RESUMEN
The relationship between epilepsy and the presence of visceral larva migrans caused by Toxocara canis in Mexican children remains uncertain; however, this relationship needs to be elucidated because these parasite larvae can invade the human central nervous system. Accordingly, this study aimed to determine the frequency and specificity of anti-T. canis antibodies in the sera of children with epilepsy to determine the relationship between this parasite and epilepsy. The sera samples of 214 children were examined: 111 children diagnosed with epilepsy and 103 clinically healthy children without neurological disorders. In the sera of each group, the presence and specificity of anti-T. canis and anti-Ascaris lumbricoides antibodies, as well as the cross-reactivity between them, were assessed using enzyme-linked immunosorbent assay and Western blotting analysis. Among the children with epilepsy, 25.2% exhibited seropositivity to T. canis. Cross-reactivity against the A. lumbricoides antigen was present in 46.8% of the children with epilepsy, whereas 11.7% of the children with epilepsy and anti-T. canis antibodies did not exhibit cross-reactivity against this antigen. The Western blotting analysis of the sera from the children with epilepsy demonstrated the presence of T. canis proteins, with molecular weights of 24, 35, 55, 70, 120 and 210 kDa, and A lumbricoides proteins with molecular weights of 70, 80 and 110 kDa. Our results revealed the presence of anti-T. canis antibodies in the children with epilepsy; furthermore, cross-reactivity tests with A. lumbricoides showed the importance of the presence of anti-T. canis antibodies in revealing the relationship between this parasite and epilepsy in children.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Epilepsia/parasitología , Larva Migrans Visceral/inmunología , Adolescente , Animales , Antígenos Helmínticos/sangre , Antígenos Helmínticos/inmunología , Western Blotting , Estudios de Casos y Controles , Niño , Preescolar , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Epilepsia/inmunología , Femenino , Humanos , Lactante , Larva , Larva Migrans Visceral/complicaciones , Masculino , México , Toxocara canisRESUMEN
Hepatitis B core antigens (HBcAg) and hepatitis B surface antigens (HBsAg) are the main structural antigens of hepatitis B virus (HBV). Both antigens are potent immunogens for experimental animals as well as in acutely infected patients. A novel formulation based on the combination of HBsAg and HBcAg has been developed as a therapeutic vaccine candidate, aimed at inducing an immune response capable of controlling the infection. An immunization schedule was conducted to evaluate the immunogenicity of this formulation after simultaneous immunization by the intranasal and parenteral routes using different schedules and doses. Humoral and cellular immune responses generated in blood and spleen were evaluated by engyme-linked immunosorbent assay (ELISA) and enzyme-liked immunospot (ELISPOT) assays respectively. A first experiment evaluated two groups of mice simultaneously immunized by intranasal (IN) and subcutaneous (SC) routes, one including alum by SC route and, in the other, the formulation was injected without adjuvant. As a result, alum adjuvant did not increase the immunogenicity under the studied conditions. In fact, the group without alum induced the most potent immune response. The immune response was enhanced by combining IN and SC immunization compared to the SC route alone. In a second experiment, mice were immunized by different mucosal routes at the same time, and compared to the simultaneously (IN/SC) immunized groups. It was demonstrated that there is no improvement on the resulting immune response by using multiple routes of immunizations simultaneously; however, the increase of the antigen dose induced a superior immune response. Interestingly, the increase of antigen dose only by SC route did not favor the resulting immunogenicity. In conclusion, the use of HBsAg transgenic mice has proven useful to optimize the formulation, avoiding the unnecessary use of alum as adjuvant as well as provided information of the role of different mucosal immunization routes and antigen dose on the resulting immune response. How to cite this article: Trujillo H, Blanco A, García D, Freyre F, Aguiar J, Lobaina Y, Aguilar JC. Optimization of a Therapeutic Vaccine Candidate by Studying Routes, Immunization Schedules and Antigen Doses in HBsAg-positive Transgenic Mice. Euroasian J Hepato-Gastroenterol 2014;4(2):70-78.
RESUMEN
The development of new adjuvants for human vaccines has become an expanding field of research in the last thirty years, for generating stronger vaccines capable of inducing protective and long-lasting immunity in humans. Instead of such efforts, with several adjuvant strategies approaching to requirements for their clinical application, limitations like adjuvant toxicity remain to be fully surpassed. Here we summarize the current status of adjuvant development, including regulatory recommendations, adjuvant requirements, and adjuvant categories like mineral salts, tensoactive compounds, microorganism-derived adjuvants, emulsions, cytokines, polysaccharides, nucleic acid-based adjuvants, and a section dedicated to particulate antigen delivery systems. The mechanisms of adjuvanticity are also discussed in the light of recent findings on Toll-like receptors' biology and their involvement on immune activation.
Asunto(s)
Adyuvantes Inmunológicos , Receptores Toll-Like , Vacunas , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/clasificación , HumanosRESUMEN
The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens; however, the mucosal immunogenicity of HBcAg has been poorly studied. In this study, we explored the immunogenicity and the immunoenhancing effect elicited by two different variants of the recombinant complete nucleocapside of HBV in mice by intranasal route. For this purpose, we used as co-administered antigen, the HBV surface protein (HBsAg) and the antibody response in sera was evaluated after each dose. To analyze the specificity of the generated antibody response, the recognition of lineal epitopes was evaluated on a cellulose membrane bearing 12 mer peptides covering the HBcAg sequence. The obtained results evidenced that the intranasal immunogenicity of both variants of HBcAg was similar and high, developing early responses of IgG. The immunoenhancing effect on the HBsAg-specific antibody response was also similar for both variants. The results of the recognition of lineal epitopes study evidenced a similar recognition pattern to all sera and vaginal lavages samples generated by the immunization of mice with the two variants of HBcAg, and also similar to a pool of human anti-HBcAg positive sera samples.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Epítopos/inmunología , Femenino , Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Humanos , Inmunoglobulina G/sangre , Ratones , Garrapatas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vagina/inmunologíaRESUMEN
The hepatitis B virus (HBV) core and surface antigens are potent immunogens in animal models and humans. They have been used in vaccine studies for prevention or therapy of HBV diseases and also as carrier molecules in new developments. In this study we explored the nasal immunogenicity of two different variants of the recombinant complete nucleocapsid (HBcAg) as well as their adjuvant effect on hepatitis B surface antigen (HBsAg). To characterize the immune response, the serum IgG antibody response was tested during one year against both antigens, and the serum and vaginal secretions were tested for recognition of linear epitopes of HBcAg for both HBcAg variants. The results obtained evidenced that the intranasal immunogenicity of both HBcAg variants was similar and high, developing early and long lasting IgG responses. A similar recognition pattern to all sera and vaginal washes samples was generated by the two variants of HBcAg, also similar to a pool of human anti-HBcAg positive sera. A synergistic effect in the enhancement of the immunogenicity for both antigens was evidenced in the combined formulation after nasal administration. Taken together, these results would be of interest in the design of more potent therapeutic and preventive vaccines complementing systemic and mucosal responses.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos contra la Hepatitis B/sangre , Humanos , Inmunoglobulina G/sangre , Ratones , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/inmunología , Mapeo PeptídicoRESUMEN
There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Hepatitis B/terapia , Células TH1/inmunología , Administración Intranasal , Animales , Formación de Anticuerpos , Enfermedad Crónica , Femenino , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/inmunologíaRESUMEN
The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Inmunidad Mucosa/efectos de los fármacos , Animales , Líquido del Lavado Bronquioalveolar/química , División Celular/inmunología , Relación Dosis-Respuesta Inmunológica , Vías de Administración de Medicamentos , Evaluación Preclínica de Medicamentos , Femenino , Anticuerpos contra la Hepatitis B/sangre , Antígenos del Núcleo de la Hepatitis B/genética , Vacunas contra Hepatitis B/genética , Inmunidad Mucosa/inmunología , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
The conjugation of synthetic peptides to carrier proteins is a widely used method for immunological studies. Different coupling agents have been described to form the conjugate with carrier proteins. In this paper, we demonstrate that the antibody response toward V3-based synthetic MAPs derived from HIV-1, JY1 isolate, conjugated to two different carrier proteins using either m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or beta-maleimidopropionic acid N-hydroxysuccinimide ester (MPS), or succinic anhydride (SA) show different behaviors. An excellent anti-JY1 response without a strong response to the coupling agent is observed in the case of succinic anhydride spacer. In contrast, MBS produces total abrogation of the antibody response with a high response toward the coupling agent.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Biosíntesis de Péptidos , Animales , Formación de Anticuerpos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Indicadores y Reactivos/farmacología , Ratones , Ratones Endogámicos BALB C , Péptidos/química , Proteínas/síntesis química , Anhídridos Succínicos/química , Succinimidas/químicaRESUMEN
Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.
Asunto(s)
Hepacivirus/química , Pichia/virología , Proteínas del Núcleo Viral/química , Virión/química , Immunoblotting , Peso Molecular , Renaturación de Proteína , Proteínas del Núcleo Viral/metabolismoRESUMEN
The ligands 4,7-diaza-2,3,8,9-dibenzo-15-crown-5 (L1), 4,10-diaza-2,3,11,12-dibenzo-18-crown-6 (L2), 4,10-diaza-2,3,11,12-di(4'-tert-butylbenzo)-18-crown-6 (L3) and N,N-di(methylenecarboxyethoxy) 4,10-diaza-2,3,11,12-dibenzo-18-crown-6 (L4) have been prepared. Partition coefficients and acid dissociation constants for these four diazadibenzocrown ether compounds were determined in water-chloroform. Their effectiveness was assessed in solvent extraction of Pb(2+) from aqueous solutions into toluene. Ligands L3 and L4 provide high selectivity for Pb(2+) over Cd(2+) and Zn(2+) in transport across plasticized cellulose triacetate membranes.
RESUMEN
BACKGROUND: The objective of this study was to analyze hospitalization costs, morbidity, disability, and mortality in patients with acquired immunodeficiency syndrome (AIDS) treated with protease inhibitors (PI). METHODS: This is a self-controlled, ambispective study of a total of 581 patients with human immunodeficiency virus (HIV)/AIDS seen at the Hospital de Infectología, Centro Médico La Raza, IMSS, in Mexico City during 1997. A total of 210 (36.14%) patients initiated protease inhibitor (PI) treatment at the onset of the study. Thirty-eight patients satisfied the inclusion criteria for this study and were analyzed retrospectively during the year prior to PI treatment, and then prospectively throughout the year on PI treatment. As concerns main outcome measures, financial costs, number of hospitalizations, number of infections, and productivity and laboratory parameters (CD4(+) counts and viral load) were analyzed during the year prior to PI treatment and then prospectively during the year on PI prescription. Our hypothesis was that the hospital costs, morbidity, disability, and mortality of patients with AIDS decreased while on PI treatment. RESULTS: During the year prior to PI prescription, the 38 patients enrolled in the study were admitted on a total of 59 occasions (1.55 hospitalizations/patient), whereas during the year on PI therapy, all 38 patients had only seven admissions (0.18 hospitalizations/patient). Hospitalization costs decreased 35% when annual PI costs for the 38 patients studied were taken into account. The number of microorganisms detected during hospitalization decreased from 24 prior to PI to five on PI. The number of disability days involved in patients on PI decreased significantly (p <0.0002). None of the 38 patients studied died during the year of follow-up under PI treatment. Mortality decreased significantly, from 116/481 (23.2%) in 1996, to 77/581 (13.2%) in 1997, to 40/740 (6.4%) in 1998. There were no deaths among the 38 patients studied during the 1-year follow-up period; when the observation period was extended 1 additional year, only one patient died (2.63%). Only six (3.48%) of the 172 PI-treated patients with AIDS not included in the study died during the same period. CD4(+) cell counts increased from 190.56 +/- 169.5 cells/mm(3) to 235.00 +/- 112.65 cells/mm(3) (p <0.05) after 12 months of PI treatment. Viral loads decreased from 5 logs to 2.4 logs at 12 months of PI treatment (p <0.001). CONCLUSIONS: Introduction of PI to antiretroviral treatment in patients with AIDS was associated with a lower rate of hospital admissions, lower costs, and a lesser number of infections/year, disabilities, and mortalities. Increase of CD4(+) cell counts and decrease in viral loads in the 38 patients were associated with decreased morbility and mortality.