RESUMEN
Adenovirus (Ad) vaccine vectors have been used for many applications due to the capacity of the Ad capsid proteins to evoke potent immune responses, but these vectors are often ineffective in the context of pre-existing anti-Ad immunity. Leveraging the knowledge that E1(-)E3(-) Ad gene transfer vectors are potent immunogens, we have developed a vaccine platform against small molecules by covalently coupling analogs of small molecules to the capsid proteins of disrupted Ad (dAd5). We hypothesized that the dAd5 platform would maintain immunopotency even in the context of anti-Ad neutralizing antibodies. To test this hypothesis, we coupled cocaine and nicotine analogs, GNE and AM1, to dAd5 capsid proteins to generate dAd5GNE and dAd5AM1, respectively. Mice were pre-immunized with Ad5Null, resulting in high titer anti-Ad5 neutralizing antibodies comparable to those observed in the human population. The dAd5GNE and dAd5AM1 vaccines elicited high anti-cocaine and anti-nicotine antibody titers, respectively, in both naive and Ad5-immune mice, and both functioned to prevent cocaine or nicotine from reaching the brain of anti-Ad immune mice. Thus, disrupted Ad5 evokes potent humoral immunity that is effective in the context of pre-existing neutralizing anti-Ad immunity, overcoming a major limitation for current Ad-based vaccines.
Asunto(s)
Adenoviridae/inmunología , Caproatos/inmunología , Proteínas de la Cápside/inmunología , Cocaína/análogos & derivados , Vectores Genéticos/genética , Complejos Multiproteicos/inmunología , Nicotina/análogos & derivados , Vacunas/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Western Blotting , Caproatos/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cocaína/inmunología , Cocaína/metabolismo , Cocaína/farmacocinética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Complejos Multiproteicos/metabolismo , Nicotina/inmunología , Nicotina/metabolismo , Nicotina/farmacocinéticaRESUMEN
2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.
Asunto(s)
Aldehídos/análisis , Arteriosclerosis/metabolismo , Aldehídos/química , Aldehídos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Aorta/química , Arteriosclerosis/patología , Caproatos/química , Caproatos/inmunología , Epítopos/inmunología , Femenino , Haptenos , Hemocianinas/inmunología , Humanos , Hibridomas , Inmunoquímica , Peroxidación de Lípido , Lisina/química , Reacción de Maillard , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , Proteínas/química , Proteínas/metabolismo , Piridonas/química , Piridonas/inmunologíaRESUMEN
X-linked immune deficient (Xid) mice fail to produce anti-phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC-specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H-Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.
Asunto(s)
Anticuerpos Antifosfolípidos/metabolismo , Sitios de Unión de Anticuerpos , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región de Unión de la Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Fosforilcolina/inmunología , Infecciones Neumocócicas/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antifosfolípidos/genética , Afinidad de Anticuerpos , Adhesión Bacteriana/inmunología , Secuencia de Bases , Caproatos/inmunología , Haptenos/inmunología , Hibridomas/química , Inmunización Pasiva , Alotipos de Inmunoglobulinas/análisis , Cadenas Pesadas de Inmunoglobulina/análisis , Cadenas Pesadas de Inmunoglobulina/genética , Isotipos de Inmunoglobulinas/análisis , Región de Unión de la Inmunoglobulina/análisis , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/análisis , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos CBA , Ratones Mutantes , Datos de Secuencia Molecular , Fosforilcolina/análogos & derivados , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/patogenicidad , VirulenciaRESUMEN
A novel form of phosphocholine (PC), p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC) coupled to keyhole limpet hemocyanin (KLH) has been compared with unencapsulated, avirulent Streptococcus pneumoniae (R36a) and with the traditional thymus-dependent form of PC, diazophenylphosphocholine (DPPC)-conjugated KLH for its vaccine potential against virulent S. pneumoniae. Immunization with any of these three PC-containing Ags protects normal mice against a lethal challenge with 10(4) S. pneumoniae, whereas only EPC-KLH provides total protection to Xid mice. DPPC-KLH and unencapsulated S. pneumoniae confer less than 40% protection in Xid mice. Passive transfer of a PC-specific hybridoma Ab made from EPC-KLH-immunized Xid mice also provided protection against lethal challenge with S. pneumoniae. Protective anti-PC Ab were capable of binding to the surface of virulent bacteria, whereas anti-PC Ab incapable of binding to the bacterial surface failed to protect. Furthermore, serum Ab from EPC-KLH immunized and protected mice bound to S. pneumoniae, whereas secondary Abs from DPPC-KLH- or R36a-immunized mice failed to bind to the bacteria. EPC-KLH is potentially a vaccine candidate for pneumococcal prophylaxis in settings of immune compromise.
Asunto(s)
Antígenos/inmunología , Caproatos/inmunología , Fosforilcolina/análogos & derivados , Fosforilcolina/inmunología , Infecciones Neumocócicas/prevención & control , Animales , Compuestos Azo/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hemocianinas/inmunología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Streptococcus pneumoniae/inmunología , Cromosoma XRESUMEN
X-linked immune deficient (XID) mice are susceptible to infection with Streptococcus pneumoniae because they fail to mount an immune response to the immunodominant phosphocholine (PC) epitope on the bacterial cell wall. It is difficult to induce PC-specific antibodies in XID mice because PC-specific B cells expressing the T15-, M167- and M603 idiotype (Id), which provide protection against S. pneumoniae, are deleted in these mice via an antigen-specific, receptor-mediated process. In addition, the standard PC hapten, p-diazophenylphosphocholine (DPPC), induces high affinity phenylphosphocholine (PPC)-specific antibodies in XID mice, which are not protective against S. pneumoniae. We have used a novel PC hapten, p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC), to induce PC-specific antibodies in XID mice. The immune response to EPC-keyhole limpet hemacyanin (KLH) is dominated by IgG1, VH1+, T15-Id-, PC-inhibitable antibodies. A small IgM anti-PC response having a consistent T15-Id+ component is also induced in XID mice, whereas normal mice produce a large IgM response dominated by T15-Id+ antibodies. The immune response to EPC-KLH remains predominantly PC-inhibitable even after multiple immunizations, while the response to DPPC-KLH becomes dominated by PPC-specific antibodies. C.CBA/N mice immunized twice with EPC-KLH are protected against 10(4) S. pneumoniae while as few as 10 bacteria are 100% lethal for the unimmunized controls. The ability of EPC-protein to induce a long-lived, PC-specific response should make this hapten a potential TD vaccine candidate for S. pneumoniae.
Asunto(s)
Fosforilcolina/inmunología , Streptococcus pneumoniae/inmunología , Animales , Compuestos Azo/inmunología , Vacunas Bacterianas , Caproatos/inmunología , Femenino , Haptenos/inmunología , Hemocianinas/inmunología , Técnica de Placa Hemolítica , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Inmunoterapia Adoptiva , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Fosforilcolina/análogos & derivadosRESUMEN
A competitive radioimmunoassay for an advanced glycosylation endproduct, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) was developed. The immunogen was prepared by coupling of 4-furanyl-2-furoyl-1H-imidazole-1-hexanoic acid to keyhole limpet hemocyanin. The antiserum obtained by immunizing guinea pigs with the immunogen exhibited high affinity binding to FFI, but no cross-reactivity was observed for structurally related compounds containing an imidazole ring or furan ring(s). By using the radioimmunoassay, the levels of FFI in bovine serum albumin incubated with glucose for varying lengths of time were measured. A time-dependent increase was obtained in the amount of acid-liberated FFI and fluorescence. The radioimmunoassay described here had satisfactory reproducibility as judged by the intra-assay precision of 3.4-6.4% and the interassay precision of 7.3-8.9%. The method allows to quantitate FFI on the modified proteins that have been implicated in the complications of diabetes and in normal aging as well.