion of B-cells in C57BL/6 mice, as demonstrated by immunophenotyping experiments. Interestingly, a conformational analysis of the low energy conformers of I and the endogenous B-cell stimulant bursin (LysHisGlyNH2) shows that these molecules can be superimposed. However, I displayed significantly enhanced physiological stability. For a number of reasons, I may be a particularly useful vaccine adjuvant.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Imitación Molecular , Oligopéptidos/química , Oligopéptidos/inmunología , Animales , Asparagina/inmunología , Células Cultivadas , Femenino , Semivida , Lisina/inmunología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/farmacología , Prolina/inmunología , Bazo , Tirosina/inmunologíaRESUMEN
Imreg (Tyr1-Gly2-Gly3) is a well-known immunostimulant. However, it possesses a short half-life. Stabilized analogues of Imreg were prepared by a regioselective insertion in which peptide bonds at position 1,2 or 2,3 were replaced by thioamide linkages. This was achieved by using new thioacylating agents based on thioacyl-fluoro-N-benzimidazolone. The synthesis and properties of these reagents are described herein. This peptide modification enhanced significantly the half-life of the thioanalogues relative to Imreg in blood. The thioanalogues and Imreg were tested in vitro in T and B cell proliferation assays and for their ability to stimulate cytotoxic T-lymphocytes (CTLs). Only thiotyrosyl glycyl glycine 11 displayed some activity as evidenced by a weak stimulation of CTLs. On the basis of this activity and the increased stability, an in vivo immunological evaluation was undertaken. Immunophenotyping of 11 revealed a significant increase in activated CTL and NK cell populations in the spleen. This expansion was also accompanied by a significant stimulation of NK cells and the B cell proliferative response. Thioanalogues of Imreg were generally nontoxic, as exemplified by 11. The latter is a promising immunostimulant which may be targeted for cancer and viral infections, where CTLs and NK cells play an important role, or as a vaccine adjuvant where stimulation of antibody-producing B cells is important.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Oligopéptidos/química , Oligopéptidos/síntesis química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Estabilidad de Medicamentos , Endopeptidasas/química , Femenino , Inmunofenotipificación , Riñón/enzimología , Riñón/ultraestructura , Células Asesinas Naturales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microvellosidades/enzimología , Oligopéptidos/farmacología , Ratas , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Water soluble analogues of the lipophilic immunostimulant, octadecyl D-alanyl-L-glutamine, BCH-527, were synthesized and evaluated for the ability to stimulate natural killer (NK) cells. One of these compounds in which the octadecyl chain of BCH-527 was replaced with a shorter chain alcohol, 6-(D-alanyl-L-glutaminylamino)hexan-1-ol, 9, displayed an in vitro stimulation of NK cells comparable to that of interleukin 2 (IL 2). However, when the hydroxyl of 9 was linked to L-fucose to yield 1-beta-[6-(D-alanyl-L-glutaminylamino)hex-1-yl]-L- fucopyranose (BCH-2537, 1), the observed stimulation of NK cells was greater than that observed with IL 2. Further evaluation of these compounds revealed that the improved in vitro activity of BCH-2537 was more pronounced in vivo. That is, while both compounds significantly increased splenic NK cells, only BCH-2537 significantly increased the activity of these cells in vivo. In terms of a structure-activity relationship, NK cell activity was sensitive to minor structural modifications. It was influenced by conservative substitutions within the dipeptide, the length of the hydrocarbon chain, and the functionality at the end of the chain. No other compound enhanced NK cell activity to the extent exhibited by BCH-2537, although a few were equipotent to 9.
Asunto(s)
Adyuvantes Inmunológicos , Dipéptidos , Fucosa/análogos & derivados , Hexanoles , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Dipéptidos/síntesis química , Dipéptidos/química , Dipéptidos/farmacología , Femenino , Fucosa/síntesis química , Fucosa/química , Fucosa/farmacología , Hexanoles/síntesis química , Hexanoles/química , Hexanoles/farmacología , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mitógenos/síntesis química , Mitógenos/química , Mitógenos/farmacología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/inmunología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Relación Estructura-Actividad , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
A series of 6-substituted purinyl alkoxycarbonyl amino acids were synthesized and evaluated for their ability to stimulate cytotoxic T lymphocytes (CTLs) and the mixed lymphocyte reaction (MLR). A few of these compounds, in particular [[5-[6-(N,N-dimethylamino)purin-9-yl]pentoxy]-carbonyl]D-arginine (BCH-1393, 4a), displayed an in vitro stimulation of CTLs comparable to interleukin 2 (IL 2). BCH-1393 increased the CTL response between 10(-9) M and 10(-5) M. Further, this potent in vitro activity was reflected as a significant increase in CTL cell number in vivo. However, immunophenotyping of some of the other equipotent compounds did not reveal a parallel relative increase in CTLs in vivo. It was difficult to formulate a rigorous structure-activity relationship based on in vitro CTL activity. Nevertheless, the activity was dependent upon the nature of the 6-substituent on the purine, the type and stereochemistry of the amino acid, and the distance and spatial freedom between the purine and amino acid as defined by the length and rigidity of the linker. These compounds were generally nontoxic, as exemplified by BCH-1393. BCH-1393 is a promising immunostimulant which may be targeted for those disease states which require an increased CTL or TH1 type response.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Aminoácidos , Arginina/análogos & derivados , Activación de Linfocitos/efectos de los fármacos , Purinas/síntesis química , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Arginina/síntesis química , Arginina/química , Arginina/farmacología , Femenino , Inmunofenotipificación , Interleucina-2/biosíntesis , Prueba de Cultivo Mixto de Linfocitos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Purinas/química , Purinas/farmacología , Bazo/inmunología , Relación Estructura-Actividad , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BCH-527, the lipophilic hydrochloride salt of octadecyl D-alanyl L-glutamine, was evaluated for efficacy against experimentally induced murine cytomegalovirus (MCMV), influenza A (H1N1) (IV-A), and Punta Toro virus (PTV) infections in mice. The compound was administered i.p. every other day for a total of 4 injections commencing 24 h previrus exposure. Doses ranged from 12.5 to 200 mg/kg per injection in the various experiments. The MCMV infection was significantly inhibited in two experiments by doses of 25-200 mg/kg, as manifested by increased numbers of survivors and decreased titers of virus recoverable from tissues. The IV-A infection was weakly inhibited, with antiviral activity seen in lowered lung scores and lung weights and less decline in arterial oxygen saturation values. The PTV infection was not inhibited. BCH-527 was stimulatory to cytotoxic T-cells, natural killer (NK) cells, macrophages, and splenic B-cells. The highest dose tested, 200 mg/kg, was inhibitory to cytotoxic T-cell activity and to some extent to NK cell and macrophage activity. These data suggest BCH-527 functions as an immune modulator in exerting the observed antiviral activity.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antivirales/farmacología , Dipéptidos/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Virus de la Influenza A , Muromegalovirus , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Femenino , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estructura Molecular , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Stearly tyrosine has been described in the literature as a candidate adjuvant for human vaccines. More recently, other stearyl esters have been reported to be adjuvant-active. This preliminary study assesses the adjuvanticity of stearyl amides, including the amide analogue of stearyl tyrosine: tyrosyl stearylamine. This study indicates that phenylalanylethanolamine O-stearate induces a significant antibody response and a favourable isotype distribution.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Fenilalanina/análogos & derivados , Estearatos/farmacología , Tirosina/análogos & derivados , Animales , Formación de Anticuerpos/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos BALB C , Fenilalanina/farmacología , Tirosina/farmacologíaRESUMEN
A new family of immunoadjuvants, long-chain stearyl esters of amino acids and peptides, are described and examined with bacterial and viral vaccines. The parent compound, stearyl tyrosine, displayed significant adjuvant activity with these vaccines. Stearyl glycyl glycine displayed superior activity with viral vaccines. A number of analogues of stearyl tyrosine were adjuvant-active. Further, these adjuvants were able to elicit a neutralizing antibody response. Stearyl tyrosine and stearyl ester analogues represent promising adjuvants for human vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Aminoácidos/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos , Ésteres/inmunología , Ésteres/farmacología , Femenino , VIH/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/farmacología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neisseria meningitidis/inmunología , Oligopéptidos/farmacología , Poliovirus/inmunología , Toxoides/inmunología , Toxoides/farmacología , Tirosina/inmunología , Tirosina/farmacología , Proteínas Virales/inmunología , Proteínas Virales/farmacologíaRESUMEN
In this present report we compare the humoral immune response induced by immunization with an HIV-1 gp160 peptide corresponding to amino acid sequence 503-535 complexed with different adjuvants. Specifically, the antipeptide, anti-HIV-1 gp160 and neutralizing antibody responses were measured in groups of mice and baboons that received peptide 503-535 conjugated to a carrier protein in either saline, alum, or stearyl tyrosine. The highest antibody responses were induced when mice and baboons were immunized with peptide adsorbed on stearyl tyrosine. These data indicate that stearyl tyrosine represents a potent candidate as a nontoxic adjuvant not only for subunit viral vaccines, but also for HIV peptides.
Asunto(s)
Adyuvantes Inmunológicos , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/biosíntesis , Precursores de Proteínas/inmunología , Tirosina/análogos & derivados , Animales , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH , VIH-1/inmunología , Inmunización , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Papio , Fragmentos de Péptidos/inmunología , Radioinmunoensayo , Tirosina/inmunologíaRESUMEN
We describe the enhancement of the antibody response against hepatitis B surface Ag by octadecyl L-tyrosine, a synthetic adjuvant designed to exert its adjuvant effect in a manner similar to that of alum because it binds soluble Ag and releases it slowly from the site of injection. Our data demonstrate that octadecyl L-tyrosine showed a significant enhancement of the antihepatitis B surface Ag response compared to that of alum in the secondary response. The most striking difference between octadecyl L-tyrosine and alum in the antihepatitis B surface Ag antibody response was the absence of IgE-specific antibodies subsequent to immunization of the Ag in octadecyl L-tyrosine. Both the optical isomers of the octadecyl esters of tyrosine were adjuvant active, however, the racemic mixture showed a significantly lowe adjuvant activity. This adjuvant has great potential to be used in humans because it is devoid of side effects as assessed by the lack of acute and chronic toxicity in mice and rats, pyrogenicity in rabbits, formation of granuloma in cats, and adjuvant arthritis in rats.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos de Superficie de la Hepatitis B/inmunología , Tirosina/análogos & derivados , Vacunas Sintéticas/inmunología , Vacunas/inmunología , Animales , Femenino , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/farmacocinética , Inmunoglobulina E/inmunología , Isotipos de Inmunoglobulinas/inmunología , Memoria Inmunológica , Ratones , Ratones Endogámicos BALB C , Tirosina/inmunologíaRESUMEN
A dipstick suitable for immunoassay procedures has been developed, and its utility demonstrated by the development of tests for the determination of immunoglobulin levels in newborn bovine blood, and for the detection of bovine antibodies against Brucella abortus. The dipstick is of simple design, consisting of three components: a polycarbonate membrane, an adhesive, and a support material. The polycarbonate membrane is a porous filter material upon which the immunoassay is undertaken. Biomolecules bind to the membrane with sufficient affinity to permit the development of useful immunoassay procedures; however, this membrane exhibits low non-specific binding of reagents. Collectively, these properties make polycarbonate a useful material for solid-phase immunoassay procedures.
Asunto(s)
Cementos Dentales , Técnicas de Inmunoadsorción/instrumentación , Cemento de Policarboxilato , Animales , Animales Recién Nacidos , Anticuerpos Antibacterianos/análisis , Brucella abortus/inmunología , Brucelosis/diagnóstico , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/inmunología , Inmunoglobulinas/análisis , Membranas ArtificialesRESUMEN
A simple, rapid, high performance liquid chromatographic (HPLC) method for the analysis of the immunoadjuvant octadecyl tyrosine hydrochloride is described. The HPLC procedure can be applied to the direct determination of amino acid reactants present as contaminants in the adjuvant (tyrosine, ethyl tyrosine) and from this information the content of octadecanol reactant can be estimated. Further, these same determinations provide a means of monitoring immunoadjuvant stability in any vaccine preparation.
Asunto(s)
Adyuvantes Inmunológicos/análisis , Tirosina/análogos & derivados , Vacunas/análisis , Cromatografía Líquida de Alta Presión , Humanos , Control de Calidad , Tirosina/análisis , Tirosina/inmunologíaRESUMEN
Preliminary studies of some of the properties of stearyl tyrosine have shown that it is non-toxic, free of adverse reactions at the sites of injection, non-pyrogenic, stable upon storage and easy to sterilize. Formalin inactivated poliovirus vaccine (IPV) adjuvanted with stearyl tyrosine hydrochloride induced significantly higher titres of antibodies in non-human primates, after two injections, than the non-adjuvanted vaccine. Furthermore, the adjuvanted vaccine, even when diluted 1:4, showed consistently higher antibody titres as well as a longer persistence of antibodies than the non-adjuvanted undiluted vaccine. These studies suggest that stearyl tyrosine is an excellent and cost effective adjuvant for IPV. Hence further investigation with this novel synthetic compound would be worthwhile to ascertain its adjuvanticity for IPV in human subjects.