RESUMEN
BACKGROUND: Prostate hyperplasia and neoplasia are major illness of men and elderly dogs. Treatment of prostate cancer requires androgen deprivation surgery or therapy to prevent metastases and alleviate pain. Recently, six DNA vaccines have entered clinical trials against prostate cancer in humans with limited success. There is a need for new therapies that delay the establishment of malignancy and prolong survival. MATERIALS AND METHODS: A plasmid DNA vaccine coding for eight gonadotrophin-releasing hormone (GnRH-I) interspersed in eight T-helper epitopes was used. Sexually mature male mice were immunized with the vaccine in hemagglutinating virus of Japanese envelope vector and boosted in nonionized surfactant vesicles in study weeks 0, 3, 6, 9, and 12. Plasma anti-GnRH-I antibody response, serum testosterone concentration, and effect on prostate were evaluated. RESULTS: Results of an indirect enzyme linked immunosorbent assay (ELISA) showed anti-GnRH-I antibody response (OD value) detected in the study week 3 (0.613 ± 0.179) with a highest response in the week 12 (1.205 ± 0.219). Serum testosterone concentration (ng/ml) in vaccinated mice was significantly reduced (P > 0.000, 0.761 ± 0.531) in the study week 24 in contrast to control serum (7.583 ± 1.251). Group average gross combined weight of prostate and seminal vesicles of vaccinated mice was significantly (P < 0.000) reduced in the study week 24 (319.75 ± 89.19 mg) in contrast to control weight (563.25 ± 108.60 mg). Sections of prostate stained with Goldner's trichrome showed profuse pink color secretion in control tubules, which however was absent in the vaccinated prostate. The lining epithelium of the vaccinated prostate was atrophied and did not enfold in its lumen. CONCLUSIONS: Immunization strategy designed with the plasmid DNA vaccine in hemagglutinating virus of Japanese envelope and nonionized surfactant vesicles can be the genetic immunization platform. This vaccine bears potentials in terms of reducing serum testosterone concentration and induction of atrophy of prostate. Targeted ablation of native GnRH-I by genetic immunization could offer leverage to vaccinologists, seeking therapeutic target to control and prevent malignancy of prostate.
RESUMEN
Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Hormona Liberadora de Gonadotropina/inmunología , Nanopartículas/química , Espermatogénesis/efectos de los fármacos , Vacunas Anticonceptivas/administración & dosificación , Animales , Anticoncepción Inmunológica/métodos , Anticonceptivos Masculinos/administración & dosificación , Anticonceptivos Masculinos/farmacología , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/química , Inmunoglobulina G/sangre , Masculino , Ratones Endogámicos BALB C , Simulación de Dinámica Molecular , Nanopartículas/administración & dosificación , Albúmina Sérica Bovina/química , Dióxido de Silicio/química , Espermatogénesis/fisiología , Testosterona/sangre , Vacunas Anticonceptivas/farmacologíaRESUMEN
Research into active immunisation against gonadotrophin releasing hormone (GnRH-I) has gained widespread acceptance as a means of controlling reproduction and behaviour of farm, companion and wild animals. Many studies describe the use of multiple copies of the self-peptide in linear alignment and conjugation with a large carrier protein to increase the immune response to the peptide. However, problems resulting from carrier protein epitope suppression have seen a diversion of interest into the use of genetic materials to elicit an optimum immune response. In this study, a 533-bp long DNA vaccine was constructed in pcDNAV5-HisB coding for 18.871 kDa GnRH-I-T-helper-V5 epitopes fusion protein. COS1 cells transfected with the vaccine construct were found to release fusion protein into culture supernatant. The vaccine construct (100 microg/mice) in saline solution administered into the anterior quadriceps muscle of ICR male and female mice stimulated antigen-specific IgG antibody responses. Testosterone levels in the vaccinated male mice were significantly (p = 0.021) reduced. A significant reduction in uterine implants were noted following mating between immunised males and control females (p = 0.028), as well as between immunised females and control males (p = 0.004). Histological examination of both the male and female gonads in study week 13 showed atrophy of the seminiferous epithelium and suppression of folliculogenesis.
Asunto(s)
Epítopos de Linfocito T/inmunología , Fertilidad/inmunología , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/inmunología , Plásmidos/inmunología , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Anticonceptivas/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Femenino , Fertilización In Vitro , Masculino , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Ovario/citología , Ovario/efectos de los fármacos , Ingeniería de Proteínas , Ratas , Ratas Sprague-Dawley , Recuento de Espermatozoides , Testículo/citología , Testículo/efectos de los fármacos , Testosterona/sangre , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Immunisation against mammalian gonadotrophin releasing hormone (GnRH-I) linked to large carrier proteins has been shown to disrupt fertility. However, various studies have shown that the carrier protein causes epitope suppression of the hapten response, resulting in short-lived immunoneutralisation, followed by a return of fertility. A range of strategies has been used to resolve this, with limited success. The aim of this study was to construct a plasmid DNA vaccine encoding GnRH-I and T-helper epitopes. A 498 bp long vaccine construct in pcDNA3.1+ was administered to male mice in conjunction with a Hemagglutinating Virus of Japanese Envelop (HVJ-E) vector or in saline solution. The vaccine efficacy was evaluated in terms of GnRH-I specific IgG antibody response, serum testosterone levels, testicular spermatogenesis and the ability to produce offspring. The vaccine appeared to induce higher anti-GnRH-I IgG antibody response and insult the fertility axis, which was characterised by a drop of epididymal sperm counts, reduction of serum testosterone levels, suppressed testicular spermatogenesis and a significant decrease in litter numbers compared to control animals. The end-point vaccine efficacy was much higher in the HVJ-E vector mediated immunisation, than in saline alone.
Asunto(s)
Epítopos de Linfocito T/genética , Fertilidad/efectos de los fármacos , Hormona Liberadora de Gonadotropina/inmunología , Vacunas de ADN/administración & dosificación , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Secuencia de Bases , Anticoncepción Inmunológica , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Femenino , Hormona Liberadora de Gonadotropina/genética , Inmunización/métodos , Inmunoglobulina G/sangre , Tamaño de la Camada/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Plásmidos/genética , Espermatogénesis/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Testosterona/sangre , Vacunas Anticonceptivas , Vacunas de ADN/inmunologíaRESUMEN
In mammals, the hypothalamic decapeptide, gonadotrophin releasing hormone (GnRH-I), is regarded as the major fertility regulating peptide. However, a range of isoforms also exists, varying only in the core region between amino acids 5-8. The physiological role of two of these, GnRH-II and GnRH-III, remains controversial, particularly with regard to fertility. The basis of the present study was to examine whether there is potential for GnRH-II and GnRH-III to be developed into highly specific vaccines, and to determine what the impact of their neutralisation would be on fertility. Computer modelling was used to predict how many common amino acids could be sequentially removed from the N-terminus, without loss of conformational structure. Sequences predicted to retain structure, were synthesised and conjugated to tetanus toxoid. Male mice were actively immunised, in study weeks 0, 2, 4 and 6 and peptide specific ELISA carried out. Mice immunised with TT-GnRH-I, TT-GnRH-II and TT-GnRH-III conjugates induced high antibody titres to the respective peptide. However, serum from TT-GnRH-I treated mice showed cross-reactivity to GnRH-II and GnRH-III peptides, and serum from TT-GnRH-II immunised mice showed cross-reactivity to GnRH-III. On the other hand, serum from only two of the TT-GnRH-III treated animals showed cross-reactivity to GnRH-II. Histological examination of the testes enabled comparative quantification of the disruption to spermatogenesis. Immunisation against TT-GnRH-I and TT-GnRH-III caused 66% and 68%, respectively, of seminiferous tubules viewed to show evidence of spermatogenesis, compared with 82% and 92% against TT-GnRH-II and untreated controls, respectively. Endocrine analysis revealed that only the TT-GnRH-I immunised animals showed significant reduction (p<0.05) in follicle stimulating hormone, while testosterone levels were reduced in the TT-GnRH-I and TT-GnRH-III treated animals. Taken together, our data suggests that GnRH-I and GnRH-III are implicated in spermatogenesis, unlike GnRH-II.
Asunto(s)
Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/inmunología , Oligopéptidos/inmunología , Ácido Pirrolidona Carboxílico/análogos & derivados , Espermatogénesis , Animales , Anticuerpos/sangre , Pollos , Simulación por Computador , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Hormona Folículo Estimulante/sangre , Histocitoquímica , Lampreas , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Péptidos/síntesis química , Péptidos/química , Isoformas de Proteínas/inmunología , Ácido Pirrolidona Carboxílico/inmunología , Túbulos Seminíferos/patología , Testículo/patología , Testosterona/sangre , Toxoide Tetánico/química , Vacunas Anticonceptivas , Vacunas de Subunidad/inmunologíaRESUMEN
PROBLEM: Recent evidence shows the existence of numerous isoforms of gonadotropin releasing hormone (GnRH), with high sequence homology and a core variable region. This raises the issue that previous GnRH distribution studies may have identified a variety of isoforms. This investigation was carried out to confirm the distribution and binding activity of GnRH-I only. METHOD OF STUDY: A monoclonal antibody (7B101D10), with specificity for the core region of GnRH-I was used to stain formalin-fixed tissue sections from adult male Sprague-Dawley rats, while a biotinylated GnRH-I sequence was used with avidin-labelled HRP to evaluate regions of GnRH-I binding. RESULTS AND CONCLUSIONS: GnRH-I expression was only found in the hypothalamus, cerebellum, anterior/fore brain and in Sertoli cells, while, binding activity was only present in the pituitary, subendocardium and subepicardium, thymic lymphocytes, peripheral blood lymphocytes and neutrophils. There was overlap in the olfactory neurons, liver (Kupffer macrophages and hepatocytes), spleen (lymphocytes and dendritic cells), myocardium and testes (spermatozoa and Leydig cells) and this may be further evidence of the paracrine/autocrine activity of a neuropeptide.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Isoformas de Proteínas/metabolismo , Receptores LHRH/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Sitios de Unión/inmunología , Ensayo de Inmunoadsorción Enzimática , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/inmunología , Inmunohistoquímica , Masculino , Isoformas de Proteínas/inmunología , Ratas , Receptores LHRH/inmunologíaRESUMEN
PROBLEM: We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH (des-1 GnRH-I), conjugated to tetanus toxoid via a carbodiimide reaction. The castration effect on the reproductive system was not consistent in all the treated animals. Therefore, we examined the possibility that conjugation to the carrier protein via the N- or C-terminal could have an effect on efficacy. METHOD OF STUDY: GnRH analogue sequences were synthesized consisting of an additional cysteine at either terminal and specific conjugation was carried out using a bifunctional linker agent. RESULTS: Conjugation of the monomer through the N-terminal proved to be a highly effective means of causing immunocastration in terms of decreased gonadotrophin and testosterone concentrations and testicular size, whereas conjugation through the C-terminal proved to be ineffective. This was reflected in the ability of the antibodies to bind native GnRH, but not the levels of the anti-GnRH antibodies. CONCLUSION: Immunoneutralization efficacy was attributed to the importance of preserving the GnRH C-terminal.