Cholera toxin modulates the systemic immune responses against Vibrio cholerae surface antigens after repeated inoculations

Fil: Fernandez-Miyakawa, Mariano E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Microbiología. Departamento de Contralor. Servicio de Vacunas Bacterianas; Argentina.Fil: Brero, María Luisa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Microbiología. Departamento de Contralor. Servicio de Vacunas Bacterianas; Argentina.Fil: Mateo, Nancy A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Microbiología. Departamento de Contralor. Servicio de Vacunas Bacterianas; Argentina.The immunomodulating properties of a low cholera toxin (CT) dose over the systemic antibody response against Vibrio cholerae antigens after a comparatively extensive period of time were evaluated. Groups of 10 mice were injected intraperitoneally three times at 0, 30 and 86 days with 500 microl of buffer or 10(8) viable recombinant V. cholerae bacteria (lacking cholera toxin A subunit) with or without 100 ng of CT. Sera were obtained from inoculated mice at 0, 14, 28, 37, 58, 80, 93, 114, 236 and 356 days after the first injection. Vibriocidal activity and IgM and IgG anti-lipopolysaccharide (LPS) or outer membrane protein (OMP) antibodies levels were estimated by ELISA in sera of inoculated mice. Anti-LPS IgG subclasses were measured 2 weeks after each immunization by ELISA. Treatment of mice with CT markedly influenced the immune response to LPS but not against OMP of V. cholerae. Simultaneous intraperitoneal administration of CT with V. cholerae resulted in marked enhancement of both IgM anti-LPS and vibriocidal titers which subsisted for a relatively extensive period of time after repeated antigen administration. No differences were observed in IgM and IgG anti-OMP titers after extended periods of time between CT and control treatments. A similar pattern of IgG anti-LPS subclasses was observed in the serum samples analyzed. These results suggest that long term CT administration modulates the IgM anti-V. cholerae LPS response and the serum vibriocidal activity

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies – IgY-technology – by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20–40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 μg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.Fil: Leiva, Carlos Leónidas. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Cangelosi, Adriana. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; ArgentinaFil: Mariconda, Virginia Laura. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; ArgentinaFil: Farace, Mariano Gabriel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Geoghegan, Patricia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Brero, María Luisa. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; ArgentinaFil: Fernandez Miyakawa, Mariano Enrique. Ministerio de Salud de la Nación. Administración Nacional de Laboratorios e Institutos de Salud; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Chacana, Pablo Anibal. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; Argentin

Structure and protective capacity of tetanus and diphtheria antibodies produced during human pregnancy and transferred to new-born

Fil: Pasetti, M F: Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Facultad de Farmacìa y Bioquímica, Universidad de Buenos Aires; Argentina.Fil: Dokmetjian, Christian. Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Facultad de Farmacìa y Bioquímica, Universidad de Buenos Aires; Argentina.Fil: Brero, María Luisa. Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Facultad de Farmacìa y Bioquímica, Universidad de Buenos Aires; Argentina.Fil: Eriksson, P V. Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Facultad de Farmacìa y Bioquímica, Universidad de Buenos Aires; Argentina.Fil: Ferrero, Fernando. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Manghi, M A. Hospital General de Niños “Pedro de Elizalde,” (1270) Buenos Aires; Argentina.PROBLEM: The structure and protective activity of antibodies against tetanus (anti‐T) and diphtheria (anti‐D), produced during human pregnancy and transferred to new‐born, was studied. METHOD: Antibody levels were measured by ELISA in non‐pregnant women (control group), primiparae, and multiparae, and in their children. The proportion of symmetric and asymmetric IgG molecules was determined and their respective protective capacity evaluated. RESULTS: The quantity of asymmetric anti‐T and anti‐D antibodies in mothers at the time of delivery was roughly four‐ and three‐fold that of the control group, respectively, dropping significantly 1 month later. A similar proportion of these antibodies was observed in the new‐born. The lower neutralizing capacity of asymmetric molecules was demonstrated in vivo. CONCLUSION: Results show that during pregnancy there is a modulation of the immune response with an increase in the production of asymmetric molecules of lower protective capacity

Whole-cell Bordetella pertussis vaccine component modulates the mouse immune response to an unrelated soluble antigen

Fil: Lavigne, María Victoria. Instituto de Estudios de la Inmunidad Humoral, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Castro, Marisa. Instituto de Estudios de la Inmunidad Humoral, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Mateo, Nancy A. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Deluchi, Silvana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Atzori, Carlos. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Piudo, Luciana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Calcagno, María. Departamento de Físico-Matemática, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Brero, María Luisa. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Manghi, Marcela. Instituto de Estudios de la Inmunidad Humoral, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires; Argentina.Several factors are involved in the selective activation of Th1 or Th2 cells, such as different physical characteristics of antigens and the type of antigen-presenting cells involved in the immune response, among others. To study the influence of a particulate antigen on Th1/Th2 cell differentiation during the immune response to another antigen, we analysed the immune response to tetanus toxoid (soluble antigen) in BALB/c mice immunized with one of the three following vaccines: tetanus and diphtheria toxoids (DT), or DT associated with whole-cell Bordetella pertussis or its soluble antigens (DTPw and DTPa, respectively). Similar total antibody levels were observed for all vaccines. DT vaccine showed a higher IgG1/IgG2a ratio than the similar values observed for DTPw and DTPa vaccines. DT- and DTPa-primed spleen cells showed a Th2 (IL-5) profile while a Th1/Th2 (IFN gamma, IL-5) profile was observed for DTPw. IL-6 was only produced by DTPw-primed cells. Besides, IL-12 levels induced by DTPw were three times higher than the ones induced by both DT and DTPa. Our findings indicate that whole-cell B. pertussis priming modifies the tetanus immune response from Th2 to Th1/Th2 type probably via inflammatory mechanisms. In addition, in the light of conflicting reports regarding the mechanisms of protection induced by DTP vaccines, we studied the pertussis immune response. Only DTPw immunization generated memory T cells capable of proliferating with B. pertussis as an in vitro stimulus. Results might indicate that these cells may not play a key role in protecting against B. pertussis when the host is vaccinated with DTPa

Cytokines induced by experimental anti-tetanus immunization. Vaccine formulation effect

Fil: Castro, Marisa. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Fil: Mateo, Nancy A. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Lavigne, Victoria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Fil: Deluchi, Silvana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Atzori, Carlos. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Piudo, Luciana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Brero, María Luisa. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Manghi, Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Several factors are involved in the selective activation of T helper 1 or T helper 2 cells, such as the type of antigen-presenting cells involved in the immune response and the different physical characteristics of antigens. The aim of this work was to evaluate if adding other antigens to tetanus toxoid modifies the original immune response. BALB/c mice were immunized with tetanus and diphtheria toxoids associated with whole-cell Bordetella pertussis (DTPw vaccine), B. pertussis soluble antigens (DTPa vaccine) or Salmonella typhi plus DTPa (DTPaSt vaccine). DTPw and DTPaSt immunization induced a T helper 1/T helper 2 (Th1/Th2) anti-tetanus response with gamma interferon and interleukin 5 production. DTPa immunization induced a Th2 response with production of interleukin 5 and interleukin 6. Only DTPw vaccine induced higher levels of IL-12 in non-immunized mice. Our findings indicate that the co-injection of whole-cell antigens such as B. pertussis or S. typhi, modifies the anti-tetanus response shifting it from Th2 to Th1 type. However, the original Th2 immune response is not modified when the vaccine consists only of soluble antigens

Cytokines induced by experimental anti-tetanus immunization. Vaccine formulation effect

Fil: Castro, Marisa. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Fil: Mateo, Nancy A. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Lavigne, Victoria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Fil: Deluchi, Silvana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Atzori, Carlos. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Piudo, Luciana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Brero, María Luisa. ANLIS Dr.C.G.Malbrán. Centro Nacional de Control de Calidad de Biológicos; Argentina.Fil: Manghi, Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral; Argentina.Several factors are involved in the selective activation of T helper 1 or T helper 2 cells, such as the type of antigen-presenting cells involved in the immune response and the different physical characteristics of antigens. The aim of this work was to evaluate if adding other antigens to tetanus toxoid modifies the original immune response. BALB/c mice were immunized with tetanus and diphtheria toxoids associated with whole-cell Bordetella pertussis (DTPw vaccine), B. pertussis soluble antigens (DTPa vaccine) or Salmonella typhi plus DTPa (DTPaSt vaccine). DTPw and DTPaSt immunization induced a T helper 1/T helper 2 (Th1/Th2) anti-tetanus response with gamma interferon and interleukin 5 production. DTPa immunization induced a Th2 response with production of interleukin 5 and interleukin 6. Only DTPw vaccine induced higher levels of IL-12 in non-immunized mice. Our findings indicate that the co-injection of whole-cell antigens such as B. pertussis or S. typhi, modifies the anti-tetanus response shifting it from Th2 to Th1 type. However, the original Th2 immune response is not modified when the vaccine consists only of soluble antigens

Development of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure anti-tetanus toxoid antibody levels in immunized guinea-pig sera as a useful alternative to the currently used toxin neutralization test (TNT) in determining the activity of the tetanus toxoid in vaccines. The ELISA was found to measure antibody levels as low as 5.8 x 10(-5) IU/ml. Furthermore, a comparison of the results from ELISA and TNT involving 132 different commercial vaccines showed a very good correlation (r = 0.94, p < 0.001) between antibody levels measured by both methods. The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in mice for assessing the activity of tetanus toxoids in vaccines

Development of an alternative method for testing the immunogenicity of diphtheria vaccines

The immunogenicity of the diphtheria component of 73 commercial vaccines from five different manufacturers was tested by the toxin neutralization test (TNT) and the enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. A comparison of the antibody levels measured by both assays showed a very good correlation (r = 0.95, p < 0.001). The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in guinea pigs. Also, the ELISA was found to measure IgG antibody levels as low as 5.5 x 10(-5) IU ml-1. To evaluate the possibility of accelerating the active immunization during the activity test of vaccines, an alternative schedule using one single human dose was assayed. A very good correlation was observed between the IgG antibody response obtained with this schedule and with the traditional programme. Therefore, the cost and the time required to perform the activity test may be considerably reduced when both the rapid immunization schedule and the ELISA are used

National production of certified reference fungal cultures

Fil: Davel, Graciela Odelsia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Mazza, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Refojo, N. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Taverna, C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Rivas, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Fernandez, J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Vivot, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Mazza, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Centro Nacional de Control de Calidad de Biológicos (CNCCB); ArgentinaFil: Mastomónaco, G. Instituto Nacional de Tecnología Industrial; Argentina.Fil: Bueno, N. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; ArgentinaFil: Molina, V. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Brero, M L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Centro Nacional de Control de Calidad de Biológicos (CNCCB); ArgentinaFil: Brero, M L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Centro Nacional de Control de Calidad de Biológicos (CNCCB); ArgentinaFil: Kornblit F. Instituto Nacional de Tecnología Industrial; Argentina.Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short- and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20±5°C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry